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.


Anisotropic graphene domains are of significant interest since the electronic properties of pristine graphene strongly depend on its size, shape, and edge structures. In this work, considering that the growth of graphene domains is governable by the dynamics of the graphene-substrate interface during growth, we investigated the shape and defects of graphene domains grown on copper lattices with different indices by chemical vapor deposition of methane at either low pressure or atmospheric pressure. Computational modeling identified that the crystallographic orientation of copper strongly influences the shape of the graphene at low pressure, yet does not play a critical role at atmospheric pressure. Moreover, the defects that have been previously observed in the center of four-lobed graphene domains grown under low pressure conditions were demonstrated for the first time to be caused by a lattice mismatch between graphene and the copper substrate. For instance, graphene domains grown under LPCVD conditions typically has a dendritic four- or six-lobed structure, whereas under APCVD conditions hexagonal domains with six-fold symmetry are dominantly formed, with their edges macroscopically oriented parallel to the zigzag directionsIn this study, we investigated a correlation between the surface diffusion of carbon adatoms and the growth of anisotropic graphene domains synthesized using a CVD method. We varied the reaction pressure in CVD synthesis, which in turn caused variations in the growth mechanism and kinetics, and found that the crystallographic orientation of a copper substrate differently influences the shape and defects of graphene domains formed on it under LPCVD and APCVD conditions. We also calculated the energy barrier for surface diffusion of carbon adatoms on different Cu lattices, and propose a surface diffusion directed growth of anisotropic graphene domains on different Cu lattices with experimental observations.We synthesised graphene domains on Cu foil by CVD method under low and atmospheric pressures, using methane as the carbon precursor gas11−1) peak, and thus the ratio of the 2D (~2680 cm−1) to G (~1580 cm−1) peak intensity (I2D/IG) and D to G peak intensity (ID/IG) was determined for each of the graphene domains produced. Their spatial dependencies (Raman maps) are given in 2D/IG values are greater than 2.0. And, D/IG, in which the higher value under LPCVD (~0.4) than APCVD (~0.2) indicates a higher defects density in the former. This was attributed to the greater likelihood of Cu sublimation under low pressure, with recent reports suggesting that copper at 1000 °C in a vacuum has an evaporation rate approaching as high as 4 μm/h, while at higher pressures, sublimation of copper is suppressed18D/IG of ~0.7, with additional Raman maps of this region provided in the D/IG value in our case is higher than that of normal CVD graphene (~0.05). This is due to the fact that partial graphene growths have more Raman active edge19D/IG value of general graphene film in our system is in the Micro Raman maps were used to further identify the uniformity of the graphene domains produced. Typically, the presence of defects is indicated by the presence of an additional D  images were obtained and are shown in Computational modeling for the diffusion of carbon adatom on the Cu (001) and Cu (102) lattice orientations was used to further explore how the Cu lattice influences the shape of the four- and six-lobed graphene domains, respectively, at low pressure . In Fig.This change in the influence of the crystallographic orientation of Cu substrate on the anisotropic growth of graphene domains with reaction pressure is due to a variation in the pressure of carbon precursor gas, which provides an explanation as to why the Cu substrate does not always have the same effect. For example, in the case of LPCVD, carbon adatoms diffuse only along those directions with a low energy barrier, which these being determined by the crystallographic orientation of Cu substrate  lattice. That is, the defects produced by this symmetry mismatch provide an activation site for further reaction, with hydrogen in particular having been identified as both an activator for the formation of surface-bound carbon and an etching reagent that affects the shape of the graphene domainsIn summary, we have demonstrated that the crystallographic orientation of Cu substrate differently influences the shape and defects of graphene domains formed on it. Under low pressure, the growth of graphene domains is governed by the underlying Cu substrate, resulting in four- and six-lobed structures on Cu (001) and Cu (102) lattice, respectively. Experimental observations of such structures were corroborated by computational modeling, which identified the preferential diffusion route of carbon adatoms on each copper lattice. In contrast, hexagonal graphene domains were found to form over the entire Cu substrate under atmospheric pressure, suggesting that growth under these conditions is determined by the intrinsic symmetry in the atomic structure of the graphene. Based on these results, the holes and multilayered spots that have frequently observed in the center of four-lobed graphene domains in the past are considered to be due to defects originated from the lattice mismatch between the intrinsic six-fold symmetry of graphene and the preferential surface diffusion of carbon adatoms on Cu (001) lattice. We believe that our research on the surface diffusion directed graphene growth can greatly improve graphene-based engineering by allowing controlled alignment of individual graphene domains, in addition to providing greater insight into producing high-quality large area graphene for use in transparent conducting electrodes, sensors, and nano-electronic devices.2 pressure of 40 mTorr was kept with 2 sccm flow. Then, 35 sccm of CH4 was introduced in the tube for 10 minutes at a total pressure of 500 mTorr. After exposure to methane, the furnace was cooled to room temperature. The cooling rate was 20 °C/sec.Graphene was synthesized by copper-catalyzed chemical vapor deposition under low and atmospheric pressure, using methane as the carbon containing precursor gas. The Cu foil was loaded in the 1 inch quartz tube of tube furnace for annealing process. The tube was evacuated, back filled with hydrogen, and heated to 1000 °C. It was annealed for 1 h. For APCVD, a methane/argon gas mixture (50 ppm methane) of 300 sccm was allowed to flow across the copper at a temperature of 1000 °C, while maintaining 20 sccm hydrogen. The precursor supply time was 15 min during graphene growth. The sample was left to rapidly cool under a hydrogen and argon atmosphere. And, for LPCVD, the temperature was maintained to 1000 °C, and a H2/Si wafer for Raman spectroscopy. A thin layer of PMMA  was spin-coated on an as-synthesized sample at 3000 rpm for 1 min. Since both Cu surfaces were exposed to CH4, graphene was grown on both sides of the Cu foil. Excessive PMMA on the back side of PMMA coated graphene was removed by acetone. Subsequently, the sample was placed in a 0.1 M aqueous (NH4)2SO4 to etch off the Cu foil. Generally, the etching process runs overnight. After the Cu foil was completely etched away, graphene with PMMA coating was scooped out from the solution and transferred it into DI water for 10 min to wash away remaining etchants (done three times). Then, SiO2/Si wafer was dipped into water and the film was picked up. The PMMA was then removed with acetone and the sample was rinsed several times with DI water.After growth, graphene was transferred by a PMMA-assisted wet-transfer method on 300 nm SiOScanning electron microscopy (SEM) images of the synthesized graphene domains were obtained using a field-emission SEM system (JEOL 7600F) operated at acceleration voltages of 10 to 20 kV. Atomic force microscopy (AFM) and electron backscatter diffraction (EBSD) analyses were performed to identify the surface roughness and crystallographic nature of the Cu foil samples, respectively. Raman spectra of the synthesized graphene samples were obtained using a laboratory-made micro-Raman spectroscopy system; the system used the 514.5 nm line of an Ar ion laser as excitation source with a power of ~1 mW. The heating effect of the laser could be neglected at this power level. The laser beam was focused onto the graphene sample using a 50× microscope objective lens . The collected scattered light was dispersed using a Shamrock SR 303i spectrometer (1200 grooves/mm) and was detected using a charge-coupled device (CCD) detector.The obtained Cu crystal structures were optimized using density functional theory (DFT) calculations carried out with Dmol package included in materials studio. The calculation of total energy and electronic structure was followed by the geometry optimization using the generalized gradient approximation (GGA) with the Perdew–Burke–Ernzerhof (PBE) functional. The density functional semi-core pseudopotential (DSPP) was employed for the Cu atoms to take relativistic effects into account, while all electrons were considered for the carbon atoms. Further, using linear synchronous transit (LST) and quadratic synchronous transit (QST) methods, a new transition state (TS) search technique for stable configurations of carbon atom is developed, in which the stable configuration is independent on selected initial configurations. Double numerical atomic basis sets augmented with the polarization function (DNP) were used to describe the valence electrons. Two Cu surfaces were simulated using six layer with 54 atoms for (001) and eight layer with 72 atoms for Cu (102), respectively. Each system was sampled with nine (3×3 × 1) in-plane k-points and a vacuum of 10 Å.How to cite this article: Jung, D. H. et al. Surface Diffusion Directed Growth of Anisotropic Graphene Domains on Different Copper Lattices. Sci. Rep.6, 21136; doi: 10.1038/srep21136 (2016).


Dendropanax morbifera Léveille is used in herbal medicine as a cancer treatment. In this study, we investigated the effects of Dendropanax morbifera stem extract (DMS) on cadmium (Cd) excretion from the blood and kidney and brain tissues of rats exposed to cadmium, as well as the effects of DMS on oxidative stress and antioxidant levels in the hippocampus after Cd exposure.Seven-week-old Sprague-Dawley rats were exposed to 2 mg/kg of cadmium by intragastric gavage and were orally administered 100 mg/kg of DMS for 4 weeks. Animals were sacrificed and Cd determination was performed using inductively coupled plasma mass spectrometry. In addition, the effects of Cd and/or DMS on oxidative stress were assayed by measuring reactive oxygen species production, protein carbonyl modification, lipid peroxidation levels, and antioxidant levels in hippocampal homogenates.S-transferase (GST) activity in the hippocampus were significantly decreased after exposure to Cd, and administration of DMS significantly inhibited these Cd-induced changes.Exposure to Cd significantly increased Cd content in the blood, kidneys, and hippocampi. DMS treatment significantly reduced Cd content in the blood and kidneys, but not in the hippocampi. Exposure to Cd significantly increased reactive oxygen species production, protein carbonyl modification, lipid peroxidation, total sulfhydryl content, reduced glutathione content, and glutathione reductase activity. In contrast, Cu, Zn-superoxide dismutase (SOD1), catalase (CAT), glutathione peroxidase (GPx), and glutathione-S-transferase activities.These results indicate that DMS facilitates cadmium excretion from the kidneys, reduces cadmium-induced oxidative stress in the hippocampus, and modulates SOD1, CAT, GPx, and glutathione- Dendropanax morbifera Léveille is mainly distributed in southwestern Korea. This tree was initially used for the production of golden varnish [Dendropanax morbifera leaves have anti-complement, anti-diabetic, and anti-atherogenic activities [ varnish , but the varnish . The poltivities , 3–5.Cadmium (Cd) is one of the most harmful heavy metals in nature and it is released into the environment through mining and smelting. The main sources of Cd exposure include production of certain types of batteries, intake of contaminated food and water, and inhalation of tobacco smoke or polluted air , 7. Cd eDendropanax morbifera stem extract (DMS) decreases 2,2-diphenyl-1-picrylhydrazyl radical levels, and its antioxidant capacity is greater than that of other organ extracts from D. morbifera[D. morbifera, significantly decreases rotenone-induced generation of reactive oxygen species in SH-SY5Y cells and mitochondrial membrane potential depletion [Several approaches have been used to promote or facilitate the excretion of Cd from the body because it has a long biological half-life (approximately 30 years in humans) and readily accumulates in tissues . Debarkeorbifera. In addiepletion . HoweverMale Sprague-Dawley rats were purchased from Orient Bio Inc. . Rats were housed in a conventional animal facility at 23°C with 60% humidity, a 12 h/12 h light/dark cycle, and free access to food and tap water. The handling and care of the animals conformed to the guidelines established in order to comply with current international laws and policies , and were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU-130522-1). All of the experiments were conducted with an effort to minimize the number of animals used and the suffering caused by the procedures used in the study.Dendropanax morbifera Léveille was purchased from a local market on Jeju Island in Korea. The plant was authenticated by two practitioners of traditional Asian medicine, and a voucher specimen was deposited with Egreen Co. Ltd. (deposition number: 2013-001). Stems from the plant samples (100 g) were chopped, blended, soaked in 2 L of 80% ethanol, and refluxed three times at 20°C for 2 h. The insoluble materials were removed by centrifugation at 10,000 × g for 30 min, and the resulting supernatant was concentrated and freeze-dried to yield a powder (yield: 4.7%). Before each experiment, the dried extract was dissolved in distilled and deionized water.Fresh n = 29 in each group): a control group, a group treated with 100 mg/kg DMS, a group treated with 2 mg/kg Cd, and a group treated with Cd and DMS. Cd and/or DMS were administered orally to 7-week-old rats once a day for 4 weeks.Cadmium chloride was obtained from Sigma-Aldrich . Animals were divided into 4 treatment groups (n = 7 in each group) were anesthetized with 100 mg/kg of Zoletil 50®  and the blood, hippocampi, and kidneys were extracted. Blood samples were allowed to clot, after which they were centrifuged for 30 min at 1,000 g to separate the serum. Hippocampi and kidneys were weighed in glass vessels, and tissues were digested by adding 3-8 mL of HNO3 at 130°C for 3 h, after which 2 mL of H2O2 was added and the samples were heated for 1 h. Serum and digested samples were transferred to polypropylene flasks for Cd determination. Cd determination was performed by using inductively coupled plasma mass spectrometry .To measure Cd concentration in the blood and brain and kidney tissues, control, DMS-, Cd-, and Cd-DMS-treated rats (n = 5 in each group) were assessed using the fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA) [The effects of Cd and/or DMS on ROS production in the hippocampus of control, DMS-, Cd-, and Cd-DMS-treated rats (DCFH-DA) . IntraceDCFH-DA) . MitochoDCFH-DA)  using bon = 5 in each group) were assessed by measuring malondialdehyde (MDA) formation using the Bioxytech MDA-586 kit . Briefly, bilateral hippocampi were collected using a surgical blade and the left part was homogenized in 20 mM PBS (pH 7.4) containing 5 mM butylated hydroxytoluene. After centrifugation of the homogenates at 3000 × g for 10 min at 4°C, the supernatants were collected. For each reaction, 10 μL of probucol and 640 μL of diluted R1 reagent (1:3 of methanol:N-methyl-2-phenylindole) were added and mixed with 150 μL of 12 N HCl. Each reaction was incubated at 45°C for 60 min and centrifuged at 10,000 × g for 10 min. The supernatant was collected and MDA formation was determined by measuring the absorbance at 586 nm. MDA data were normalized to the protein concentration of each sample.The effects of Cd and/or DMS on lipid peroxidation levels in control, DMS-, Cd-, and Cd-DMS-treated rats (n = 5 in each group) by the Levine method [3 L/mol cm), and results were expressed as nmol/mg of protein.To elucidate the effects of Cd and/or DMS on protein modification, carbonylation of proteins as a result of oxidative stress was determined in control, DMS-, Cd-, and Cd-DMS-treated rats (e method . The coln = 7 in each group). Briefly, the right part of the hippocampus (matching the right part used for measurement of lipid peroxidation) was homogenized in 10 mM Tris buffer containing 1 mM EDTA and 1 mM PMSF. The homogenates were centrifuged at 600 × g for 10 min, and then centrifuged at 13,000 × g for 20 min at 4°C.To elucidate the effects of Cd and/or DMS on Cu, Zn-superoxide dismutase 1 (SOD1), catalase (CAT), and glutathione peroxidase (GPx), the activity of these enzymes was measured in control, DMS-, Cd-, and Cd-DMS-treated rats . The gel was exposed to a fluorescent light source until the bands showed maximum resolution.SOD1 activity was measured by monitoring its capacity to inhibit the reduction of ferricytochrome ridovich . SOD1 acridovich . Briefly2O2 degradation in 10 mM of potassium phosphate buffer (pH 7.0) according to Aebi’s method [2O2 consumed per min and the specific activity is reported as units per mg of protein.CAT activity was assayed at 25°C by determining the rate of Hs method . An extit-butyl-hydroperoxide as a substrate according to the method of Maral et al. [GPx activity was assayed by measuring nicotinamide adenine dinucleotide phosphate (NADPH) oxidation with l et al. . Brieflyn = 5 in each group) were investigated using the tissue samples obtained for the measurement of protein carbonyl levels. Glutathione-S-transferase (GST) activity was determined spectrophotometrically using 1-chloro-2,4-dinitrobenzene as a substrate [The effects of Cd and/or DMS on glutathione-related enzymes in the hippocampus of control, DMS-, Cd-, and Cd-DMS-treated rats (ubstrate . Glutathubstrate .All data are expressed as mean ± standard error of the mean (SEM). To determine the effects of Cd and DMS, differences between the means were statistically analyzed using two-way analysis of variance (ANOVA) with repeated measures and Bonferroni’s post hoc test.The ICP-MS method was used to determine Cd concentrations in serum, kidney, and hippocampal tissues. Cd concentrations in serum, kidney, and hippocampal tissues were similar in the control and DMS-treated groups. In the Cd-treated group, Cd concentrations in the serum and kidney were significantly higher than those in the control group. In contrast, there was no significant difference in the Cd concentration in the hippocampus between the control and DMS-treated groups, although the level was increased 1.57-fold in the control group. In the Cd-DMS-treated group, Cd concentrations in the serum and kidneys were significantly lower than those in the Cd-treated group (Table DCF fluorescence levels were similar in the control and DMS-treated groups. DCF fluorescence was significantly increased 1.71-fold) in the Cd-treated group in comparison with that of the control group. However, the MDA level of the Cd-DMS-treated group was significantly decreased to 74.4% of that of the Cd-treated group  in comparison with that of the control group. The protein carbonyl level of the Cd-DMS-treated group was significantly decreased to 57.4% of that of the Cd-treated group Figure B.MDA levels in hippocampal homogenates were 25.8 and 25.4 nmol/mg protein in the control and DMS-treated groups, respectively. The MDA level of the Cd-treated group was 2.2-fold higher than that of the control group. However, the MDA level of the Cd-DMS-treated group was significantly lower (39.1 nmol/mg protein) than that of the Cd-treated group  level, and the activities of GR and GST were similar in the hippocampal homogenates. In the Cd-treated group, TSH level and GR activity were significantly decreased to 53.5% and 37.8% of those of the control group, respectively. In contrast, GST activity in the Cd-treated group was significantly increased to 147.7% of that of the control group. In the Cd-DMS-treated group, TSH level and GR activity were significantly increased to 172.9% and 174.7% of those of the control group, respectively. GST activity in the Cd-DMS-treated group was significantly decreased to 58.5% of that of the Cd-treated group (Figure Cd causes toxicity by disturbing the cellular homeostasis of essential metal ions such as copper and zinc. In addition, Cd affects antioxidant enzymes, which use copper and zinc, through oxidation or peroxidation in the brain . In contDendropanax morbifera Léveille significantly decreases rotenone-induced generation of reactive oxygen species in SH-SY5Y cells [Cd can affect the integrity and permeability of the vascular endothelium, penetrate the blood-brain barrier, and accumulate in the brain –28. In t5Y cells .In this study, we also determined the effects of DMS and/or Cd on the activity of SOD1, CAT, and GPx in the hippocampus because Cd has been shown to inhibit antioxidant enzymes –38. We fWe focused on glutathione-related enzymes because the increase in activity after DMS treatment in Cd-exposed rats was most prominent for GPx, among the antioxidative enzymes. Exposure to Cd significantly decreased GR activity, TSH level, and GSH level, while GST activity was significantly increased. The decrease of GR activity, TSH level, and GSH level after Cd exposure could be responsible for increased ROS levels and subsequent lipid peroxidation and protein carbonylation in the brain, because GSH can directly scavenge ROS or act as a substrate for GPx and GST in the detoxification of hydrogen peroxide . DMS admThe administration of DMS promotes the excretion of Cd from the kidney and ameliorates Cd-induced increases in ROS, lipid peroxidation, and protein carbonyl levels, which are modified by oxidative stress. In addition, DMS efficiently attenuates Cd-induced deficits in SOD1, CAT, GPx, and glutathione-related enzyme activities in the hippocampus.


Graphene, whose absorbance is approximately independent of wavelength, allows broadband light–matter interactions with ultrafast responses. The interband optical absorption of graphene can be saturated readily under strong excitation, thereby enabling scientists to exploit the photonic properties of graphene to realize ultrafast lasers. The evanescent field interaction scheme of the propagating light with graphene covered on a D-shaped fibre or microfibre has been employed extensively because of the nonblocking configuration. Obviously, most of the fibre surface is unused in these techniques. Here, we exploit a graphene-clad microfibre (GCM) saturable absorber in a mode-locked fibre laser for the generation of ultrafast pulses. The proposed all-surface technique can guarantee a higher efficiency of light–graphene interactions than the aforementioned techniques. Our GCM-based saturable absorber can generate ultrafast optical pulses within 1.5 μm. This saturable absorber is compatible with current fibre lasers and has many merits such as low saturation intensities, ultrafast recovery times, and wide wavelength ranges. The proposed saturable absorber will pave the way for graphene-based wideband photonics. Ultrafast lasers that produce pico- to femto-second optical pulses have many potential applications, such as in basic scientific research, materials processing, electronic components, metrology, telecommunication, and medicine1234678910111213et al. proposed using an ultrathin graphene sheet as an SA in an ultrafast fibre laseret al. incorporated graphene flakes into polyvinyl alcohol (PVA) and demonstrated the use of a graphene–PVA nanocomposite film as an SA1120212225Graphene, a single sheet of carbon atoms forming a honeycomb crystal lattice, exhibits a variety of exceptional electronic and photonic properties1516et al.To enhance the interaction of graphene with the evanescent field of propagating light, it is imperative to explore new operating schemes of graphene-based SAs with a nonblocking configuration. When a layer of graphene is wrapped around a microfibre , the allThe microfibre, with a minimum diameter of ~6 μm, is fabricated via a flame-brushing technique and integrated onto a hollow glass substrate, as shown in x0 is the position of the microfibre at the minimum diameter. The circles are the experimental data, which are fitted with a polynomial function of degree 4 (red solid curve). The polynomial equation that produces the best fit is D = 5.8465 − 0.02548 · Δx + 0.27112 · Δx2 − 4.54967 · 10−4 · Δx3 + 9.33575 · 10−4 · Δx4, where D is the diameter of the microfibre and Δx = x − x0 is the relative position of the microfibre. The minimum diameter of the GCM is approximately 6 μm. −1 exhibits a single Lorentzian feature with a narrow full width at half maximum (FWHM) of ~30.6 cm−1, and the intensity of the 2D band is much larger than that of the G band, with a 2D-to-G intensity ratio of ~3, as shown in 313234350), the nonsaturable absorption (αns), and the saturation intensity (Isat) are approximately 4.37%, 95.27%, and 1.51 GW/cm2, respectively.Pair/P) for the fundamental mode  along the diameter of the microfibre , respectively. The central wavelength of the guided wave is 1531.3 nm. The inset illustrates the cross-sectional intensity distribution in a 10-μm diameter microfibre. These figures were calculated using COMSOL. In the calculation, the peak power of the pulse wave is 200 W, which is consistent with the experimental results. One can see from D. For instance, the power densities are approximately 20, 3, and 0.038 MW/cm2 for D = 6, 10, and 30 μm, respectively. Therefore, the light–graphene interaction is enhanced significantly in the GCM via a tightly confined evanescent field guided along the surface of the microfibre. The energy fraction in the air, Pair/P, increases exponentially with decreasing D. For example, Pair/P ≈ 22.7% and 77.6% for D = 1 and 0.5 μm, respectively, as shown in A schematic of the GCM-based fibre laser is shown in λ, is 2.08 nm. The sidebands at 1527.28, 1528.81, and 1533.82 nm are typical of soliton-like pulse formations, which originate from intracavity periodic perturbations2-shaped fit. Assuming a sech2 temporal profile, the deconvolution yields a pulse duration of 1.21 ps. The time bandwidth product (TBP) is 0.322, which is near the value of the transform-limited sech2-shaped pulses. 6 contrast), indicating low-amplitude fluctuations and good mode-locking stabilityThe threshold pump power for CW lasing is approximately 5 mW. Self-starting mode-locking operation starts at the proper pump power when the polarization controller is adjusted appropriately. Once the stable laser is obtained, no further polarization controller adjustment is needed. D, is changed from ~1 to ~30 μm. We can see from D > 30 μm, the light–graphene interaction is so weak  that the mode locking fails. When D is less than 1 μm, a considerable fraction of the energy of the pulse is from the microfibre, in which case slight roughness on the surface of the microfibre can cause large losses. As a result, the loss that occurs in the GCM prevents the mode-locking operation of a laser.In the experiments, the average power of pulses in the laser cavity is ~0.45 mW, corresponding to a peak power of ~200 W. The experimental observations demonstrate that the proposed fibre laser can generate ultrafast pulses when the diameter of GCM, The experimental observations demonstrate that if the GCM-SA component is excluded from the proposed fibre laser, it fails to mode-lock; thus, the CW rather than ultrafast pulses is generated. Although the polarization effects can affect the characteristics of pulses, they hardly play such a role in emitting pulses. Both theoretical and experimental results indicate that the polarizer plays the key role in the nonlinear polarization rotation techniqueThe loss that occurs in the GCM is dependent on the diameter of the taper and the length of the graphene. In this work, the loss is approximately 1 dB. Although the taper fibre is not protected, the proposed laser can work well over the long term and deliver pulses consistently in our ultraclean laboratory. Outside the ultraclean laboratory environment, the taper fibre must be protected appropriately. Because the diameter of the taper is approximately 6 μm, the damage threshold is sufficiently high.3 solution for ~10 h. After the Cu is etched completely, transparent PMMA/graphene sheet floats on the surface of the solution. The PMMA/graphene sheet is then transferred into distilled water five times and is immersed for approximately 2 h each time to remove the remaining FeCl3 etchant. Subsequently, a microfibre, which is integrated onto a hollow glass substrate in advance, is placed under the centre of the PMMA/graphene sheet. When the microfibre is lifted together with the hollow substrate from the water, the PMMA/graphene spontaneously wraps around the microfibre , an autocorrelator, a 6-GHz oscilloscope, a radio-frequency (RF) analyser, and a 10-GHz photodetector are used to measure the laser output performance.How to cite this article: Liu, X. M. et al. Graphene-clad microfibre saturable absorber for ultrafast fibre lasers. Sci. Rep.6, 26024; doi: 10.1038/srep26024 (2016).


PD-L1 is an immune checkpoint molecule expressed by a variety of tumors, including ovarian, which binds to circulating PD-1 expressing effector T cells allowing for tumor escape from the immune system. PD-L1 blockade prevents PD-L1/PD-1 interaction and is currently explored as therapy of solid tumors. Ovarian cancer patients receive combination cisplatin/taxane chemotherapy as standard of care. Chemo-induced effects on tumor PD-L1 expression have been only partially addressed. We studied here the effect of platinum/taxane exposure on PD-L1 expression in vitro and in vivo.Human (OVCA 420 and OVCA432) and mouse (2F8) ovarian cancer cell lines were exposed to increasing doses of cisplatin and paclitaxel for different time periods. PD-L1 expression was analyzed with flow cytometry and Western blot. Through continuous exposure in vitro of mouse 2F8 ovarian cancer cells to increasing doses of cisplatin we have derived a new cisplatin-resistant line (2F8-Cis). In vivo, we have challenged n=37 mice IP with 0.8 million 2F8 cells. Tumor-bearing mice were treated with cisplatin, anti-PD-L1 antibody, both drugs, or isotype control every two weeks for three doses starting at day 14 post-inoculation. Tumor- and ascites-derived cancer cells were analyzed with flow cytometry.Exposure of OVCA420 and OVCA432 to cytotoxic doses of cisplatin or paclitaxel trigger PD-L1 up-regulation. Similarly, 2F8-Cis cells show increased cell surface PD-L1 compared to parental 2F8 cells, providing the rationale for combination therapy with PD-L1 blockade. In vivo treatment of mice with aggressive 2F8 tumors respond well to cisplatin and anti-PD-L1 individually with increased survival . At necropsy, anti-PD-L1 therapy significantly reduced tumor burden . Tumor cells cultured from cisplatin-only treated mice expressed higher levels of PD-L1, in line with our in vitro results. A higher percentage of PD-1 expressing cells were found amongst the tumor cells in these cultures versus cisplatin/anti-PD-L1 treated mice. Although high dose anti-PD-L1 immediately following cisplatin administration can control tumor burden (0.48 g), it does not significantly prolong survival (median 29 days). We are currently testing an alternative therapeutic schema exploring a lower anti-PD-L1 dose and a different timing post-chemo.Tumor cells upregulate PD-L1 in response to chemotherapy exposure and combination PD-L1 blockade in conjunction with chemotherapy effectively controls tumor burden. Optimization of timing and dosage for this combination therapy will likely increase its therapeutic benefit.