Intron retention (IR) occurs when an intron is transcribed into pre-mRNA and remains in the final mRNA. We have developed a program and database called IRFinder to accurately detect IR from mRNA sequencing data. Analysis of 2573 samples showed that IR occurs in all tissues analyzed, affects over 80% of all coding genes and is associated with cell differentiation and the cell cycle. Frequently retained introns are enriched for specific RNA binding protein sites and are often retained in clusters in the same gene. IR is associated with lower protein levels and intron-retaining transcripts that escape nonsense-mediated decay are not actively translated.The online version of this article (doi:10.1186/s13059-017-1184-4) contains supplementary material, which is available to authorized users. Alternative splicing (AS) affects up to 95% of multi-exonic genes in humans . The thrWe recently discovered that IR combined with the NMD pathway is not a by-product of faulty splicing but rather a major driver of the cellular differentiation of granulocytes . In thishttps://github.com/williamritchie/IRFinder) and a database of IR in over 2000 human samples is freely available at IRBase (http://mimirna.centenary.org.au/irfinder/database/).Here we have developed a significantly enhanced program in terms of sensitivity and speed for detecting retained introns and filtering samples that are inappropriate for IR analysis in terms of library preparation and quality. This novel program was validated using quantitative RT-PCR (RT-qPCR) against retained introns and a NMD knockdown experiment. IRFinder correctly identified IR events and measured the ratio of retained introns to correctly spliced introns with great accuracy. Using IRFinder, we analyzed 3435 human samples, of which 2573 were suitable for analysis. We found that IR occurs in hundreds of genes in all tissues analyzed, affects over 80% of all coding genes, and is associated with cell differentiation and cell cycle. Retained introns in the same genes are frequently adjacent to each other and intron-retaining genes cluster closely on the genome, suggesting a global mechanism that regulates multiple introns simultaneously. By analyzing mass spectrometry and ribosome sequencing data we discovered that intron-retaining genes have lower protein output and that IR transcripts that escape NMD are not actively translated. Finally, by comparing introns that are frequently retained amongst the 2573 samples with introns that are rarely retained, we discovered a distinct primary sequence signature amongst frequently retained introns that is enriched in RNA binding protein sites. These proteins modulate the level of IR and thus the level of repression of frequently retained introns. Our program to analyze IR from sequencing data is available at GitHub  were suitable for analysis in this study. This was mainly due to the incorrect use of the term “mRNA-Seq”, which was frequently used in whole RNA experiments and CHIP-Seq experiments. Our approach also uses a series of programmatically fast steps written in C++ that automatically detect and trim adapters from sequencing reads. Of the 3435 initial samples we analyzed, 3096 (91%) still had adapters in the sequencing reads despite having been already processed by an adaptor-trimming algorithm.IRFinder was capable of estimating IR events with low coverage or low mappability as confirmed by RT-qPCR .The IRFinder algorithm and instruction manual are available at GitHub (http://www.ebi.ac.uk/ena). These were annotated as mRNA-Seq experiments in the archive with over 50 million reads each. The IRFinder quality control filter (see “Methods”) labeled 1201 of these as unfit for further analysis because they were either not RNA-seq experiments or not enriched for poly(A) tailed mRNAs. We searched for retained introns in 2573 remaining samples. We focused on introns that were retained in more than 10% of transcripts (IR ratio >0.1) with at least a coverage of three reads across the entire intron after excluding non-measurable intronic regions (see “Methods”). We found that 87.9%  of all multi-exonic protein coding genes with sufficient coverage had retained introns in at least three samples in our dataset. None of these 16,307 was retained in all of the samples and the majority (95%) of introns were retained in less than 7% of samples, suggesting that the IR events we detected were specific to tissue or cell types. All samples had over 100 retained introns with a median of 926 retained introns  and found that the average IR ratio was homogenous within each tissue , we calculated the distance between genes with retained introns Fig. c and howhttp://mimirna.centenary.org.au/irfinder/database).We created an online database of IR calculated in these samples, which is available at IRBase . These data were coupled with mRNA sequencing and quantitative translation initiation sequencing (QTI-seq), which uses lactimidomycin to preserve initiating ribosomes and puromycin to deplete elongating ribosomes . These dWe previously discovered that IR was essential for granulocyte , erythrop = 2.5 × 10−5). We also found numerous genes involved in neurogenesis with high levels of IR amongst which were the neural transcription factors SOX15 and POU4F1. SOX15 is known to be highly expressed in undifferentiated cells and repressed upon neuron differentiation [hiPSCs can be differentiated into highly homogenous neurons within 4 days by overexpressing the transcription factors Neurogenin-1 and Neurogenin-2 . This alntiation . In agrentiation . In agreWe then analyzed another set of data where the authors used genetically matched hESCs and hiPSCs derived from fibroblasts using the Sendai virus reprogramming method to prove that these cells are equivalent at a gene transcription level . To asse+ T cells. IR has recently been shown to be an integral regulator of T-cell activation [+ T-helper cells that were induced to differentiate toward a TH2 subtype [p = 2E-17 and p = 8E-6 after Benjamini–Hochberg correction, respectively  consisting of a shRNA-mediated knockdown (KD) of RNA binding proteins in HepG2 cells. We used IRFinder to calculate IR before and after KD of proteins for which the motifs were enriched in frequently retained introns , treated with DNaseI (Life Technologies), and converted to cDNA using Superscript III (Life Technologies). Quantification of intronic expression shown in Additional file To compare the efficiency of IRFinder with other existing tools that can detect IR events, we measured the average change in expression of retained introns predicted by IRFinder, MISO , and DEXUpf2fl/fl) and Upf2 knockout  mouse (GSE26561) was downloaded from the Gene Expression Omnibus (GEO). The single-end reads from both samples were mapped to the transcriptome annotation version 73 of Ensembl mouse genome GRCm38 using STAR (version and options described in IRFinder methods).Total RNA-seq of liver tissue from wild-type  less than 0.05 in the DEXSeq report was recognized as differentially retained.DEXSeq 1.14.0 with default parameters was applied to determine differential intron usage between the wild-type and UPF2 knockout samples. Each intron with q value  the Bayesian factor was above 19 (the likelihood that this intron is retained is 19 times higher than that of not being retained); 2) at least one read covered this intronic region; 3) at least ten reads covered the two flanking exons of this intron.


Moreover, anti-hPD-L1 treatment also led to profound inhibition of Treg expansion and shifting of myeloid cell profiles, showing bona fide induction of multilateral anti-tumor responses by anti-hPD-L1 blockade. Thus, this hPD-L1 mouse model system would facilitate the pre-clinical investigation of therapeutic efficacy and immune modulatory function of various forms of anti-hPD-L1 antibodies.Huge efforts have been devoted to develop therapeutic monoclonal antibodies targeting human Programmed death-ligand 1 (hPD-L1) for treating various types of human cancers. However, thus far there is no suitable animal model for evaluating the anti-tumor efficacy of such antibodies against hPD-L1. Here we report the generation of a robust and effective system utilizing hPD-L1-expressing mouse tumor cells to study the therapeutic activity and mode of action of anti-human PD-L1 in mice. The model has been validated by using a clinically proven hPD-L1 blocking antibody. The anti-hPD-L1 antibody treatment resulted in potent dose-dependent rejection of the human PD-L1-expressing tumors in mice. Consistent with what have observed in autochthonous mouse tumor models and cancer patients, the hPD-L1 tumor bearing mice treated by anti-hPD-L1 antibody showed rapid activation, proliferation and reinvigoration of the cytolytic effector function of CD8 Recently monoclonal antibodies targeting immune checkpoint molecules have achieved unprecedented success in clinic for the treatment of a broad range of the most prevalent human cancers12313412313PD-L1 was originally identified and cloned as a B7 family of co-stimulatory/co-inhibitory molecule, called B7-H114812Human peripheral lymphoid cells101115+CTLs and repressed Treg cell enrichment, as well as facilitated the expansion of tumor infiltrating myeloid cells in tumor tissues. Taken together, we construct an hPD-L1 tumor animal model and we hope this model could contribute to quick pre-clinic antibody screening. Additionally, this model could also be used for pre-clinic screening of other immune checkpoint inhibitors.In this study, we constructed an hPD-L1 expressing MC-38 tumor animal model and observed an evident anti-tumor effect by treating with MPDL-3280A, the hPD-L1 monoclonal antibody. Flow cytometry analysis revealed antibody treatment increased the frequency and the cytotoxicity of infiltrated CD8in vivo is in favor with the general public. This study reported a human PD-L1 tumor model which was constructed by replacing mouse PD-L1 with human PD-L1 in a common transplantable mouse tumor cells , MC-38-hPD-L1 cells in vitro  to mouse PD-1 (mPD-1) by ELISA. The OD value indicated that the hPD-L1 bound to mPD-1 very well . Further+T cell infiltration. To address whether this model also hit this point, the phenotype of immune cells in tumor microenvironment were analyzed by flow cytometry. The frequency of CD8+T cells and the ratio of CD8 to CD4 T cells were both remarkably up-regulated  assay  assay . ConsistT) assay . To summ+, Ly6G+or Ly6C+myeloid cells in tumor tissues by flow cytometry. FACS data revealed CD11b+myeloid cells were not affected by anti-hPD-L1 antibody treatment. Amazingly, the frequency of CD11b+Ly6G+cells was slightly increased at day15 but significantly increased at day21. Similarly, though the frequency of CD11b+Ly6C+was not affected at day15, it was significantly increased at day21 . All experiments were carried out in accordance with the approved guidelines.http://crispr.mit.edu), cloned into sgRNA expression vector (FG-BB-U6-sgRNA) at BbsI restriction site, and confirmed by sequencing. CRISPR/Cas9 knockout was performed by co-transfection of the anti-mPD-L1 sgRNA construct with Cas9 expression plasmid (FG-hEF/HTLV-Cas9-PGK-Puro-WPRE) carrying a puromycin selection marker and into the parental MC-38 cells using Lipofectamine 3000 (Invitrogen), and the sgRNA sequence is 5′-GCTTGCGTTAGTGGTGTACT-3′. The transfected cells were transient selected with puromycin for 2 days, then expanded and cloned to screen for target gene knockout by flow cytometry and TA cloning-sequencing. For TA cloning-sequencing analysis, genomic DNA was extracted from the pooled cells by DNeasy Blood&Tissue kit (Qiagen), forward primer is 5′-TGGTTCCTTTTAAACAAGACTGGG-3′, and reverse primer is 5′-CGCACCACCGTAGCTGATTA-3′. The sgRNA targeted DNA fragment was amplified by Taq DNA polymerase (NEB) and cloned into pTZ57R vector using Ins TAclone PCR Cloning Kit (Fermentas Thermo). Numbers (5 clones) of individual resulting TA plasmid clones were sequenced, and the sequencing results were analyzed by ClusterW software. Frameshift Indels in mouse PD-L1 gene were identified by Blast (NCBI), and functional knockout was confirmed by flow cytometry for the absence of PD-L1.C57BL/6 derived mouse colon tumor cell line MC-38 was originally obtained from ATCC (ATCC#CRL-2638). Short-guide RNAs (sgRNA) specific to mouse PD-L1 (CD274) was designed according to online design tool  carrying human PD-L1 ORF was constructed and verified by sequencing. High titer recombinant lentivirus particles were produced from HEK293T cells by calcium phosphate transfection with the third generation of lentiviral packaging plasmids according to Qin The plate was coated with mPD-L1-Fc or hPD-L1-Fc protein in high pH coating buffer overnight at 4 °C. Added diluted biotin labeled mPD-1 protein into each well with dilution buffer for 1 h at room temperature after incubated by blocking buffer. Added diluted Neutavidin-HRP (1:5000 dilution) to wells and incubated for 1 hr in room temperature. Read OD value of the plate at 450 nm by automatics plate reader .We performed flow cytomtry analysis to determine whether mouse PD-L1 could bind to human PD-L1. Briefly, 1.0*10^6 MC-38-hPD-L1 cells were incubated with 5.0 μg/ml or 2.5 μg/ml mouse PD-1 protein for 25 min after the cells were blocked by FcR antibody. Next, these cells were washed by staining buffer for 3 times and then stained by anti-Fc-APC for 20 min. These cells were subjected to flow cytometry analysis. All of these proteins and antibodies are obtained from Mabspace Biosciences (Suzhou) Co., Ltd.Cell proliferation activity was determined by Cell Counting Kit 8 (CCK8) purchased from Beyotime  according to the manufacturer’s instruction. Briefly, the 96 well plate was seeded with 20,000 cells per well and cultured in 100 μl 10% FBS RPMI-1640 medium for 24 h. We next added 10 μl CCK8 reagent and incubated for 4 h. The plate was measured by automatics plate reader .7 cells/mL (2 × 106 cells/mouse). Tumor growth was monitored and measured every two days using digital caliper , and the tumor volume was calculated according to the following formula: M12*M2 /2 . When the tumor volume reached 100 mm3 (day 7), mice were treated with 1.0 mpk, 3.0 mpk or 10.0 mpk of anti-hPD-L1 antibody (the antibody was synthesized by MabSpace Biosciences (YW243.55.S70 or MPDL3280A as disclosed in the US Patent No. 8217149) or PBS vehicle control by intraperitoneal (i.p.) injection, and the second dose of treatment was given 4 days later. The mice were sacrificed for immune phenotype and function analyses either 4 or 10 days after the second dose of treatment.Female C57BL/6 mice were purchased from Vital River Laboratories . All mice were maintained in a barrier facility at Soochow University. To establish subcutaneous tumor, C57BL/6 mice were inoculated with various tumor cell lines in the right side axillae with 0.2 mL/mouse of the cells adjusted to 1.0 × 10Flow cytometry was performed with Life Attune Nxt FACS machine . Fluoro-chrome conjugated anti-mouse CD45 , CD3 , CD4 , CD8 , Foxp3 , Ki-67 , Granzyme B , Ly-6C , Ly-6G , mPD-L1 , hPD-L1  and IFN-gamma (XMG1.2). Granzyme B and IFN-gamma staining was performed with intracellular staining kit from BD Bioscience. While Ki-67 and Foxp3 staining was done with nuclear protein staining kit from eBioscience . FACS data were analyzed using FlowJo software .5 tumor tissue cells were added to the plate per well in 100 μl and then incubated the plate at 37 °C for 12 h in a 5% CO2 incubator. Discard the cell suspension and wash every micro-well by Elispot wash buffer. Added the diluted detection antibody at 2.0 μg/ml in 100 μl and incubated the plate at room temperature for 2 h. After washing with PBS, the plate was incubated with 100 μl streptavidin-HRP conjugate for 1 h at room temperature. Then washed the plate for 3 times by Elispot by wash buffer and added 100 μl final substrate solution for antibody detection. Stop the substrate reaction by washing wells with DI water. Enumerate spots of every micro-well by automatical ELISPOT plate reader (Celluar Technology Ltd. USA).The IFN-γ ELISPOT assay was performed according to manufacturer’s protocol from BD life science. Briefly, the ELISPOT plate was coated with anti-mouse IFN-γ capture antibody diluted in PBS at 5.0 μg/ml overnight at 4 °C. 2.0*10Statistical analysis was performed for all experiments using Student’s t-test. Values of p inferior to 0.05 were considered significant. All tests were done using Prism 6 software (Graph Pad Prism6).How to cite this article: Huang, A. et al. A human programmed death-ligand 1-expressing mouse tumor model for evaluating the therapeutic efficacy of anti-human PD-L1 antibodies. Sci. Rep.7, 42687; doi: 10.1038/srep42687 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Supercapacitors, as one of the energy storage devices, exhibit ultrahigh capacitance, high power density, and long cycle. High specific surface area, mechanical and chemical stability, and low cost are often required for supercapacitor materials. Graphene, as a new emerging carbon material, has attracted a lot of attention in energy storage field due to its intrinsic properties. Polymers are often incorporated into graphene for a number of enhanced or new properties as supercapacitors. In this paper, different polymers which are used to form composite materials for supercapacitor applications are reviewed. The functions, strategies, and the enhanced properties of graphene and polymer composites are discussed. Finally, the recent development of graphene and polymers for flexible supercapacitors are also discussed. Origina2(SO4)3] . With th2(SO4)3] . The rol2(SO4)3] .Fig. 8SE−1 [−1 in HCl and H2SO4 electrolytes, respectively [−1, and RGO/PEDOT composite electrode has a specific capacitance of 209 Fg−1 with 87% capacitance retention over 2000 cycles [Pristine PEDOT supercapacitors have a specific capacitance in the range from 70 to 130 Fg−1 , 60, 61 ectively . Wen et 0 cycles . The rolGraphene and its derivatives can have great influence on the morphologies, electrical properties, and the structural stabilities of the CPs when they are made into composites, and thus lead to drastic enhancement of the electrochemical properties of CPs. However, for a graphene/CPs supercapacitor, there are many factors which can determine the ultimate supercapacitor performance  such as polymerization method, intrinsic properties of individual CPs, electrolyte, dispersion/aggregation of the graphene in the composites, different properties of graphene used, etc. Generally speaking, for the aforementioned three types of CPs , there are distinct advantages and disadvantages of using individual CPs to form the composite. PEDOT has the largest molecular molar mass, hence, the specific capacitance of the graphene/PEDOT composites supercapacitor is normally smaller than the ones formed with PANI or PPy. On the other hand, the larger particles formed in the PPy film make it less porous than PANI or PEDOT, thus the cycling performance of PPy is normally the worst among these three CPs. In addition, the conductivity comparison of these CPs is PEDOT > PPy > PANI , which c−1 at the current density of 0.3 Ag−1, respectively. Figure Zhang et al. compared the supercapacitor performance of the composites formed of RGO nanosheets with PANI, PPy, and PEDOT polymers using similar polymerization and mixing methods . The speTable -1 for the 100-nm-thick graphene film. There are other types of flexible supercapacitor electrodes based on graphene materials, such as graphene paper [The recently developed flexible electronic devices such as flexible displays, curved smartphones, flexible implantable medical devices, and wearable electronic devices imply that flexible devices are beginning to emerge as the leading revolution in next generation of electronics. Several advantages of flexible electronic devices compared to conventional electronic devices include lighter weight, wearability, bendability, environmental friendliness, reduced cost, etc. In order to match the fast growth of flexible electronic devices, energy storage systems that are light, thin, and flexible should also be developed. Recently, considerable attempts have been made to develop flexible supercapacitor electrode based on carbon materials including activated carbon , 66, carne paper , 78, grane paper , 80, andne paper –83. Howe3PO4 gel was used as the solid electrolyte in this device, and gold was sputter coated on one side of the electrode as the metal current collector [Graphene/conducting polymers (CPs) composite film, due to its flexible nature, becomes a viable option for flexible supercapacitor applications as it can also adapt to the roll-to-roll manufacture. Graphene/CPs film can be assembled with solid electrolyte to form a flexible supercapacitor device which can be subject to high degrees of bending or twisting without losing the device integrity. Meanwhile, as it is discussed previously, CP itself can provide pseudocapacitance due to the redox reaction, increasing the specific capacitance, as well as the capacity of the device. Figure ollector . Polymerollector .Fig. 12SBending test is usually necessary to examine the structural and electrical integrity of the flexible supercapacitor device subject to different bending angles and cycles. Bending or twisting can cause stress on the films which may lead to material loss, material fatigue  or even material failure . CV and/or charge-discharge curves are monitored at several of bending angles during the bending cycles , 86–90 tThe emergence of graphene has undoubtedly changed the scope of supercapacitor field due to its outstanding electrochemical properties along with other unique properties such as large surface area, high electrical conductivity, light weight, and mechanical strength. The recent development of graphene and polymer composites showed very promising features of these composite materials for supercapacitor applications. Compounded with binder polymers, graphene would compensate the undesired features of insulating polymers . On the other hand, when graphene makes composites with conducting polymers, it would provide mechanical support for the framework of the polymers and thus greatly improve the cycling performance as well as the specific capacitance. In addition, the flexible nature of the graphene/polymer film makes it possible for flexible, wearable, conformable energy storage devices. Despite the innovative ideas and techniques that have been demonstrated for the graphene/polymer supercapacitor device with unique features not possessed by current state-of-art technology, there remain a lot of challenges for graphene/polymer composite-based supercapacitors to reach their full potentials. One of the main challenges is to find a feasible way for the low-cost mass production of graphene/polymer supercapacitor electrode without compromising the micro/nanostructures of graphene due to the restacking or aggregation. Many studies have demonstrated the compatibility of large-scale coating or roll-to-roll manufacture capability of graphene/polymer supercapacitor electrodes, however, there is still a need to have a rational design of the porous structures inside the supercapacitor electrodes to form hierarchical interconnected porous microstructures and avoid the formation of dead volume or the collapse of the porous microstructures . Further


The magnetic circular dichroism and the Faraday rotation are the fundamental phenomena of great practical importance arising from the breaking of the time reversal symmetry by a magnetic field. In most materials, the strength and the sign of these effects can be only controlled by the field value and its orientation. Furthermore, the terahertz range is lacking materials having the ability to affect the polarization state of the light in a non-reciprocal manner. Here we demonstrate, using broadband terahertz magneto-electro-optical spectroscopy, that in graphene both the magnetic circular dichroism and the Faraday rotation can be modulated in intensity, tuned in frequency and, importantly, inverted using only electrostatic doping at a fixed magnetic field. In addition, we observe strong magneto-plasmonic resonances in a patterned array of graphene antidots, which potentially allows exploiting these magneto-optical phenomena in a broad THz range. Time reversal symmetry breaking gives rise to magnetic circular dichroism and Faraday rotation in graphene. The authors use terahertz magneto-electro-optical spectroscopy to demonstrate that electrostatic doping at a fixed magnetic field allows inversion of magnetic circular dichroism and Faraday rotation. Terahertz waves are actively explored for fundamental research and applications1345678111214151617181920214237Here we combine MO THz spectroscopy with electrostatic gating and graphene patterning. This allows us to observe several new phenomena, including an electrostatic control of the THz cyclotron frequency, a purely electrostatic inversion of the magnetic circular dichroism (MCD) and the Faraday rotation (FR) and strong gate-tunable magneto-plasmonic resonances in periodic antidot arrays. We argue that these effects potentially allow a broad spectral tunability and enable novel functionalities in graphene-based passive terahertz devices.n (positive for electrons and negative for holes) with magneto-transport and MO measurements in a perpendicular magnetic field B (positive towards the light source). The THz transmission T(ω) and the FR spectra θF(ω) were obtained while graphene was illuminated by a linearly polarized light with the angular frequency ω. The Fabry–Perot interference in the substrate was suppressed intentionally to simplify the spectra analysis. From these measurements, we extracted the transmission spectra for the right-hand (RH)/left-hand (LH) circular polarizations, T±(ω)We first discuss the intrinsic MO terahertz effects in continuous (unpatterned) graphene. A basic device used in our experiments is a large-area CVD -graphene field-effect transistor (g-FET) sketched in Vg in all our devices has a characteristic peaked shape /TCNP(ω), which are representative of the optical absorption in graphene , τ is the scattering time and ± refer to RH/LH circular polarizations. A relation between σ± and T± that takes the substrate into account is given in where Before we analyse the dependence of the Drude parameters on the doping and the magnetic field, we stress that the Dirac-fermion theory  predictsandvF is the Fermi velocity, ħ the reduced Planck's constant and e the (positive) elementary charge. This behaviour is different from the case of conventional 2DEGsn, while the cyclotron frequency is n-independent. Notably, where τ extracted by fitting the zero-field extinction curves (where ωc=0) are strongly doping dependent  for the LH circular polarization, where the CR is observed in this case, are shown in B .In a magnetic field, the terahertz spectra are dominated by the cyclotron resonance (CR) . The extinset of  and a ren changes sign. The inversion of the MCD is thus due to the fact that the polarization where the CR is observed is defined by the doping type. This new effect that enables novel applications (as discussed below) is thus directly related to the unique possibility of ambipolar doping control in grapheneA) as in continuous graphene and two additional peaks (B and C) due to Bragg scattering of graphene plasmons on the periodic structure. Our previous study revealed that peak B is mostly due to the -order Bragg reflection, while peak C has a mixed – character. Importantly, they both demonstrate a As demonstrated above, the non-reciprocal MO effects in unpatterned graphene are especially strong at low frequencies (below 5–7 THz). However, in the higher THz region the Drude response is less efficient. In order to broaden further the tuning range of the magneto-teraherz response, one can use the new physical properties arising from the excitation of magneto-plasmons  in graphene nanostructures23343536fermions 41415 thaA comparison of A shifts upwards in the LH polarization while keeping the intensity constant. Although this resembles the behaviour of the CR peak in continuous graphene . Most notable is a strong increase/decrease of their intensity for the LH/RH polarization, accompanied by field-induced shifts as indicated by the dashed lines in B, where opposite shifts are observed in the two polarizations. Associated with these spectacular magneto-plasmonic effects are the resonant MCD and FR structures  between the experimental and the simulated frequencies. Nevertheless, overall our simulations based on the Dirac-fermion theory prove to be quantitatively accurate also in patterned graphene.To give a further physics insight into the origin of the observed MO resonances, we show in μ≈3,500 cm2 V−1 s−1 in our present g-FETs is largely limited by the substrate (SiO2/Si), and high magnetic fields (up to 7 T) are needed to attain reasonably good device specifications452 V−1 s−1 at room temperature was recently obtained in h-BN-encapsulated CVD graphene using a dry-transfer techniqueOur results indicate that combining terahertz spectroscopy, electrostatic gating, magnetic field and pattering reveals interesting properties of the Dirac fermions, such as a broadband electrostatic control of the cyclotron frequency and relativistic magneto-plasmons, and give rise to qualitatively new phenomena, such as a purely electrostatic control of the magnetic effects (MCD and FR). Importantly, the experiments were done close to room temperature, in a striking contrast to the existing 2DEG studies3839As we demonstrated, the unusual properties of Dirac fermions allow a wide tuning of the working (cyclotron or magneto-plasmon) frequency, potentially in the entire THz range, by the doping, magnetic field and patterning parameters. Moreover, the possibility to invert the MCD and the FR electrically opens the door to completely new functionalities, such as electrically switchable isolators, and to combining several basic functions in a single device.Importantly, in the present case the MO effects occur in an atomically thin layer, therefore the device thickness is limited only by the substrate and external optical elements . This potentially allows a miniaturization of the graphene-based elements, in a stark contrast with conventional ones, where light has to travel a centimetre-scale distance. The scope of the MO terahertz applications of graphene is certainly not limited to free-space wave propagation, as in the present experiments, and we anticipate that its remarkable properties will be employed in miniaturized chip- and THz waveguide-integrated2 that served as a back-gate dielectric. Au/Ti electrodes were deposited outside the illuminated region (5 × 5 mm2) allowing transport measurements and back-gate control simultaneously with MO studies. The carrier concentration was calculated using the formula n=α, where α=7.2 × 1010 cm−2 V−1 is the gate capacitance and Vg, CNP is the gate voltage corresponding to the maximum resistivity. A perpendicular magnetic field up to 7 T was applied using a superconducting split-coil system. The sample was illuminated by linearly polarized light and the terahertz transmission and FR spectra using the technique presented in ref. Large-area monolayer CVD graphene was grown on copper and transferred to a weakly doped Si substrate with a 300 nm layer of SiOFinite-element electromagnetic simulations of antidot arrays were performed using the COMSOL software. The graphene layer was modelled as a surface current in the boundary conditions. In order to achieve convergence, the mesh element size in the vicinity of graphene was much smaller than the magneto-plasmon wavelength.The data that support the findings of this study are available from the corresponding author upon request.How to cite this article: Poumirol, J.-M. et al. Electrically controlled terahertz magneto-optical phenomena in continuous and patterned graphene. Nat. Commun.8, 14626 doi: 10.1038/ncomms14626 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Notes and Supplementary References


The spectral singularity existing in PT-synthetic plasmonic system has been widely investigated. Only lasing-mode can be excited resulting from the passive characteristic of metallic materials. Here, we investigated the spectral singularity in the hybrid structure composed of the photoexcited graphene and one-dimensional PT-diffractive grating. In this system, both lasing- and absorption-modes can be excited with the surface conductivity of photoexcited graphene being loss and gain, respectively. Remarkably, the spectral singularity will disappear with the optically pumped graphene to be lossless. In particular, we find that spectral singularities can exhibit symmetry-modes, when the loss and gain of the grating is unbalanced. Meanwhile, by tuning the loss (gain) of graphene and non-PT diffraction grating, lasing- and absorption-modes can also be excited. We hope that tunable optical modes at spectral singularities can have some applications in designing novel surface-enhanced spectroscopies and plasmon lasers. Beyond some non-Hermiticity threshold, typically called the exceptional point, PT-symmetric systems can display an abrupt phase transition and the corresponding eigenspectra become complex. Recently, the concept of PT-symmetry has been fruitfully extended to wave optics. Photonic platforms are ideally suited to implement the structures with PT-symmetry, which is directly translated into a requirement for the arrangement of elements with balanced gain and loss25. Various intriguing optical phenomena have been revealed in PT-symmetric systems, such as asymmetric light propagation9 and invisibility11, Bloch oscillation of energy12, coherent perfect laser absorber17, PT-symmetric metasurfaces21, single-mode laser23, reversing the pump dependence of a laser24 and loss-induced suppression and revival of lasing25.Since the pioneering work of Bender 30. Besides the exceptional point, the spectral singularity32 existing in the PT-plasmonic system, which is related to scattering resonance of the non-Hermitian Hamiltonian and manifests itself as giant transmission and reflection with vanishing bandwidth, also has unique features. For example, hugely anisotropic optical scattering33, three-dimensional light confinement34, and loss-induced super scattering36.More recently, PT-symmetric systems built using plasmonic elements have attracted increased attention. The strong interaction between surface plasmons and electromagnetic field can enhance the extraordinary properties associated with exceptional points38. For example, nanopatterned graphene can be used as an active medium for infrared electro-optic devices40. Meanwhile, loss induced amplification of graphene plasmons41, regulation of energy distribution in graphene42, and singularity-enhanced sensing based on the PT-graphene metasurface43, as characteristics of exceptional point behaviors, have been demonstrated theoretically. However, characters of spectral singularities in graphene-based PT systems has not been discussed.On the other hand, there is a rapid progress in the field of graphene plasmonics motivated by the unique electrical and optical properties of graphene47. Such diverse surface dynamic conductivity responses can be used to manipulate optical modes of spectral singularities in graphene-based PT-systems. When the grating possesses perfect PT-symmetry, the spectral singularity will present the feature of lasing-mode (electric fields concentrate mainly on the gain element) or absorption-mode (electric fields concentrate mainly on the loss element) with the loss of optically pumped graphene being negative or positive, respectively. It is noted that spectral singularities are vanished if the loss of optically pumped graphene becomes zero. In particular, the spectral singularity may exhibit symmetry-modes , when the loss and gain of the grating is unbalanced. In this case, the spectral singularity on the lasing or absorption-mode also appears with the loss and gain for the grating exceeding the value of the corresponding symmetry-modes.In this work, we systematically investigate characteristics of the eigenmode at spectral singularity when the electromagnetic field, in terahertz (THz) regime, is incident on an optically pumped monolayer graphene underneath the one-dimensional gain-loss diffractive grating. The intrinsic loss of graphene can exhibit negative, zero, and positive values resulting from the population inversion produced by cascaded optical-photon emissionuFe and holes uFh of the same absolute value uFe = −uFh = uF. Since the relaxation time for intraband transition is much faster than the recombination time for electron-hole pairs, the population inversion can be achieved with optical pumping. In this condition, the complex intraband and interband conductivities, 48:e is the electric charge, T is the temperature, and T = 3 K and quasi-Fermi-level is uF = 100 meV. As can be seen, the loss of graphene 49. The amplifying view of a unit cell is clearly presented in the inset of Fig. p = 8 um. The sizes of the grating along x- and y-axis are 2.0 um and 1.8 um, respectively. The relative permittivities of the gratings are given by Fgain and Floss being the non-Hermiticity coefficients of the system. It is noted that the whole structure possesses the PT-symmetry, only when distributions of gain/loss elements are balanced (Fgain = Floss) and the dynamic conductivity of graphene becomes zero . Only two modes are considered here with eigenfrequencies  locating within 6–8 THz. Although the system has no loss or gain element, the imaginary part of eigenfrequency is still non-zero resulting from the existence of radiation loss, which is largest at the Brillouin center and vanished at Brillouin boundaries. As we turn on the gain and loss (Fgain = Floss = 0.05), the gap of the real part of eigenfrequency at the Brillouin boundary will be closed and the imaginary part separated, giving rise to the exception point (red arrow), as shown in Fig. Firstly, we proceed to investigate the interaction between the PT-diffraction grating and lossless monolayer graphene , as shown in the inset of Fig. F, as shown in Fig. In order to observe the evolution of eigenmode with the variation of non-Hermiticity coefficient, in Fig. kx = 0 in a complex frequency plane are shown in Fig. F being increased. The black dot line represents the condition that the real part of the surface conductivity of graphene is positive . The evolutions of eigenfrequencies for the states at F = 0.06613 and F = 0.06455, respectively. It is presented that the giant transmittance and reflectance appear, and corresponding near-field distributions are shown in Fig. The spectral singularity manifests itself as giant transmission and reflection with vanishing bandwidth. In Fig. Fgain = Floss = F, and the optically pumped graphene. The evolutions of the eigenfrequencies for the states at kx = 0 in a complex frequency plane are shown in Fig. F being increased. When the surface dynamic conductivity of graphene is active 1, 2, 3) and each possesses different eigenmodes, shown in right insets of Fig. 1, 2) possess symmetric eigenmodes before the eigenfrequency passes through the inflection points (marked by the blue arrows). Beyond the inflection points, the lasing-mode, which previously left away from the lasing threshold, will approach to it again and the corresponding spectral singularity reappears. This phenomenon follows the main characteristics of the quasi-PT-systems with exceptional points, like loss-induced suppression and revival of lasing15, and reversing the pump dependence of a laser14. It is noted that spectral singularities only exhibit symmetry-modes, when the distributions of the loss and gain for the diffractive grating are unbalanced. Moreover, in Fig. Finally, we will investigate the interplay between a non-PT diffraction grating, 2Fgain = 2Floss = F. Characteristics of the corresponding spectral singularities in a manner analogous to the above case are shown in Fig. 1, 2) possess symmetric eigenmodes and another one presents features of the absorption-mode. Similarly, the giant transmission and reflection are also excited at frequencies of the corresponding spectral singularities.We also consider the condition, when non-Hermiticity coefficients satisfied the relationship of In conclusion, we have demonstrated numerically that eigenmodes at spectral singularities can be conveniently tuned by a suitable variation of the loss and gain in the graphene-based quasi-PT systems. When the diffractive grating, which assisted graphene plasmonics excitation, has the perfect PT-symmetry, the spectral singularity can present the feature of lasing- or absorption-modes, which is decided by the intrinsic property of loss or gain characteristic for the surface conductivity of the pumped graphene. These spectral singularities vanished if the surface resistance of graphene becomes zero. In particular, the spectral singularity may exhibit symmetry-mode only with the asymmetric distribution of the loss and gain for the diffractive grating. Furthermore, with the increasing of non-Hermiticity coefficients, the spectral singularities with asymmetric eigenmodes reappeared. In contrast to the previous method to control the near-field by modifying the geometrical parameters of the plasmonic structures, we can tune the near-field distributions around the graphene-based PT-system by just varying the surface dynamic conductivity of graphene. We hope that our finding may have some applications in designing novel surface-enhanced spectroscopies and plasmon lasers.All full wave numerical simulations and dispersion relations were done using finite element method (Comsol Multiphysics). In the simulations, the graphene is modeled as a two-dimensional surface with complex conductivity. The mesh size inside the graphene layer is 0.5 nm, which is fine enough for convergence.


We also prove that multiple carriers exist in vertically aligned graphene.We investigated how to control the growth of vertically aligned graphene on C-face SiC by varying the processing conditions. It is found that, the growth rate scales with the annealing temperature and the graphene height is proportional to the annealing time. Temperature gradient and crystalline quality of the SiC substrates influence their vaporization. The partial vapor pressure is crucial as it can interfere with further vaporization. A growth mechanism is proposed in terms of physical vapor transport. The monolayer character of vertically aligned graphene is verified by Raman and X-ray absorption spectroscopy. With the processed samples, d Graphene is a 2D carbon allotrope with honeycomb monolayers. This kind of unique structure exhibits amazing electronic, photo-electric, magnetic, mechanical, and chemical properties3675678Vertically aligned graphene1216171819201416171920211511122729Although the properties of vertical graphene have been studied well, its growth on SiC is hard to control and its growth mechanism is not clear, which will hinder its further development and application. To promote the potential application, it is necessary to determine how process parameters (such as temperature and pressure) affect the graphene growth. Here, we investigated the growth of vertically aligned monolayer-like graphene on C-face SiC. The influence of temperature, annealing time, temperature gradient, pressure, and crystalline quality of SiC substrates was studied. Based on the growth, the magnetism and transport properties in as-grown and Cu-implanted vertical graphene were explored.To confirm the temperature threshold required to grow the vertically aligned graphene, the substrates were annealed at different temperatures for 2 hours between 1400 °C and 1900 °C. No graphene was obtained using temperatures of 1400 °C to 1600 °C. Vertically aligned graphene begins to grow when the temperature reaches 1700 °C. With further increasing temperature, graphene growth speeds up. We find that the growth rate increases with temperature. The height of the obtained graphene in different samples is listed in Usually, epitaxial planar graphene is compact and it will prevent the vapor release, so planar graphene cannot grow too thick. However, the situation for vertically aligned graphene is unclear. Can tunnels inside the vertical graphene increase or decrease the sublimation rate? The vertically aligned graphene was prepared with annealing times varying up to 40 hours, as shown in When the C-side is faced up with the initial setup (6 mm of susceptor thickness and 175 mm of mount height), the C-side faces towards the temperature maximum and the vapor cannot escape as it usually moves from high to low temperature. So no graphene can grow during annealing.When the C-side is faced down with the initial setup, the substrates cross the position of temperature maximum, the vapor can escape easily and then vertically aligned graphene can be obtained.When the susceptor thickness or the mount height increases, the substrates move away from the temperature maximum, the temperature gradient increases and graphene can grow faster.When the susceptor thickness or the mount height continuously increases, the substrates move further away, the temperature drops and then the growing rate decreases, considering that the difference is dramatically increased between the sample temperature and the measured temperature.The crystal growth is sensitive to the temperature distribution in the crucibleTherefore, altering the position of the substrates is an important means to control the growth of vertically aligned graphene.The substrates were loaded both in the middle and at the edge of the susceptor to grow graphene simultaneously for 8 hours. The height of the graphene grown in the middle is 22 μm compared with 58 μm at the edge. As the heat is generated only on the outer lateral surface of the susceptor and the lid, the edge will have both higher temperature and higher temperature gradient than the inner part, which can accelerate graphene growth. However, the direction of temperature gradient is not perpendicular to the substrate surface, which may result in a height inhomogeneity in lateral direction. It is also noticed that, the substrates are located in the middle of the susceptors when graphene grew with different susceptor thicknesses, while they are at the edge of the susceptors when different mount heights are applied. Therefore, the former graphene heights are smaller than the latter ones because of the relatively low temperature and relatively gradual temperature gradient See .The residual pressure in the chamber also affects the growth of vertically aligned graphene. For this investigation, we used a mechanic pump instead of the turbo molecular pump. The pressure maintained at about 9 mbar. With the susceptor thickness of 6 mm and the mount height of 178 mm, the graphene height reduces from 15.7 μm to 9.4 μm. It can be understood that when the pressure increases, the vapor partial pressure around the substrates rises and the convection decreases the temperature gradient, both of which will suppress the sublimation. If several pieces of substrates are annealed at one time, the height of the obtained graphene will also decrease. For instance, the height was 28 μm after annealing 8 pieces for 8 hours, compared with 40 μm for annealing only 1 piece, as the total Si-evaporation from 8 pieces raises the Si-partial pressure higher than that of 1 piece and it will suppress the sublimation on each single sample. This result reveals the evacuation of the vapor created from the surface of substrates is critical to the growth of vertically aligned graphene.It is known that the arrangements of vertical graphene arrays are different when grown on substrates with different crystallographic orientation2, Si2C, Si and C) can be transported rapidly with the proper temperature gradient discussed above (Some may deposit on the inner surface of the lid.), the sublimation will continue. As the saturation vapor pressure of Si is higher than that of C at the same temperature, carbon will be left and will reconstruct. The reconstruction of C atoms resulting in vertically aligned graphene is more complicated. Considering the difference between the temperature, the temperature gradient, and the pressure utilized to prepare planar graphene and those of vertically aligned graphene, the vaporization rate could be the key factor in determining the structure after reconstruction. When the vaporization is high enough, the initial C atom sheets will vertically stand due to the strong gas flow. So graphite or planar graphene cannot form but only the low density of vertical graphene makes the crystallization possible. The guide of the high speed gas flow, the annealing under a high temperature, and the etching of Si atoms will further improve the standing thin film into vertically aligned graphene. The schematic is shown in According to the results above, the growth of vertically aligned graphene can be understood in terms of SiC single crystal growth using the physical vapor transport method with a single wall crucible343537G and W2D) from 24 to 29 cm−1 and from 47 to 53 cm−1 shows the similarly high quality of all samples. The 2D peaks are symmetric and well match a Lorentzian line, so the interaction between the adjacent layers is weak and graphene layers exhibit a monolayer behavior. It is heeded that the difference between measured samples is not only limited to their graphene height. They were prepared under different conditions: the 241 μm sample has grown for 40 hours; the 40 μm sample used 1700–1800 °C for 10 hours; and the Si-face of the 1.3 μm sample was up during the process  and the carrier concentration of about 3 × 1018 cm−3, and the hole of 1 × 104 cm2/(Vs) and 1 × 1019 cm−3 according to a two-band model454 cm2/(Vs) while the concentration reduces slightly to 2 × 1018 cm−3. The hole Hall mobility and concentration almost remain unchanged after implantation. It is speculated that the impurity band of Cu is located a little bit below the major negative carrier so Cu doping can shift the Fermi level towards the Dirac cone and thus increase the mobility.In as-grown samples, only diamagnetism is observed. After implantation, the magnetism after removing the diamagnetic background is shown in 0 magnetism of 0.97 μB per Cu at 300 K is revealed in Cu-implanted vertical graphene. The negative magnetoresistance due to weak localization after implantation is observed. Vertical graphene possesses both electron and hole as major carriers, whose Hall mobility is more than 1 × 104 cm2/(Vs). In implanted samples, its electron Hall mobility is further improved to 9 × 104 cm2/(Vs). The results will be helpful to extend the research area and the potential application of vertically aligned graphene.In conclusion, the growth control of vertically aligned graphene on C-face SiC has been investigated. The influence of processing conditions is demonstrated. A temperature higher than 1700 °C is indispensable and the growth will accelerate with temperature increase. The graphene height is basically proportional to the annealing time. Proper temperature gradient and good crystalline quality of SiC substrates can further associate the vaporization of substrates and promote the growth. Pressure does not directly affect the growth while the partial pressure of vapor is crucial as it can interfere with further vaporization. The growth mechanism is proposed in terms of physical vapor transport. The monolayer character is verified by Raman and XAS spectra regardless of the graphene height, which indicates the robustness of the growth method. With the obtained samples, the d3.We used commercially available SiC substrates (Beijing TankeBlue) to grow vertically aligned graphene. Without special notation, the substrates are conductive C-face 4H-SiC of dummy level with dimensions of 10 × 5 × 0.33 mm−6 mbar. SiC substrates with C-side facing up are loaded into a graphite susceptor with a lid. The susceptor is placed in the middle of the chamber supported by a graphite tubular mount  with an induction coil and a turbo molecular pump, which can reach temperatures as high as 2000 °C and a vacuum of ~10ar mount . The tem14 to 1 × 1016 cm−2 are labeled according to their fluence. Cu distribution is demonstrated in inset of 20 cm−3 for the sample with the Cu fluence of 1 × 1016 cm−2.Some vertical graphene samples were implanted with Cu ions after annealing. The incident energy and angle were 190 keV and 7°, respectively. The samples with the Cu fluence varying from 1 × 10−1. X-ray absorption spectroscopy (XAS) was performed at beamline 6.3.1 of the Advanced Light Source using the total electron yield mode at room temperature. Rutherford backscattering (RBS) spectrometry with ~1.7 MeV He ions was employed to determine the composition distribution. The magnetic properties were measured by a vibrating sample magnetometer equipped with a commercial superconducting quantum interference device  with a sensitivity limit of 10−7 emu in the field range of 5 kOe at 300 K. The transport measurements were executed using a current of 1 mA under the magnetic field of 50 kOe at 5 K with a Lakeshore system.Scanning electron microscopy (SEM) micrographs were captured using an S-4800 field-emission SEM (Hitachi) operated at an acceleration voltage of 10 kV. Room temperature Raman measurements were carried out at a LabRAM (HORIBA Jobin Yvon) using a 532 nm Nd:YAG laser as excitation source with a resolution of ~0.5 cmHow to cite this article: Liu, Y. et al. Controllable growth of vertically aligned graphene on C-face SiC. Sci. Rep.6, 34814; doi: 10.1038/srep34814 (2016).


However, its universal validity has not been very well verified experimentally. By functionalization of approximately pure amino groups on graphene basal plane, a spin-generalization efficiency of ~1 μB/100 NH2 was obtained for the first time, thus providing substantial evidence for the validity of inducing magnetic moments by sp3-defects. As well, amino groups provide another potential sp3-type candidate to prepare magnetic graphene.Inducing magnetic moments in graphene is very important for its potential application in spintronics. Introducing sp Generalically H  or hydroically H ,5,6; andically H ,7, cuttiically H , nanoribically H  or nanomically H , or subsically H . From th defects ?3-type defects has been reported [3-defect may be not a good choice to experimentally confirm the universal validity for inducing magnetism in graphene. Amino group (NH2) provides a better choice than the hydroxyl group. In fact, atomic-scale control of the distribution of H atoms to paint magnetism on graphene has been achieved by scanning tunneling microscopy (STM) . Graphenreported . Howeverreported  or potasreported  because 3-type defects on the graphene basal plane. It has been reported that illuminating graphene under white light in ammonia atmosphere is a feasible way to form amino-type sp3-defects on the graphene basal plane [3-type graphene suitable for measurement on a superconducting quantum interference device (SQUID) without importing original 3-D metals, as long as graphene is not originated from GO and can be massively produced—Parvez et al. provided a good way to solve such a problem by electrolytic exfoliation of graphite [In fact, there are a lot of ways to introduce highly atomic N in graphene ,16; howeal plane ,18. By sgraphite . Graphengraphite ,21.3-type N-doped graphene (sp3-NG). Our results demonstrate that almost all the N atoms are bonded to graphene basal-plane carbon atoms in the form of amino groups, and these amino-type sp3-defects can effectively introduce magnetic moments in graphene with an efficiency of ~1 μB/100 NH2. We firstly experimentally proved the universal validity of inducing magnetic moments by amino-type sp3-defects and provide another potential candidate to prepare magnetic graphene, which is regarded as significantly crucial in the application of graphene spintronics. Furthermore, by using such sp3-type magnetic graphene to introduce localized magnetic moments on graphene, controlling the spin scattering to control the magnetoresistance as dilute F-doped sp3-functionalized graphene [In this study, we illuminated electrolyzed graphene (EG) with a decent few-layer ratio in ammonia to successfully obtain spgraphene  is hopefgraphene  can be eThe graphene sheets were obtained through electrolytic exfoliation  of comme3-NG was obtained by irradiating EG for 30 min with light from a 500 W high-pressure Hg lamp in NH3 (99%) atmosphere at a rate of 80 sccm [3 atmosphere to get nitrogen-doped graphene (NG), respectively.Amino-functionalized sp 80 sccm . For com−8 emu , and all data was corrected for the diamagnetic contribution by subtracting the corresponding linear diamagnetic background at room temperature. The 3-D impurity elements of all the samples are measured by inductively coupled plasma (ICP) spectrometry .The morphologies of the samples were characterized using transmission electron microscopy , and the X-ray photoelectron spectroscopy (XPS) measurements were performed on ESCALAB 250Xi  using an Al Kα radiation. Raman spectra were performed on Renishaw inVia  using a laser excitation of 532 nm. The magnetic properties of the samples were measured using SQUID magnetometer with a sensitivity less than 10Shown in −1, respectively. The shape and height of 2-D peak is similar to the previous report [2-carbon-based structure of graphite or graphene since the D peak is prominent (DI/GI = 0.44), which demonstrates that during the electrolysis a lot of defects had been induced on the graphene sheet. By the determination of XPS measurement (DI/GI = 0.32) and oxygen content (5.5 at %), which means a lot of oxygen groups were removed and the pristine sp2-aromatic structure was restored to a certain extent. From −1, that is, with respect to 1580 cm−1 of pristine graphene, such blue shift means a lot of non-crystalline fractions still exist after heating [3-NG), the defect ratios are dramatically increased . Note although the nitrogen contents of NG and sp3-NG are very close (3-NG is more prominent and the slight red-shift (~2 cm−1) of G peak of NG imply they may have different N-doping types. Since in-plane N atoms implanting into graphene will lead to the red shift of the G peak, we may guess the N-doping type of sp3-NG is out of plane, that is, amino sp3-type [3-NG is very alike to lightly sp3-functionalized F-doping few-layered graphene [The main features in the Raman spectra a of carbs report , typicals report . Obvioussurement , we foun heating . It alsory close , the D psp3-type . Moreovegraphene . The fol3-NG, so we know that by thermal or light treatment EG in ammonia, the nitrogen atoms can be successfully inserted into the carbon skeleton of graphene. To identify the difference of the N-bonding environments of these two kinds of nitrogen doping, we carefully deconvolute the fine-scanned N 1s spectra of NG and sp3-NG  [3-NG, aside from a similar weak graphite-N subpeak, NG presents another two subpeaks which sit at ~398.3 and 400 eV, typically identified as pyridinic- and pyrrolic-N, respectively. As is known, pyridinic-, pyrrolic- and graphite-N are all in-plane in graphene sheet while amino-N is out of plane to form sp3-defect, so we name the illuminated N-doped graphene as sp3-NG. As shown in 3-NG is almost purely amino-N functionalized with a total N content of 2.9 at %. Naturally, the different types of N bonding will lead to different physical properties of N-doped graphene.From the XPS spectra in d sp3-NG b. Fairly (NH2–C) , accompaM) and the different applied magnetic field (H) of the typical samples under the temperature of 2 K. EG, TG, NG and sp3-NG are all diamagnetic, which means the intrinsic diamagnetism of graphene is predominant in these samples. However, the diamagnetic degrees are different. The diamagnetic measurements of TG . We plotted ΔM under different H in M of EG, NG and sp3-NG, both O and N heteroatoms have successfully introduced magnetic moments in graphene. To fit the ΔM–H curves with Brillouin functionBk is the Boltzmann constant, N is the number of present magnetic moments, S is the spin angular momentum number, and g is the Landau factor assumed to be 2, we found that, all EG, NG and sp3-NG can be well fitted by using SM of EG, NG and sp3-NG are 0.045, 0.032 and 0.082 emu/g, respectively.All the samples were compressed into a diamagnetic plastic bag for magnetism measurement. To make the results as accurate as possible, we used at least 20 mg samples for measurement of each run. tent see , indicat defects . Corresp−4 emu/g, which is far less than the magnetization we got (10−2 order of magnitude). Note that we burned at least 30 mg sample each time for ICP measurement, which avoided the influence from the uneven distribution of 3-D metals. Thus, we can further study the magnetic sources without considering possible extrinsic factors. Previous reports [zp orbitals of carbon atoms are partly occupied to change the symmetric structure of π electron system. That is, uncompensated electron spins are generated. During the electrolysis, due to the decomposition of water, many oxygen atoms are physically adsorbed on or even form hydroxyl groups to bond to the graphene basal plane [3-NG has an improvement of magnetization to about twofold of EG, indicating the N atoms in sp3-NG are effective to induce magnetic moments. Clearly, the different types of N atoms resided on NG and sp3-NG bring forth such different effects.To analyze the magnetic sources of these samples, first, we must make certain the magnetic moments are not originated from 3-D contaminants. Through ICP measurement, the possible impurity contents are listed in  reports ,27,28 inal plane , thus in3-NG were schematically represented in According to the XPS deconvolution results b, possib3-type point defect on graphene basal-plane to introduce the magnetic moment of 1 μB  [3-defects sitting on the same sublattice can contribute large spin clusters  [3-NG samples since its ΔM exhibit good spin-1/2 paramagnetic behavior  [3-type defect, the amino group exhibits the universal effectiveness of inducing magnetic moments in graphene.As for an isolated amino-N, it creates a spB dimer) ,13, willbehavior b, so we /1000 F) . We trie3-type defect, the amino group can introduce magnetic moments in graphene with an efficiency of 1 μB/100 NH2. As is known, the existence of localized magnetic moments is a prerequisite for magnetic coupling to induce ferromagnetism in graphene, which is deemed promising to design a spin field-effect transistor (SFET). Although the as-prepared amino-functionalized graphene is still diamagnetic, the validity of inducing magnetic moments of amino groups provides the imagination space to achieve ferromagnetic graphene. Moreover, unlike F atoms producing lots of holes on graphene basal plane to damage the integrity of graphene film when generating sp3-defects [3-type defect was verified experimentally, laying a solid foundation for graphene magnetism theory, and paving the way for its potential applications in spintronics.In summary, we have prepared almost purely amino-functionalized graphene by white-light illumination of EG. As a sp-defects , the pho


The immune escape or tolerance of cancer cells is considered to be closely involved in cancer progression. Programmed death-1 (PD-1) is an inhibitory receptor expressed on activating T cells, and several types of cancer cells were found to express PD-1 ligand 1 (PD-L1) and ligand 2 (PD-L2).In the present study, we investigated PD-L1/2 expression in papillary renal cell carcinoma (pRCC).p value = 0.084 in Chi-square test). The frequency of high PD-L1 expression cases was higher in type 2 (23%) than in type 1 (11%), and the frequency of high PD-L1 expression cases was higher in grade 3/4 (21%) than in grade 1/2 (13%). However, no significant association was found between PD-L1 expression and all clinicopathological factors in pRCC.We found PD-L1 expression in 29 of 102 cases, but no PD-L2 expression was seen. PD-L1 expression was not significantly correlated with any clinicopathological factor, including progression-free survival and overall survival. The frequency of PD-L1-positive cases was higher in type 2 (36%) than in type 1 (22%) pRCC; however, there was no significant difference in the percentages of score 0 cases  contains supplementary material, which is available to authorized users. MET gene　activation, which is known to promote proliferative activity and cell survival, is frequently observed in pRCC, and MET inhibitors have become a new type of therapeutic agent for patients with advanced pRCC [Renal cell carcinoma (RCC) is a common cancer of the kidney, and the three most frequent histological subtypes are clear cell RCC , papillary RCC pRCCc, 10 to 20%), and chromophobe RCC 5%) 0 to 20%,. Recent % 0 to 20ced pRCC .Anti-cancer immune responses were considered to play important roles in preventing cancer progression in RCC ; howeverWe reviewed 102 cases of pRCC that were excised at Kumamoto University, the University of Occupational and Environmental Health, and Kyushu University between 2001 and 2014. All samples were obtained with informed consent from patients in accordance with the study protocols that were approved by the review board of each university . Tissue samples of primary site were fixed in 10% neutral buffered formalin and were embedded in paraffin as per a routine method. Nuclear grade and T classification were assessed according to the World Health Organization classification. Patient characteristics, such as age, gender, Fuhrman grade, pathological TNM stage and follow-up data were retrospectively collected.Rabbit monoclonal antibodies against PD-L1 (clone E1L3N) and PD-L2 (clone D7U8C) were purchased from Cell Signaling Technology . Briefly, after samples were reacted with primary antibodies, they were incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibodies . Can Get Signal Solution  was used to dilute the antibodies for enhancing the immunoreaction. Reactions were visualized using the diaminobenzidine substrate system (Nichirei). Two pathologists (TM and YK), who were blinded to information about the samples, evaluated the immunostaining of PD-L1/2.Two lymphoma cell lines  were obtained from RIKEN Cell Bank , and cell block specimens were used for confirmation of specificities of anti-PD-L1/2 antibodies. Cells were fixed in 10% neutral buffered formalin and cell block samples were embedded in paraffin.p < 0.05 was considered to be statistically significant.Statistical analysis was carried out with StatMate III . The simultaneous relationships between multiple prognostic factors for survival were assessed using the Cox proportional hazards model with a stepwise backwards reduction. A value of At first, we confirmed the specificities of anti-PD-L1 and PD-L2 monoclonal antibodies used for immunostaining using cell block samples of two cell lines Fig. . In totap value = 0.084 in Chi-square test, Fig. p value = 0.47 in Chi-square test, Fig. p value = 0.42 or 0.36 in Chi-square test respectively, Fig. Next, we classified the PD-L1 staining patterns into three scores according to the percentage of PD-L1-positive cancer cells: score 0, negative or less than 2%; score 1, 2 to 30%; and score 2, more than 30% (Fig. Expression of PD-L1 is associated with a poor clinical course in colorectal cancer, lung cancer, ovarian cancer, and ccRCC –22. In tPD-L1 expression is also known to be detected in infiltrating leukocytes, including macrophages ; howeverAlthough PD-L2 expression in cancer cells has been reported in ovarian cancer , there aIn the present study, PD-L1 expression was observed in 28% of the pRCC cases. The frequency of score 2 was high in type 2 or higher nuclear grade cases. Although PD-L1 expression appeared to be related to worse overall survival, there was no significant correlation between PD-L1 expression and clinical prognosis. Further studies are necessary to evaluate if PD-L1 expression might be useful for predicting the efficacy of anti-cancer immunotherapy using immuno-checkpoint inhibitors.


PD‐1) and programmed cell death‐ligand 1 (PD‐L1) have been identified as novel targets of immunotherapy of lung cancer. In present study, we evaluated the metabolic characteristics of lung cancer by using 18F‐fluorodeoxyglucose positron emission tomography/computed tomography (18F‐FDG PET/CT) with regard to PD‐L1 protein expression. PD‐L1 protein expression was evaluated by immunohistochemistry with the antibody clone SP142 in 579 surgically resected primary lung cancer patients. Cases with less than 5% tumor membrane staining were considered negative. We examined the association between the frequency of PD‐L1 protein expression and the maximum standardized uptake value (SUVmax) in preoperative 18F‐FDG PET/CT. The cut‐off values for SUVmax were determined by receiver operating characteristic curve analyses. The SUVmax was significantly higher in nonsmall cell lung cancer (NSCLC) patients with PD‐L1 protein expression compared with those without PD‐L1 protein expression (P < 0.0001). However, there was no correlation between SUVmax and PD‐L1 protein expression in patients with neuroendocrine tumors (P = 0.6545). Multivariate analysis revealed that smoking, the presence of pleural invasion, and high SUVmax were independent predictors of PD‐L1 positivity. PD‐L1‐expressing NSCLC had a high glucose metabolism. The SUVmax in preoperative 18F‐FDG PET/CT was a predictor of PD‐L1 protein expression in patients with NSCLC.Programmed cell death‐1 ( Correlations between FDG uptake and biologic features of cancers such as proliferation, histologic type, tumor differentiation, and hypoxia were reported EGFR mutations/ALK rearrangements were also reported 18F‐FDG PET/CT.This translational study examined PD‐L1 protein expression in primary lung cancer patients who had undergone surgical resection and investigated the association between PD‐L1 protein expression and the maximum standardized uptake value (SUVmax) in preoperative 18F‐FDG PET/CT before surgery. Four hundred and forty‐one patients with ADC and 103 patients with SCC until December 2015, and four patients with large cell carcinoma (LCC), 16 patients with small cell lung carcinoma (SCLC) and 15 patients with large cell neuroendocrine carcinoma (LCNEC) until June 2016 were included in this study. A total of 579 paraffin‐embedded specimens were retrieved from the registry of the Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University. Patients with a history of SCC of the head and neck or esophagus were excluded from this study because of the possibility of metastatic SCC from these cancers. Patients who received neoadjuvant therapy were also excluded because of inconsistency in the expression of PD‐L1 on tumor cells before and after neoadjuvant chemotherapy EGFR mutation status were also examined by subset analysis. EGFR status was determined in tumor tissues using the peptide nucleic acid‐locked nucleic acid polymerase chain reaction clamp method  in 377 specimens We retrospectively examined patients with primary lung cancer who underwent complete surgical resection at the Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University. Among them, we selected patients who were investigated with chest CT and Chest CT scanning was performed in a supine position during inspiratory breath‐hold using various multi‐detector row scanners: Aquilion 4 (Toshiba), Aquilion 64 (Toshiba), Aquilion ONE (Toshiba), Aquilion ONE Vision (Toshiba), SOMATOM Plus4 Volume Zoom (Siemens), Briliance CT (Phillips), and Briliance iCT (Phillips). Imaging parameters for thin‐section CT were as follows: tube voltage 120 kVp; tube current 100–500 mA; scan field of view 320–360 mm; and slice thickness 2 mm. A real exposure control (Toshiba) of automatic exposure control (Siemens and Phillips) was added in each study. All CT data sets were transferred to a Picture Archiving and Communication System, which was accessible for workstations  with a specialized application for the lungs.®, Biograph mCT) from the vertex to the proximal thigh were performed for attenuation correction, and for determining the precise anatomic location of lesions before acquisition of PET images. CT scans were reconstructed by filtered back projection into 512  ×  512 pixel images with slice thickness of 5 mm to match the PET scan. FDG uptake in lesions was evaluated using SUVmax, calculated by dedicated workstation for each scanner.In each patient, 185 MBq FDG was intravenously administered after fasting for at least 4 h. Scans were conducted from the middle of the thigh to the top of the skull 60 min after FDG administration. FDG‐PET/CT images were obtained by integrated PET/CT scanner  or Biograph mCT . All emission scans were performed in three‐dimensional mode, and acquisition time per bed position was 3 min for Discovery STE and 2 min for Biograph mCT. We reconstructed PET images using the ordered‐subset expectation–maximization method (VUE Point Plus) with two full iterations of 28 subsets for the Discovery STE and iterative True‐X algorithm and TOF (Ultra HD‐PET) with two full iterations of 21 subsets. The True‐X algorithm incorporates an additional specific correction for the point‐spread function. The full‐width at half‐maximum values of the Discovery STE and Biograph mCT were 5.2 and 4.4 mm, respectively. A low‐dose 16‐slice CT  and a low‐dose 32‐slice CT . Carcinoma cells showing membranous staining for PD‐L1 were evaluated as positive cells. The proportion of PD‐L1‐positive cells was independently estimated as the percentage of total carcinoma cells in whole sections by three investigators . If the independent assessments did not agree, the slides were reviewed by all three investigators together to achieve consensus. The consensus judgments were adopted as the final results. Cases with <5% tumor membrane staining were considered negative in this study. Sections from human placentas were used as positive controls.18F‐FDG PET/CT using Student's t‐test. Correlations between the proportion of PD‐L1‐positive carcinoma cells and SUVmax in preoperative 18F‐FDG PET/CT were assessed using Spearman's correlation coefficient test. All statistical analyses were performed by JMP Statistical Discovery Software . P values < 0.05 were statistically significant.Associations between PD‐L1 protein expression and patient characteristics were analyzed using Fisher's exact test, and univariate and multivariate analyses of the relationship between PD‐L1 protein expression and other patient characteristics were performed by logistic regression analysis with backward elimination method. Cut‐off values for SUVmax were determined by receiver operating characteristic curve analyses. We examined the association between the frequency of PD‐L1 protein expression and SUVmax in preoperative A total of 579 patients with primary lung cancer  who underwent complete surgical resection were included in the present study (Table P < 0.0001), (B) ADC/SCC/LCC (P < 0.0001), (C) ADC (P < 0.0001) and (D) SCC (P = 0.0044) (Table P = 0.6545) , (B) ADC/SCC/LCC , (C) ADC  and (D) SCC   Table , Fig. 2.) Figure . Spearma) Figure .We examined the association between PD‐L1 protein expression and other patient characteristics, especially SUVmax. We determined the preferable SUVmax cut‐off level using receiver operating characteristic curve analyses Fig. . Multiva18F‐FDG PET/CT is a significant biomarker for metabolic characteristics of cancer cells, and correlates with important features such as proliferation, histologic type, tumor differentiation, and hypoxia EGFR mutations/ALK rearrangements was also reported We examined the relationship between PD‐L1 protein expression and other patient characteristics. Glucose metabolism was generally higher in patients with PD‐L1 protein expression than those without PD‐L1. Additionally, multivariate analysis revealed that smoking, the presence of pleural invasion, and high SUVmax in PET/CT were predictors of PD‐L1 protein expression in patients with lung cancer, especially NSCLC. Glucose metabolism of cancer cells measured on 18F‐FDG PET/CT is valuable for evaluating PD‐L1 protein expression in NSCLC, a mechanistic link between FDG uptake and PD‐L1 protein expression remains unclear. The mechanism of FDG uptake within lung cancer cells involves glucose metabolism, hypoxia, angiogenesis, and the mammalian target of rapamycin (mTOR) signaling pathway; mTORC1 activity affected the amount of FDG uptake within lung cancer cells 18F‐FDG PET/CT could provide useful information on the metabolic state of the tumor microenvironment including tumor‐related activated immune cells and tumor cells.Although We recently examined PD‐L1 protein expression in 417 surgically resected ADC specimens 18F‐FDG PET/CT. The data obtained might help identify patients with PD‐L1 expression who would benefit from antiPD‐1/PD‐L1 antibodies. The second limitation was that we investigated the association between PD‐L1 expression on tumor cells only and SUVmax. PD‐L1 protein is expressed by tumor cells and immune cells such as macrophages The present study had several limitations. First, this was a single institutional retrospective study and not a trial‐based correlative study; however, 579 patients were examined for associations between PD‐L1 protein expression and SUVmax in preoperative Immunohistochemistry is the main method for examining PD‐L1 protein expression, which requires biopsied or surgical specimens from patients, and invasive procedures such as transbronchial lung biopsy, CT‐guided biopsy, or surgery. However, this study suggests PD‐L1 protein expression can be predicted by medical examination and radioisotope examination, which are noninvasive or minimally invasive and can be used even if specimens from patients are unavailable. Thus, SUVmax could be used as a noninvasive tool to assess the tumor microenvironment including the frequency of PD‐L1 protein expression to predict benefit from antiPD‐1/PD‐L1 inhibitors. Standardized methods to define the cut‐off value for SUVmax should be established using studies with larger sample sizes.18F‐FDG PET/CT was a predictor of PD‐L1 protein expression in patients with NSCLC.In conclusion, PD‐L1‐expressing NSCLC was related to high glucose metabolism. The SUVmax in preoperative The authors declare no conflicts of interest in association with the present study.Figure S1. Representative images of computed tomography (CT), 18F‐fluorodeoxyglucose positron emission tomography/CT (18F‐FDG PET/CT) and immunohistochemistry in a patient (A‐C) with programmed cell death‐ligand 1 (PD‐L1) protein expression in LCC. The maximum standardized uptake value (SUVmax) is 18.9. LCC: large cell carcinoma. Scale bar: 50 μm.Click here for additional data file.Figure S2. The maximum standardized uptake value (SUVmax) according to programmed cell death‐ligand 1 (PD‐L1) protein expression in patients with neuroendocrine tumors (SCLC/LCNEC). There was no correlation between SUVmax and PD‐L1 protein expression (P = 0.6545). SCLC: small cell carcinoma, LCNEC: large cell neuroendocrine carcinoma.Click here for additional data file.Figure S3. Association between the proportion of programmed cell death‐ligand 1 (PD‐L1)‐positive carcinoma cells and the maximum standardized uptake value (SUVmax) in the analyses of (A) overall , (B) ADC/SCC/LCC , (C) ADC  and (D) SCC . ADC: adenocarcinoma, SCC: squamous cell carcinoma, LCC: large cell carcinoma.Click here for additional data file.Figure S4. Representative images of receiver operating characteristic (ROC) curves in the analyses of (A) ADC and (B) SCC. ADC: adenocarcinoma, SCC: squamous cell carcinoma, AUC: area under curve.Click here for additional data file.Table S1. Association between PD‐L1 protein expression and clinicopathological factors in patients with adenocarcinoma.Click here for additional data file.Table S2. Association between PD‐L1 protein expression and clinicopathological factors in patients with squamous cell carcinoma.Click here for additional data file.Table S3. The frequency of PD‐L1 protein expression according to smoking history and SUVmax in preoperative 18F‐FDG PET/CT.Click here for additional data file.


EP 16461554.4) is related to a method of analysing a solid substrate by means of Secondary Ion Mass Spectrometry (SIMS). It comprises the steps of providing a graphene layer over the substrate surface and analysing ejected secondary anions through mass spectrometry analysis. The graphene layer acts as a kind of filament that emits a lot of secondary electrons during the experiment which significantly increases the negative ionization probability and thus the intensity of the SIMS signal can be more than two orders of magnitude higher than that of a similar sample without graphene. The method is particularly useful for the analysis of surfaces, 2D materials and ultra-thin films. The intensity of dopants and contamination signals can be enhanced up to 35 times, which approaches the detection limit of ~10 The sample is bombarded with a primary ion beam that leads to the sputtering of the surface. Small parts of the sputtered particles are ionized (secondary ions) and the sample composition can be determined by means of mass spectral analysis. During the sputtering subsequent layers of the sample are removed and thus it is possible to obtain information about changes in the composition as a function of depth, thus creating a depth profile.SIMS is a very precise analytical technique for determining the elemental composition of a sample12. For most materials it is reported to be in the range of 1015–1016atoms/cm311, sometimes even as good as 1012atoms/cm312. These optimum detection limits, however, are achieved during the dynamic SIMS (dSIMS) mode. In this mode a very dense ion beam is used and a substantial amount of material is sputtered simultaneously, allowing more ions to be detected. Drawbacks include very poor depth resolution and limitation to thick materials.SIMS is well known for its excellent detection limits of trace elements15, in which a low density ion beam ensures that ions are emitted only from monolayers 1 to 3. This mode, however, has physical limitations. Because a relatively small amount of matter is sputtered, and even less ionized, there are not enough secondary ions extracted from the sample to achieve as optimum detection limit as the dynamic mode does. In most cases it is difficult to exceed a limit of 1 ppm (5 * 1016atoms/cm3 or 109atoms/cm2).Surfaces, 2D materials and ultra-thin films are analysed ideally in a special mode called static SIMS (sSIMS)19, whereas cesium enhances the formation of negative ions20. For some elements the difference can be up to four orders of magnitude. Although most elements can be detected as both positive and negative ions, each element usually gives better results in terms of detection limit in either positive or negative mode. In some experiments, oxygen flooding can further enhance the secondary ion yields of some negative ions23. Using these techniques yields acceptable results, but in the cases of surfaces, 2D materials and ultra-thin films, it is often not enough to reach the desired detection limit, particularly in the case of trace elements.There are many ways to enhance the ionization probability and thus the detection limit. It has been noted that using oxygen as primary ions significantly enhances the formation of positive ions31. More specifically, SIMS has been used to map the surface of the graphene layer25, to analyse the mass spectra of graphene27, and to create a depth profile of single-layer graphene grown on substrates that do not contain carbon29. Furthermore, SIMS has been used for the depth profiling of multilayer graphene of a thickness of more than 3 nm on SiC30 and, recently, of hydrogen-intercalated graphene on SiC having a theoretical thickness of 0.69 nm31. All these methods employed SIMS for characterising the graphene layer, while the composition of support material underneath was not analysed.Several SIMS measurements on graphene (Gr) have already been reportedIn this work we present Graphene Enhanced Secondary Ion Mass Spectrometry (GESIMS) - a new technique for enhancing the detection limit in the analysis of thin materials. It has been found that providing a graphene layer on a substrate significantly reduces the emission of matter from the substrate but at the same time increases the negative ionization probability. Destruction of a small part of the graphene layer in a dynamic SIMS mode significantly reduces the initial strong blocking effect of graphene, while the enhancement of the ionization probability of the sputtered material is still observed. Under these conditions the ionization probability greatly prevails, resulting in an SIMS signal with an intensity that is unexpectedly high compared with a sample without graphene and thus able to to reach better detection limits.A−/A+  per incident ion. In turn, secondary ion yield is defined as the product of the partial sputter yield (number of emitted species A per incident ion) and ionization probability. The presence of graphene on a substrate surface clearly reduces the partial sputter yield of a material from the substrate, since for given conditions it is expected that sputtered species are emitted from the ~3 top monolayers of material and graphene acts as a barrier to emission. If despite this fact a significant increase in the SIMS signal intensity is still observed, the only possible explanation is that the presence of graphene drastically increases the negative ionization probability. Fig. The basic concept of GESIMS method is presented on Fig. YGr and Y denote partial sputter yield for a sample with and without graphene, respectively, while αGr and α denote ionization probabilities . Under optimum conditions YGr < Y and αGr > α, whereby the latter effect is much stronger, so that GF > 1 and thus the intensity of the SIMS signal can be enhanced.To quantify the effect, a gain factor can be defined as follows:Although increasing the intensity of the matrix element signal is of interest from a scientific viewpoint, it does not seem to have much practical application in SIMS-based surface analysis. As already mentioned above, the actual benefit is the enhancement of the detection limit of trace elements and thus new sets of samples have been considered and tested. Each of the sets consisted of a pair of samples, one having graphene transferred onto its surface, and the other - without graphene - serving as a reference. Signal enhancement was observed for all of them and the maximum gain that was reached during the experiments has been presented in Table GF = 1).As already mentioned, the effect was not observed at the beginning of the experiment, when a fresh graphene layer was transferred on a sample. This is not surprising since graphene is a strong material and the blocking effect was expected to be high. Thus the reduction in the partial sputter yield was higher than the increase of the ionization probability. To overcome it, the primary beam intensity had to be increased to several hundred pA for a short time. Under such conditions dynamic SIMS mode was achieved and the entire surface was slowly eroded. When only a small part of the graphene sheet was destroyed, the blocking effect was significantly reduced while the enhancement of the ionization probability was still observed. Figure It was essential in these experiments for the uniformity and the intensity of the beam to be well defined and reproducible and thus it was not surprising that the shape of all curves and the position of the maximum were similar for all measured samples as they depended on the partial destruction of the graphene layer and not to the type of the dopant and the substrate. Only the actual enhancement was material related (both the substrate and the type of dopant) and thus each sample had a different maximal gain factor.−8 mbar for two hours. Figure The result is fully reproducible - Fig. The best results, as presented in Table 18 atoms/cm3) and low (1 * 1016 atoms/cm3) dopant concentration . As can be clearly seen for Sample A, the intensity of the SIMS signal was significantly increased and thus a better detection limit was achieved (3.02 * 1015 instead of 4.86 * 1016 atoms/cm3). In the case of Sample B, the concentration was too low to be measured using a standard sSIMS condition, whereas it was possible for the GESIMS method. As can be seen, the enhancing effect does not depend on the dopant concentration and thus GESIMS method is quantifiable.When the optimal conditions are reached, the beam intensity should be decreased and sSIMS measurement can be performed. Figure 2+ primary beam with positive ion detection. As shown on Fig. YGr/Y ratio.The same procedure was used for a O+ ions and the gain factor change was obtained just like it was shown on Fig. + beam was switched off and a very brief sSIMS measurement with O2+ beam was performed so that the YGr/Y ratio could be determined as shown on Fig. YGr/Y ratio increased rapidly from ~0.27 to ~0.43 close to the optimal condition which apparently led to a significant increase of the gain factor.A new experiment was devised for AlGaAs substrate with Si dopant: the sample was sputtered in dSIMS mode with Cs2 which normally required an electron flood gun for charge compensation. The intensity of the Cs+ primary beam was once again set to 200 pA and it turned out that a thin layer of the amorphous carbon did not alter the experiment significantly: the charging effect was visible from the very beginning and the flood gun was needed to perform the analysis. However, in case of the graphene layer we were able to sputter the sample without the flood gun up to four an a half minutes before the charging effect occurred. It corresponded well with the time frame of the enhancement effect  and yet it was still able to provide enough electrons for charge compensation. We assumed that in a similar way the GESIMS effect could be explained: during the SIMS experiment a high voltage was applied to the sample holder (up to 5 kV) and thus the graphene layer acted as a kind of filament which emitted an excess of electrons during the ion bombardment leading to the enhanced ionization probability.From Fig. applying a graphene layer over the substrate surface;annealing the graphene-coated substrate at elevated temperature and reduced pressure;sputtering of the graphene-coated substrate in dSIMS mode, which leads to the partial destruction of the graphene layer;detecting and analysing ejected secondary anions by mass spectrometry analysis in sSIMS mode.GESIMS is a process for measuring and analysing thin substrates that involves the following steps:This method cannot be used for depth profiling of thick materials because the graphene layer will be destroyed within the first moments of the measurement and the enhancing property will be lost. Furthermore, the dSIMS analysis itself offers a high detection limit. It cannot be used for a high resolution imagining neither, since the presence of the partially destroyed graphene layer will reduce the spatial resolution. However, in the case of very thin materials, sSIMS reaches its physical limit and any enhancement is beneficial. GESIMS requires more preparation and optimization than the standard sSIMS analysis but improves by one order of magnitude the detection limits of trace elements, which may prove to be invaluable for the further development of 2D materials.The effect does not take place for samples covered with a thin layer of amorphous carbon. It has been noted that the graphene layer has different electric properties during the SIMS experiment - even a significantly destroyed layer can provide enough electrons to compensate the charging effect which is typically present for insulating materials. This behaviour can suggest the explanation of the GESIMS effect: since a high voltage is applied to the sample holder the graphene layer acts as a kind of filament and emits a lot of secondary electrons during the ion bombardment and thus the negative ionization probability is increased.A pair of samples was created for every experiment described in this work, one having graphene grown or transferred onto its surface, and the other - without graphene - serving as a reference.et al.33. As a substrate, (100)-oriented Ge layers deposited on Si (100) wafers were used. Methane gas in the mixture of Ar and H2 in the ratio of 200:1 was used as a carbon precursor. Growth was preceded by the substrate’s annealing in a pure hydrogen atmosphere in order to reduce native oxides in-situ. During the growth process, 800 mbar of pressure were sustained.In the case of germanium substrate, graphene films were synthesized in a commercially available system by the CVD method as described by Paternak μm thick copper foils by the CVD method using a Black Magic Pro system (Aixtron). To grow graphene on copper foils the following procedure was used: copper samples were first pretreated at 960 °C under an Ar gas flow and then a H2 gas flow at 20 mbar of pressure. The purpose of this step was to improve the quality and enlarge the grain size of Cu substrates as well as to remove oxides from the copper surface. Next, methane was introduced into the reactor at an adequate flow rate for a few minutes. During this step, the synthesis of graphene was observed. Finally, the copper substrates covering the graphene films were cooled to room temperature in an Ar atmosphere. To transfer graphene onto target substrates, we used the high-speed electrochemical delamination method34.In other samples, graphene films were synthesized on 35 As described in aforementioned procedures, the presence of the graphene layer was confirmed by Raman spectroscopy measurements performed in back-scattering geometry, using a Renishaw inVia Raman Microscope, with a ×100 objective and a 532 nm Nd:YAG laser as an excitation source.−10 mbar. A Cs+ primary beam with negative ion detection was used for most measurements. Impact energy was varied between 1 keV and 13 keV but the highest energy was used for the most of the experiments. The intensity of the primary beam was 100–2000 pA and 0.5–10 pA for the dynamic and static SIMS mode, respectively. An O2+ primary beam with positive ion detection was used for a few experiments to show that graphene do not enhance the positive ionization probability. Impact energy was varied between 2 keV and 5 keV and the intensity of the primary beam was 300–3000 pA and 3–10 pA for the dynamic and static SIMS mode, respectively. Primary beams were rastered over 250 × 250 μm2 and the analysis area was limited to (200 × 200) μm2. Mass interferences were verified prior to analysis and adequate mass resolving power was used for each experiment.In this work all SIMS measurements were performed employing the CAMECA SC Ultra instrument under ultra-high vacuum (UHV), usually of 4 × 1037.Beam on the sample in the SC Ultra tool has a square shape and owning to the “variable rectangular shape concept” forms a homogeneous spot. The primary beam at the working point in the SC Ultra is formed by two stencils - well-shaped apertures. While the first one is used to choose the most intense and homogeneous part of the ion beam, the second one changes the size of the spot. This innovation provides high sensitivity for all measured elements and a high dynamic range with a very low sputter rate12 ions/cm2). This ensures that with a high probability every ion will impact on undamaged surface and not on an area that has already received ion impacts. In this mode, the emission is essentially limited to a few topmost monolayers and thus it is very surface-sensitive. It makes it an ideal mode for the analysis of surfaces, 2D materials and ultra-thin films because there is no interference with the lower layers. In dynamic SIMS mode the emission is theoretically still limited to the topmost monolayers but for higher ion doses it is expected that the whole surface will erode in time and thus the lower layer will be gradually exposed. In this way it is possible to obtain information on the variation of the composition of material below the initial surface, thereby creating so-called depth profiles. In this work, however, the dynamic SIMS mode was used to destroy only partially the graphene layer in order to expose the underlying substrate, which was subjected for further analysis. It was a critical step to ensure good reproducibility of the results and therefore a very homogeneous ion beam was required to perform GESIMS analysis.Secondary ions that are detected in SIMS analysis usually originate from the top three monolayers of the bombarded sample. For static SIMS analysis, very low ion doses are used (typically in range of 10