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


Fluorographene, formally a two-dimensional stoichiometric graphene derivative, attracted remarkable attention of the scientific community due to its extraordinary physical and chemical properties. We overview the strategies for the preparation of fluorinated graphene derivatives, based on top-down and bottom-up approaches. The physical and chemical properties of fluorographene, which is considered as one of the thinnest insulators with a wide electronic band gap, are presented. Special attention is paid to the rapidly developing chemistry of fluorographene, which was advanced in the last few years. The unusually high reactivity of fluorographene, which can be chemically considered perfluorinated hydrocarbon, enables facile and scalable access to a wide portfolio of graphene derivatives, such as graphene acid, cyanographene and allyl-graphene. Finally, we summarize the so far reported applications of fluorographene and fluorinated graphenes, spanning from sensing and bioimaging to separation, electronics and energy technologies. At the same time, Cheng et al. N-methyl-2-pyrrolidone (NMP) N,N-dimethylformamide (DMF) 2O2) with chlorosulfonic acid (HSO3Cl) Exfoliation of multilayer materials is a well-established approach, which is successfully applied, e.g., for production of graphene nanosheets. In the liquid-phase exfoliation process, a solvent is used as an intercalating agent, weakening the van der Walls interactions 2 and XeF2 to 24% and 100% degree of fluorination Mechanical exfoliation A represeca. 600 °C, as SEM analysis revealed.Finally, another reported method for delamination and expansion of GrF is thermal exfoliation. A partially fluorinated graphite was thermally exfoliated by inserting the solid sample in a preheated furnace at 800 °C, and further used for film formation and electrochemical testing 2.2sp2-hybridized carbons of graphene lattice, forming C-F sp3 bonds and producing fluorinated graphenes. For the direct gas-fluorination of graphene, one of the most common fluorinating agents is xenon difluoride (XeF2) gas and it was utilized for the fluorination of graphene at various temperatures under an inert atmosphere 2, providing a stoichiometric material C1.0F1.0 fully fluorinated on both sides, due to the effective etching on the Si substrate by reactive gas 2) gas, another important fluorination agent, was applied to fluorinate graphene and graphene derivatives at various temperatures and pressures 2Fluorination strategies are based on the model by which graphene  is placed into a reactor with fluorination agents, which are used as precursors for the generation of active intermediates with the aid of either thermal, pressure, or irradiation initiation B. These 4622/Si substrate and then, it decomposed under laser irradiation, generating active fluorine radicals, which reacted with carbon lattice and formed C-F bonds, providing fluorinated graphene C4F 3 in liquid Br2Plasma fluorination is an alternative way for fluorination of graphene, targeting to the synthesis of controllable fluorinated graphene materials. According to this technique, generated fluorine radicals adsorb onto carbon lattice and create different C-F bonds. The fluorine-based compounds CF26, SF4, and MoF62 and to improve the controllability of the C-F bonding character. At that point, it is worth mentioning that the approach above has been also used for the preparation of fluorographane through treatment of graphane with F23-etherate 6) All the methods discussed above employ graphene or bulk GrF for the preparation of fluorinated graphene or fluorographene. However, in the last years, there has been an increasing number of publications regarding the fluorination of GO, synthesizing fluorinated graphene oxide (FGO). Direct gas-fluorination has been successfully applied to the fluorination of GO, using as precursors F3sp2/sp3) ratio significantly affect the electronic and optical properties of fluorinated graphenes. Already a small amount of fluorine atoms in the structure give rise to a band gap opening 1F1 ratio, whereby other geometrical configurations differ only slightly from values calculated for the chair conformation The incorporation of fluorine atoms onto carbon lattice and the C/F (C 0W0@PBE level for FG showed an excitonic peak at 5.14 eV In order to study photoluminescence (PL) of FG, material dispersion in acetone was excited by 290 nm at room temperature −1 and 1345 cm−1. According to Fourier transform infrared (FTIR) spectroscopy, FG exhibits a strong band at 1260 cm−1, which is attributed to the covalent C—F bond stretching −1 and 1084 cm−1, corresponding to the stretching vibration of the C—F covalent bonds and the semi-ionic stretching vibration of C—F bonds, respectively −1, ascribable to the semi-ionic C-F bond, progressively transforms to covalent C—F bond, which is exhibited as a strong band at 1211 cm−1−1, due to the local wagging motion of the C-F bond. In the case of fluorine dimer, IR signal appeared at 1150 cm−1, originated by the local stretching motion of the bond formed by the C—C atoms connected to the fluorines.Another significant proof for the optical transparency of FG is the absence of Raman signals. That happens because the energies of the lasers, which are used for Raman spectroscopy, are lower than the band gap 2 (12%) and C-F3 (2%) species were also present, owing to defects at free edges 2 (∼289.9 eV), C—F2 (∼292.1 eV), and C—F3 (∼293.7 eV) −1, while in the fully fluorinated structure it amounts to 112.3 kcal mol−119F magic angle spinning (MAS) NMR studies on fluorinated graphite showed that the strength of the C—F bond, or its ionic/covalent character, could be also probed with NMR, according to which lower chemical shifts point to higher covalent character X-ray photoelectron spectroscopy (XPS) is a powerful technique providing information about elemental composition and binding conditions of elements in FG. According to XPS data, C—F type was the major bond (86%) in this material, while smaller fractions of C-F1F1 was found to be 2.48 Å, slightly larger than for graphene (2.46 Å). The value of the lattice parameter is in good agreement with the theoretical calculations (α = 2.48 Å), and it corresponds to the chair conformation with AA stacking sequence, the most stable structure sp3 C—C bonds, which are larger than the sp2-type bonding of graphene. The C—C and C—F bond lengths and the repeating unit of FG are shown in −2−2) than graphite Scanning and transmission electron microscopy (SEM and TEM) provide significant information about the layered morphology of FG see B, while In order to utilize FG for bioapplications, it was necessary to examine its biocompatibility and potential toxicity in cells. For that reason, extensive studies were carried out 4®), such as chemical stability in many solvents and high hydrophobicity xFy products evolve from the material Regarding the chemical behavior of FG, it was considered as an inert material, presenting similar properties to GrF and polytetrafluoroethylene  + KF + 1/2 I2. Additionally, graphene was produced by reduction of FG, utilizing either triethylsilane or zinc nanoparticles 2F(semi-ionic) + CH3C(O)CH3(l) → HF + 2C(s) + C2F + CH3C(O)CH3(l)As already mentioned, defluorination of FG is an important approach for its transformation to graphene. For that aim, a significant number of methods have been developed. They are mainly classified into two groups: (a) chemical and (b) thermal reduction. Robinson et al. Regarding the nucleophilic substitution of FG and the preparation of covalent functionalized graphene derivatives, N-, O-, S- and C-nucleophiles have been applied for that purpose see . Ethylensp2 carbons of the graphene lattice, according to [1 + 2] cycloaddition reaction On the other hand, the modification through the formation of C—C bonds between carbons of FG and carbons of the organic units had not received the proper attention and only the last years some remarkable works were reported. In the first work, chlorocarbene, generated in situ by chloroform after treatment with NaOH, attacked electrophilic carbons of FG, detaching fluorine atoms from FG and forming chloro-terminated cyclopropanic rings with the newly formed 52. At that time, there were no reports on the use of single sheets of FG. Nevertheless, the layered nature, low surface energy, and facile exfoliation of GrF plays a significant role in lubrication −1−1GrF and organosiloxane derivatized GrF were studied as solid lubricants and lubricant additives since ’60 s and ’70 s In the case of batteries as well, intercalation and even reactions  FG has also potential in electrocatalytic applications. Theoretical studies suggest that partially fluorinated graphene can act effectively as an efficient metal-free catalyst for oxygen reduction reaction (ORR) −1. This was achieved through preparation of hydroxyl-substituted fluorographene sp2-based magnetism in 2D systems and particularly room temperature sustainable magnetic ordering, pave the way for the future organic spintronic applications.Theoretically it has been indeed predicted that spin density can emerge in FG and be modulated by changing the degree of F coverage 2 and its separation from N2 have been predicted by molecular dynamic simulations upon fluorination of a pore rim in graphene 2O, SO2, H2S, and CO2) over N2, O2, and CH4. Therefore, applications are envisioned on separation of CO2 and SO2 from flue gases, purification of natural gas, and removal of H2O from air. A significant advantage is predicted for porous FG, related to the fact that gas molecule separation is not dependent on size-exclusion mechanism, but on interaction strengths. Therefore, there is no need for contiguous and unbroken films, but only large accessible surface area is required. In the same area of environmental and separation applications, fluorination of edge carbon atoms of a pore in a graphene sheet has been found by MD simulations very advantageous for extremely high water flux rates and very efficient salt rejection Owing to its hydrophobic surface properties, FG found application for anti-corrosion and self-cleaning coatings FG has also found applications in the field of biomedicine. In 2012, Wang et al. Finally, in 2012, two independent reports appeared studying the non-linear optical properties of FG 6Fluorographene displays sharply different properties than graphene because it lacks conjugated network of π-conjugated electrons, which makes this material a wide band gap insulator. Fluorographene was born rather recently, because it was isolated in 2010, but its bulk parent graphite fluoride has been known for more than one hundred years and since the seventies GrF has been used as industrial lubricant. The industrial usage of GrF makes also easily available FG, which can be prepared from GrF by exfoliation technologies. Despite its youth, FG found several important applications in coating, batteries, separation technologies, and electrochemical sensing. Recent discoveries documenting high-reactivity of FG open new doors to a wide range of graphene derivatives. Chemical transformation of FG is easy and scalable and leads to high-yield and selective covalent graphene derivatives, like cyanographene and graphene acid. Such graphene derivatives enable reactive functional groups homogeneously distributed over the graphene surface, which can be further utilized for covalent grafting of other functionalities, e.g., carbon dots, biomacromolecules, polymers etc., to graphene. Such graphene derivatives may also function as a versatile platform for countless applications, e.g., in selective electrochemical sensing, light harvesting, catalysis etc., therefore a significant attention should be paid to synthesis and testing of these materials. The chemical processability, high-thermal stability, low surface energy, and availability make FG an extraordinary material among other 2D materials, because its properties can be turned into numerous marketed technologies in the near future.


The analogue of electromagnetically induced transparency in optical ways has shown great potential in optical delay and quantum-information technology due to its flexible design and easy implementation. The chief drawback for these devices is the bad tunability. Here we demonstrate a tunable optical transparency system formed by graphene-silicon microrings which could control the transparent window by electro-optical means. The device consists of cascaded coupled ring resonators and a graphene/graphene capacitor which integrated on one of the rings. By tuning the Fermi level of the graphene sheets, we can modulate the round-trip ring loss so that the transparency window can be dynamically tuned. The results provide a new method for the manipulation and transmission of light in highly integrated optical circuits and quantum information storage devices. Since it has been proposed, the electromagnetically-induced transparency (EIT) effect has shown great potential in various applications, including slow light control, quantum information processing and sensing technology124567891078Graphene, a new material that has been available for only a decade, has generated great interest due to its exceptional properties such as high carrier mobility14161720222324In this work, we propose and experimentally demonstrate the EIT-like effect on graphene-silicon cascaded micoring resonators in which the transparency peak can be dynamically tuned via electrically control. By applying increasing voltage to the graphene/graphene capacitor which has been covered on one of the ring waveguides, regular changes of the EIT-like spectrum have been observed.EF), which will in turn alter the rate of interband transitions and, subsequently, the optical transitionsThe structure of our device is depicted in 2O3 was employed to prevent potential carrier injections from the bottom graphene layer into the silicon2 upper cladding were deposited are shown in 2O3, obtained using the same ALD method. To apply voltage to the capacitor Ti/Au (10/100 nm) contacts were used for both layers of graphene by electron-beam evaporation. We then provided 1 μm-thick SiO2 upper cladding deposited by plasma-enhanced chemical vapor deposition to avoid the loss caused by metal contact.The device was fabricated on commercial SOI wafers, having a silicon thickness of 220 nm and a buried-oxide thickness of 3 μm. The bus waveguide and the two cascaded ring resonators were fully etched, with the depth of 220 nm and a width of 450 nm, to guide single-mode transverse electric (TE) light . The radVb is added, the change of the carrier concentrations in graphene can be expressed asV0 is the offset voltage caused by natural doping, ε0 and ε are the permittivities of free space and of Al2O3, respectively, and t is the thickness of the Al2O3 between the two graphene layers, e is charge of the electron. Thus, the Fermi level of graphene will be shift as ħ is the Plank constant divided by 2π and vF is the Fermi velocity. When the bias voltage dependent Fermi level EF(Vb) is close to the Dirac point, interband absorption will occur as electrons are excited by the incoming photons (EF(Vb) is greater than half of the photon energy of incident light  of the graphene-silicon waveguide, which is mainly decided by the Fermi level-dependent dielectric constant (εg) of graphene. The dielectric constant εg can be obtained through ω is the frequency, tg is the thickness of the graphene layer, and σ is the optical conductivity of graphene. The overall mode in the graphene-silicon waveguide displays a Gaussian distribution. The graphene layers only capture very few portions of the fields and the fields outside the graphene layer cannot feel εg variationsεg′) of graphene which changes with the voltage-dependent Fermi level and is similar to the resonance frequency shifts observed in optical plasmon resonance with grapheneThe tunability of the graphene-ring resonator based EIT-like device is investigated by applying different bias voltages. For zero bias voltage, Fermi level is closed to the Dirac point and the top ring has relative high loss and the EIT-like effect is consequently weak , correspεg′ and εg″, of graphene can be obtained from its optical conductivity (σ), which can be derived from the Kubo formula in a complex form consisting of interband and intraband contributions2930The real and imaginary parts of the complex dielectric constant, τ2 is the relaxation time associated with electron interband relaxation and cooling of hot phonons313233τ2 has been measured on a picosecond scale33neff is proportional to graphene’s dielectric constant εg in both real and imaginary partsneff) represents the loss in the waveguide and it is the same for neff) which has a proportional relationship with the wavelength shift of the transmission curve (EF(Vb) and so does Re(neff), so the transparency curve has a red shift (EF(Vb) and so does Re(neff), so the transparency curve has a blue shift  at this voltage. Furthermore, the red and blue shifts of the transparency window have been well explained in terms of the real part of the dielectric constant of graphene. Our results will provide important guidelines for the design and use of tunable optical EIT devices which have promising applications in slow light, quantum information processing and sensing technology, and provide a new method for the manipulation and transmission of light in highly integrated optical circuits. Furthermore, compare with the mechanical tuning of the EIT-like effect, the tuning speed of the device could be as high as the one obtained with modulators2223242O3 was used to prevent potential carrier injections from the bottom graphene layer into the silicon. 200 nm thick polymethyl methacrylate (PMMA) was spin-coated onto the graphene sheet which was grown by chemical vapour deposition (CVD) on Cu film and baked at 200 °C for 1 min. The copper foil was then removed using FeCl3 solution and the graphene sheet, coated with PMMA, was transferred onto the device. Finally, the PMMA was removed using acetone and the residue was rinsed with isopropanol to obtain a clean graphene surface. To remove the unwanted part of graphene, the deep-ultraviolet lithography and oxygen plasma were used. Before deposing the sandwich 20 nm Al2O3, 10 nm Ti and 50 nm Au were deposited by electron-beam evaporation and patterned by a lift-off process. This was done to protect the bottom graphene from the sandwich Al2O3 etching process for electric contacting. Owing to the hydrophobic nature of the graphene basal plane, it is difficult to depose Al2O3 directly on it. For this reason, before deposing Al2O3, a 1 nm aluminum layer was evaporated using electron-beam evaporation and immediately oxidized into Al2O3 in ambient air2O3 as the seed layer, the sandwich Al2O3 was then deposited on the device. In order to contact the electrode with the bottom graphene layer, which was fully covered by Al2O3, reactive ion etching (RIE) was used to remove a patch of Al2O3 from the Ti/Au (10/50 nm) which had been previously deposed. The transfer procedures used for top graphene layer was the same with the bottom one. The final device was then covered with 1 μm-thick SiO2 deposited by plasma-enhanced chemical vapor deposition (PECVD) and drilled by RIE to contact the two graphene layers with the electrode.The cascaded ring waveguides were fabricated in the SOI wafer with a silicon thickness of 220 nm and a buried-oxide thickness of 3 μm. E-beam lithography (EBL) and inductively coupled plasma (ICP) etching were used to fabricate the 220 × 450 nm waveguides and the 300 × 300 nm gratings. The region for graphene transfer (the etching era for the top ring) was broadened to obtain a good coverage of graphene. Before the transfer of the bottom graphene, 5 nm isolation layer deposited (ALD) AlHow to cite this article: Wang, Y. et al. Tunable optical analog to electromagnetically induced transparency in graphene-ring resonators system. Sci. Rep.6, 38891; doi: 10.1038/srep38891 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In-depth study of the intron retention levels of transcripts provide insights on the mechanisms regulating pre-mRNA splicing efficiency. Additionally, detailed analysis of retained introns can link these introns to post-transcriptional regulation or identify aberrant splicing events in human diseases.e.g. U12-type vs U2-type) and its plotting functions allow visualization of the distribution of the retention levels of the introns. Statistical methods are adapted from the DESeq2, edgeR and DEXSeq R packages to extract the significantly more or less retained introns. Analyses can be performed either sequentially (on single core) or in parallel (on multiple cores). We used IntEREst to investigate the U12- and U2-type intron retention in human and plant RNAseq dataset with defects in the U12-dependent spliceosome due to mutations in the ZRSR2 component of this spliceosome. Additionally, we compared the retained introns discovered by IntEREst with that of other methods and studies.We present IntEREst, Intron–Exon Retention Estimator, an R package that supports rigorous analysis of non-annotated intron retention events (in addition to the ones annotated by RefSeq or similar databases), and support intra-sample in addition to inter-sample comparisons. It accepts binary sequence alignment/map (.bam) files as input and determines genome-wide estimates of intron retention or exon-exon junction levels. Moreover, it includes functions for comparing subsets of user-defined introns (ZRSR2 gene. Intron retention events caused by ZRSR2 mutation that we discovered using IntEREst (DESeq2 based function) show considerable overlap with the retained introns discovered by other methods (e.g. IRFinder and edgeR based function of IntEREst). Our results indicate that increase in both the number of biological replicates and the depth of sequencing library promote the discovery of retained introns, but the effect of library size gradually decreases with more than 35 million reads mapped to the introns.IntEREst is an R package for Intron retention and exon-exon junction levels analysis of RNA-seq data. Both the human and plant analyses show that the U12-type introns are retained at higher level compared to the U2-type introns already in the control samples, but the retention is exacerbated in patient or plant samples carrying a mutated The online version of this article (10.1186/s12859-018-2122-5) contains supplementary material, which is available to authorized users. Alternative pre-mRNA splicing is a cellular process in eukaryotes that generates multiple transcripts from a single gene. Of the various types of alternative splicing (reviewed by Hamid and Makeyev ) intron e.g. U4atac and U12 snRNAs, U11/U12-65K and ZRSR2 proteins) show, among other splicing defects, a further increase in IR levels of the U12-type introns [A well-established example of IR involves U12-type introns , which are spliced less efficiently compared to the U2-type (major) introns . The cla introns –19.i.e. Intron–Exon Retention Estimator that is based on the intron retention analysis used in Niemelä et al. [e.g. differential intron retention test based on the “exon usage test” provided by DEXSeq [p-values based on the null hypothesis that IR does not vary across the analyzed sample groups. The resulting p-values estimated for each intron allow subsequent identification of introns that show statistically significant difference of IR between the sample groups. IntEREst also provides tools for plotting the distribution of retention levels of the introns of interest within single or multiple samples. In addition, large datasets that demand significant computation time can be analyzed in parallel on multiple computing cores. IntEREst is available as a Bioconductor package and together with the manuals are accessible through https://bioconductor.org/packages/release/bioc/html/IntEREst.html.Various alternative splicing analysis tools have been developed –22, howeä et al. . IntEREsä et al.  values ay DEXSeq , 26, dify DEXSeq , or exacy DEXSeq , 29. Thee.g. U12-type and U2-type introns.IntEREst is an R package that supports various functions to measure the retention levels of the introns, perform statistical differential intron retention analysis across various samples, and plot the distribution of retention levels of different types of introns across various samples. The main design aim of IntEREst has been to support analysis of relative low level IR values (>10%) that are more challenging to implement with the existing software  but are i.e. interest and interest.sequential) accept a .bam read alignment file and a reference as inputs, and output the raw  and normalized number of fragments mapping to each exon or intron. The reference includes coordinates of exons and introns together with their annotations, such as gene and transcript names, and intron type identifier. The reference can be built using the referencePrepare function supported by IntEREst. Note that the intron identifiers used in our analysis are U12- and U2-type introns, but the application of IntEREst is not limited to the comparison of these intron types. Other classifications can be defined by the user and the retention levels of the introns can be plotted and compared across the user-defined classes. The functions in the IntEREst package that are specific to the comparisons of the U2- vs U12-type introns, e.g. u12Boxplot, u12DensityPlot, u12Index start with “u12”.The RNAseq read summarization functions  interest.sequential, that runs sequentially on a single computing core. These functions use the bpiterate function from the BiocParallel R Bioconductor package [m reads at a time (by default m = 1 million) to comply with the limitations of the memory usage in the running environment. When running interest.sequential, the mapped reads are analyzed as batches of m reads (or read pairs if the isPaired parameter is set TRUE) at a time on a single computing core. With interest it is possible to analyze n batches of m reads (i.e. m×n reads or read pairs) simultaneously while they are distributed over n computing cores and repeat this process until all reads have been analyzed.IntEREst features two functions that estimate the raw and normalized intron retention levels: 1)  package  to read interest.sequential and interest support two distinct analysis modes: 1) intron-exon junction estimation and 2) exon-exon junction estimation. It is possible to configure the analysis to include only the reads that map to intron-exon or exon-exon junctions, however with default settings reads that map entirely to the intronic or exonic regions are also included in the calculation of retention level estimates. For a typical intron-exon junction estimation analysis, we recommend to collapse the overlapping exonic coordinates across various splicing isoforms in the reference to avoid any biases in the IR calculation that may be introduced by the read counts of alternative exons, or by exonic regions overlapping with sequences annotated as introns in other transcripts. To improve the running time and avoid repetitive processes, in exon-exon junction analysis mode, we recommend using a filtered reference resulting from the unionRefTr function. This function identifies all repeating exons and uses only a single copy of each. Moreover, because repetitive sequence elements may bias the read mapping and thus affect the IR estimates, the read summarization functions support the possibility to exclude the repeat regions and reads that map to such regions. The default normalization method applied in the read summarization functions is Fragments Per Kilobase per Million mapped fragments (FPKM) however, it is scaled at transcript level (formula 1). For every intron i of gene g with I introns, if the length of the intron is Lig and the number of fragments mapped to the intron is Xig its normalized retention value will be FPKMig:The summarization functions lfc that estimates the log2 FC of the retention levels across two various conditions, moreover it includes a function psi to measure the Ψ values, i.e. the percent spliced in, for all studied introns. We have adapted several statistical tests from multiple sources for intron retention and exon-junctions analysis: DESEq2 [i.e. DEXSeqInterest function) differs from the others as it uses the differential exon usage method to perform gene-wise comparisonsIntEREst provides a function : DESEq2 , edgeR [: DESEq2 , 22, and: DESEq2 , 26. AllZRSR2 gene that encodes an integral protein component of the minor spliceosome. Moreover, the original analysis of the dataset reported that the ZRSR2 mutations in the patient samples led to increased retention of primarily U12-type introns while the U2-type introns were reported to be less affected [ZRSR2 gene (referred to as ZRSR2mut), 4 were diagnosed with MDS but lacked the ZRSR2 mutations (referred to as ZRSR2wt), and 4 were healthy individuals (HEALTHY).To demonstrate the application of IntEREst in comparing retention levels of various types of introns across several samples, we reanalyzed the RNAseq data from myelodysplastic syndrome (MDS) patients and control subjects included in Madan et al.  study. Saffected . The datannotateU12 function that uses Position Weigh Matrices (PWM) extracted from the U12DB database [interest function. Initially, by using the interestResultIntEx function a result object was built that includes information of both intron retention and exon-exon junction levels . Of the 510 U12-type introns in the data, 269 (i.e. 52.7% of the U12-type introns) showed significant up-regulation of IR in the ZRSR2mut samples when compared to the controls, while none of the U12-type introns showed a significant reduction in IR . Rather, the median log2 fold-change of the U2-type introns was approximately zero whereas the median log2 fold-change of U12-type introns was ~1.5  for all U12-type introns was about 1% as compared to 0.6% with U2-type introns  and 6 samples (3 roots and 3 shoots referred to as RGH3wt) that lack the mutation. The results of the Maize data analysis mirror our findings with the MDS data. Analogous to MDS data, the RGH3mut samples showed increased IR with ~46% of U12-type introns, while only a ~0.46% of the U2-type introns showed an increase in IR  in the recognition of U12-type introns [Together, our results suggest that IntEREst provides reliable quantification of differential IR events; Specifically, our results are not only consistent with the well-documented increased retention levels of U12-type introns , 11, 31, introns , 34.i.e. dedicated software for IR analysis. Note that all comparisons described in the following are based on the introns that were available in the both references used by the compared counterparts.We evaluated the performance of the IntEREst in two ways using the MDS benchmark dataset. First, we carried out internal analysis comparing IntEREst results in conjunction with different statistical analysis packages implemented in IntEREst. Subsequently, we carried out comparison with both, the published results of the MDS analysis  and IRfii.e. DESeq2, GLM function of edgeR and DEXSeq, referred hereafter as IntEREst-DESeq2, IntEREst-edgeR and IntEREst-DEXSeq, respectively. The DESeq2 and edgeR have been previously reported to result in somewhat dissimilar results in differential gene expression analysis [interest in its two running modes) using interestResultIntEx. Subsequently, we used deseqInterest and glmInterest functions (i.e. the IntEREst functions based on DESeq2 and edgeR-GLM) to analyze the change of IR relative to the change of the junction levels of their flanking exons. We used an adjusted p-value  threshoWe found that there is a significant overlap both with upregulated and downregulated introns between the IntEREst-DESeq2 and IntEREst-edgeR Fig , with a Comparison of the IntEREst-DESeq2 to IntEREst-DEXSeq revealed a considerable overlap between the two methods Fig. . Howeveri.e. 235) overlapped with the introns discovered by the IntEREst-DESeq2  of the significant IR events reported by Madan et al. [We further compared our IR results with the original analysis of the MDS dataset by Madan et al. . We founTogether, our results revealed that the different methods implemented in IntEREst are able to identify a highly overlapping set of high-confidence differentially retained introns. Additionally, each method also identified IR events that are unique to a particular method. This provides the flexibility to select an approach best fitting to the particular research questions.i.e. DESeq2Interest ) and repeated this 10 times. As expected, the results reveal that increasing numbers of biological replicates lead to a discovery of an increased number of statistically significant IR events  in each sample, followed by analysis with IntEREst-DESeq2. In our analyses we assumed that the quality and read coverage is equal in all the individual MDS datasets. As a result, we observed that an increase in the sequencing library size leads to a discovery of increasing numbers of introns showing statistically significant deviation in the IR levels. However, the slope of increase of the number of discovered IR events decreases and levels off at the highest library sizes  used in this study is not sufficient to estimate the optimal required for IR discovery, we note that library sizes with more than 35M mapped reads start to approach the point where the improvements in detecting novel IR events are marginal. In sum, we believe that IntEREst is a reliable tool in R/Bioconductor environment for detailed intron retention analysis of RNAseq datasets.IntEREst is implemented as an R package freely available at the Bioconductor repository.Project name: IntEREstArchived version: 1.2.2https://github.com/gacatag/IntEREst/Project home page: https://bioconductor.org/packages/release/bioc/html/IntEREst.htmland Operating system(s): Platform independentProgramming language: ROther requirements: R v 3.4 or higherLicense: GPLAny restrictions to use by non-academics: No restrictionsAdditional file 1:HTML file including all the running scripts and supplementary figures and tables. (HTML 789 kb)Additional file 2:Tab delimited text file that includes the differentially retained introns, when comparing the ZRSR2mut samples to the controls using the IntEREst-DESeq2. (TSV 634 kb)


Dear Editor,Blockade of PD-1/PD-L1 signaling pathway by monoclonal antibodies (MAbs) to release the anti-tumor activity of pre-existing tumor specific T cell immunity has initiated a new era for tumor immunotherapy. Administration of anti-PD-1 MAbs (nivolumab and pembrolizumab) in either monotherapy or in combination with anti-CTLA-4 MAbs or traditional chemotherapy has achieved a tumor regression rate of 30%–50% in dealing with melanoma, non-small cell lung cancer, etc. . Subsequently, crystal screen was performed with the durvalumab-scFv/PD-L1 complex proteins, and well-diffractable crystals grew in 3.5 mol/L sodium formate, pH 7.0 (See more details in supplementary information).In the present study, we expressed the two-Ig-domain PD-L1 and single chain Fv fragment (scFv) of durvalumab as inclusion bodies in The complex structure of durvalumab-scFv/PD-L1 was determined by molecular replacement at a resolution of 2.3 Å (Table S1). The binding of durvalumab to PD-L1 involves both of its heavy chain (VH) and light chain (VL)  and the durvalumab-scFv/PD-L1 complex was conducted to elucidate the molecular basis of durvalumab-based PD-1/PD-L1 intervention. The binding of durvalumab to PD-L1 shows a stereo clash with that of PD-1 Fig. . The comKD = 0.667 nmol/L, 1.75 nmol/L, and 0.83 nmol/L, respectively  and/or dissociation rate constant  compared to that of durvalumab  and atezolizumab . On the other hand, the binding affinity of avelumab to PD-L1 is much higher than the other three MAbs (KD = 0.0467 nmol/L) with highest Ka and lowest Kd value (Table S3). Taken together, the binding kinetics of durvalumab is much similar to that of atezolizumab, while binding properties of BMS-936559 and avelumab are distinct from the above two MAbs. The differential binding profiles of these anti-PD-L1 MAbs would have profound effects on their pharmacokinetics. However, whether the distinct binding epitopes and kinetics of these MAbs would affect therapeutic efficacy remains to be elucidated. Escape from anti-PD-1 or anti-PD-L1 could be induced from multiple aspects, including selection of poor-immunogenic tumor cells, immune evasion via up-regulation of alternative inhibitory molecules, etc.  under accession code 5XJ4.Atomic coordinates have been deposited in the Protein Data Bank  and the National Basic Research Program (973 Program) (NO. 2013CB531502). We also thank Yuanyuan Chen and Zhenwei Yang from Institute of Biophysics, Chinese Academy of Sciences for their technical support in the SPR assay. G.F.G. is a leading principle investigator of NSFC Innovative Research Group (Grant No. 81621091).Supplementary material 1 (PDF 371 kb)Below is the link to the electronic supplementary material.