Several clinical trials have shown that immune treatment focus on programmed death-1 and programmed death-ligand 1 (PD-L1) yields a good clinical efficacy in advanced non-small cell lung cancer (NSCLC). We investigated whether the PD-L1 expression was related to clinicopathologic and molecular characteristics in patients with surgically resected NSCLC.EGFR, KRAS, NRAS, PIK3CA, BRAF, HER2 mutations and ALK, ROS1, RET fusion genes. The PD-L1 expression was evaluated by immunohistochemistry and staining of 5 % or more was scored as positive expression. Survival analysis was evaluated using the Kaplan–Meier method. Multivariate regression was performed using the Cox proportional hazards model.Between December 2008 and 2013, formalin-fixed, paraffin-embedded samples were obtained from patients with lung adenocarcinoma at Zhejiang Cancer Hospital. RT-PCR was used to analyze EGFR mutation , followed by EML4–ALK rearrangement , KRAS , HER2 , ROS1 rearrangement , PIK3CA , RET rearrangement , BRAF , and NRAS mutations . Twenty-four (6.2 %) patients carried coexisting mutations. PD-L1 expression was detected in 48.3 % (186/385) of all the patients. PD-L1 positive patients more frequently carried coexisting mutations , followed by single-gene  and pan-negative mutations . PD-L1 expression decreased disease-free survival (DFS) in univariate analysis (P = 0.014). Multivariate analysis revealed that PD-L1 expression was not an independent risk factor for poor DFS and overall survival (OS) .Mutations were detected in 76.6 % of the 385 patients tested: PD-L1 overexpression is more frequently observed in oncogene-mediated lung adenocarcinoma, especially with coexisting mutation subtypes. PD-L1 expression is not a prognostic factor in surgically resected lung adenocarcinoma patients. Lung cancer is the leading cause of cancer-related death in China . The staBlockade of immune checkpoints in cancer with monoclonal antibodies has recently emerged as a promising approach to the treatment of solid tumors. Programmed death 1 (PD1), which belongs to the CD28 family of proteins, is a T cell surface receptor that regulates T cell activation and proliferation. Its ligand, programmed death-ligand 1 (PD-L1), is frequently expressed in many types of carcinomas –14. ReceThis study focused on patients with completely resected lung adenocarcinoma and evaluated the association of PD-L1 expression with clinicopathologic parameters and driver genes, as well as its prognosis value in Chinese patients.The Ethics Committee of Zhejiang Cancer Hospital approved this study and written informed consent was obtained from each participant.A total of 385 adenocarcinoma patients underwent resection between December 2008 and 2013 in Zhejiang Cancer Hospital. Histological typing was determined according to the 2004 World Health Organization classification . Tumor-nProteintech Group Inc., Chicago, IL, USA, Catalog number: 66248-1-Ig) was 1:100 in Dako antibody diluent; slides were incubated with this antibody overnight at 4 °C. Then, the slides were incubated with Ventana Omni Mapanti-rabbit secondary antibody for 60 min. AVentana Chromo MapKit was used for antibody detection, and then the slides were counterstained with hematoxylin. Next, the slides were dehydrated and cover slipped as per normal laboratory protocol. Two independent pathologists (Wei Wu and Guoping Cheng) assessed the expressions.Immunohistochemical (IHC) staining of PD-L1 expression was performed on 4-6 μm thick formalin-fiated, paraffi-embedded tissue. The concentration of rabbit primary antibody that reacts to PD-L1 The concentration of rabbit primary antibody that reacts to PD-L1 (PD-L1 immunostaining results were classified into two groups based on the degree and intensity of staining: (1) negative, when staining was absent or detected in <5 % of the cells; and (2) positive, when membranous staining was present in ≥5 % of the cells.We used another antibody  to confirmed the PD-L1 expression in 102 patients. The PD-L1 immunostaining criterion is same with the former antibody.Genomic DNA or RNA was extracted from tumor tissues according to standard protocols . Briefly, the isolated RNA samples were used for reverse transcription into cDNA using Revert Aid First Strand cDNA Synthesis Kit . Either genomic DNA or cDNA was used for PCR amplification and sequencing. EGFR, HER2, KRAS, NRAS, BRAF, and PIK3CA were PCR amplified using genomic DNA. Cycle sequencing of the purified PCR products was carried out with PCR primers using the commercially available ADx Mutation Detection Kits .The ALK, ROS1, and RET fusion mRNA was detected by PCR with fusion gene detection kit . In brief, total RNA was extracted with QiagenRNeasy FFPE Kit. The mRNA was reverse-transcribed to cDNA at 42 °C for 1 h. β-actin was used as the internal control. The RT-PCR conditions were as follows: an initial denaturation at 95 °C for 5 min, followed by 95 °C for 25 s, 64 °C for 20 s, and 72 °C for 20 s to ensure the specificity; and 31 cycles at 93 °C for 25 s, 60 °C for 35 s, 72 °C for 20 s were performed for data collection and sensitivity analysis. All of the positive genes including mutations or fusions were confirmed with Sanger sequencing. All the experiments were performed according to the user manual as described previously .P < 0.05 was considered statistically significant. The median follow-up was 54 months (6.5–65) and the last follow-up date was July 31, 2015.The Chi squared test was used to evaluate the relationships between different driver genes and PD-L1 expression. Survival curves of pathologically confirmed samples were calculated using the Kaplan–Meier method until death or last follow-up. Multivariate analysis was performed using the Cox regression model. Statistical analysis was performed with the SPSS 18 software . Patients’ clinical profile is presented in Table EGFR, KRAS, NRAS, PIK3CA, BRAF and HER2 mutations and ALK, ROS1, RET fusion genes. This analysis included EGFR mutations , followed by EML4–ALK rearrangements , KRAS , HER2 , ROS1 , PIK3CA , RET , BRAF , and NRAS , and 24 coexisting mutations (6.2 %). All the nine genes were negative in 90 patients, defined as pan-negative. The details of coexisting mutations are listed in Table All the patients were analyzed for P < 0.001).The PD-L1 membrane expression was detected in 186 of the 385 lung adenocarcinoma patients (48.3 %) Figs. . The relP = 0.337).Another antibody  was used in 102 patients to detect the PD-L1 expression. The same trend of PD-L1 expression difference existed in patients with different gene abnormality. The PD-L1 positive patitnets was more frequently carried coexisting mutations , followed by single-gene positive  and pan-negative mutations (P = 0.014)   and PD-L1 expression negative were significantly risk factors for tumor recurrence or metastasis Figs. , 4, whilIn multivariate analysis, only early stage suggested lower risk for DFS, while PD-L1 expression was not correlated with recurrence or metastasis. Early stage was an independent and favorable prognostic factor for OS Table .Table 5MThis study shows that PD-L1 is overexpressed in 48.3 % (186/385) of lung adenocarcinoma patients and this overexpression is more frequently seen in patients with coexisting mutations, but less frequently in patients with pan-negative genes. The PD-L1 overexpression is not a prognostic factor for overall survival. To the best of our knowledge, this is the first study with the largest number of patients correlating the nine common driver genes in lung adenocarcinoma and PD-L1 expression.PD-L1 expression and driver genes [PD-L1 positive status was significantly associated with EGFR mutations . Mu et al. observed no significant correlation between PD-L1 expression and EGFR/ALK status in stage I NSCLC patients [PD-L1 expression and EGFR status in lung adenocarcinoma [EGFR mutations or ALK rearrangements, and other driver genes were not well investigated. Last but not least, racial differences may play an important role in the controversial results.Several studies have reported the association between er genes , 24. Theer genes  observedpatients . Similararcinoma . TherefoIn the present study, PD-L1 overexpression was more frequent in patients with coexisting mutations than in pan-negative patients. One explanation is that the genetic differences affected epigenetics, which may alter the expression of tumor-associated self-antigens, which in turn, affected tumor antigenicity. Increased number of driver genes reflects a higher level of neoantigens, which alters the immune microenvironment and increases the PD-L1 expression .Because of heterogeneity of tumors, the efficacy of chemotherapy or molecular targeted treatment is relatively limited, combination treatment with different anti-cancer mechanisms drugs hold much potential in this area. Previous studies demonstrated that EGFR and ALK genes could induce PD-L1 expression to facilitate evasion of the host anti-tumour immune response, suggesting an active role for these genes in remodelling the immune microenvironment , 28. In The role of PD-L1 in predicting the prognosis of NSCLC was controversial in previous studies . Some stOur study limitations are as follows. One major limitation was its retrospective nature. Second, only 24 patients with coexisting mutations were included, and the small sample size may influence the results of the current study. Third, different antibodies were used in different anti-PD-1 or PD-L1 therapies in clinical trials currently. The choice of antibody and the threshold for positivity might influence the results of different studies. Only one antibody and 5 % threshold were used in the present study. Different anti-PD-L1 antibodies may need to be validated in the same sample in future studies.In conclusion, we demonstrated the expression of PD-L1 in over 48 % of lung adenocarcinoma patients and the expression was associated with coexisting driver genes. PD-L1 expression is not associated with overall survival in patients with completely resected NSCLC.


Here, we report a patient with sarcoidosis who developed edematous erythema and interstitial lung disease. At the initial visit, clinically amyopathic dermatomyositis (CADM) with rapidly progressive interstitial lung disease (RP-ILD) was suspected because he had progressive dyspnea but no muscle weakness. The presence of anti-CADM-140/MDA5 autoantibodies was immediately assessed to facilitate a precise diagnosis, with negative results. Thereafter, skin and transbronchial lung biopsies revealed noncaseating granuloma with Langhans giant cells in both specimens, leading to a diagnosis of sarcoidosis. In this case, clinical features of skin and lung were unable to distinguish DM (including CADM) from sarcoidosis, but the lack of anti-CADM-140/MDA5 antibody was useful for differentiating CADM with RP-ILD mimicking sarcoidosis from bona fide sarcoidosis. Sarcoidosis is a disorder of unknown etiology affecting multiple organs and is characterized by the formation of granulomatous lesions. It has many clinical manifestations including skin and pulmonary symptoms. In light of reported cases of sarcoidosis coexisting with dermatomyositis (DM) –6, it isμL, red blood cell count of 517 × 106/μL, hemoglobin 17.0 g/dL, and platelets at 22.0 × 104/μL. Although serum AST was 56 IU/L, ALT was 57 IU/L, LDH was 260 IU/L, creatine kinase (CK) was 34 IU/L, and aldolase was 6.1 IU/mL, within thenormal range. Serum C-reactive proteins, KL-6 and SP-D, were elevated to 1.05 mg/dL, 1758 U/mL, and 279 ng/mL, respectively. Rheumatoid factor, antinuclear autoantibodies, and anti-Jo-1 antibodies were absent. Bacterial culture examination of sputum and thetuberculin skin test were negative. SpO2 (in room air) at rest was decreased to 92%. Chest radiography and CT indicated diffuse fine nodular or reticular shadow in both lung fields that suggested interstitial lung disease (ILD) (5 cells/mL with increased lymphocytes (49%) and an elevated CD4/CD8 ratio (8.40). TBLB and skin biopsy from the area of anterior chest erythema revealed infiltration of lymphocytes and epithelioid cells into the tissues and formation of noncaseating granuloma with Langhans giant cells. No caseous necrosis or malignant cells were seen. This is consistent with the pathological findings of sarcoidosis  revealed mediastinal lymphadenopathy, emphysematous change, and granular/nodular shadow on both lung fields. Blood chemistry revealed an elevation of serum LDH and serum KL-6. Because his respiratory symptoms were progressive, he was referred to our university hospital for further examination and treatment of the skin and respiratory symptoms. At the first visit, he had dyspnea on exertion and facial erythema as well as erythema on his anterior chest and back . He was se (ILD) . Electrocoidosis . GalliumThis is a case report of sarcoidosis in a patient with lung and skin manifestations mimicking CADM and RP-ILD. Our case highlights the difficulty of diagnosing patients who manifest clinical features indistinguishable from DM (including CADM), particularly skin and pulmonary symptoms. Symptoms of both diseases sometimes closely resemble each other and it is difficult to distinguish them. Moreover, patients with both DM and sarcoidosis have been reported –6, althoInitially, this patient was suspected of having CADM and RP-ILD due to edematous erythema of the face and trunk and progressive dyspnea on exertion. RP-ILD accompanied by DM, especially CADM, is a life-threatening complication and it is well recognized that respiratory symptoms in these patients often progress rapidly and resist treatment even if intensive immunosuppressive therapy is given. However, it is also known that early intervention with high-dose corticosteroids and immunosuppressive agents can rescue patients from this life-threatening condition. On the other hand, sarcoidosis therapy usually does not require the type of intensive treatment that is necessary for DM and RP-ILD. In this regard, it would be important to differentiate sarcoidosis from DM in the early stages as quickly as possible. Anti-CADM-140/MDA5 antibody is a recently discovered myositis-specific autoantibody . The preIn summary, we report a sarcoidosis patient who developed edematous erythema and ILD mimicking CADM and RP-ILD. In this case, measurement of anti-CADM-140/MDA5 antibody was useful to distinguish sarcoidosis from CADM with RP-ILD. Successful treatment with PSL alone achieved complete remission of the sarcoidosis. This case emphasizes the importance of being aware of other conditions mimicking DM and RP-ILD, as well as the usefulness of measurement of anti-CADM-140/MDA5 antibody.


Improving the informed consent process in clinical research is of constant concern to regulatory authorities in the field and presents a challenge for both the specialists and patients involved. Informed consent is a process that should adequately match the complexity of clinical research. In analyzing the behaviour of 68 patients during the informed consent process related to the clinical research performed at Neomed Clinical Center in Brasov, we found that many patients do not ask any questions (35.3%). From those who do, part of the questions  referred to general aspects (addressed the form but not the gist) of the clinical trial, some  referred to specific aspects of the clinical trial they will attend and others  unrelated to the clinical trial. These results suggest a lack of interest, awareness, and understanding of the information presented in the informed consent form. The possible underlying causes of this attitude and its bureaucratic, ethic, and legal implications are discussed. Advances in the design and targets of clinical research should translate to changes in the informed consent process that would guarantee that subjects always understand the potential costs and benefits of participating in a clinical research project. Clinical trial participation offers subjects the promise to meet two of their most important expectations: an improvement, or even a cure, for their condition, and minimal costs associated with their treatment (possibly even free treatment). An improvement in the general condition of the patients during the study, even marked by brief episodes of mild adverse reactions, mask the procedural shortcomings in obtaining informed consent full awareness. The lack of the patient's awareness in giving consent could manifest itself when participants experience a decline in their general condition due to the development of the disease, associated conditions, or adverse reactions. Awareness is also important in long-term effects of the treatment, even if the patient's condition has improved, as adverse reactions could appear across shorter or longer period of time. The informed consent process begins with a conversation between the clinical investigator and the subject, who is presented with the option to participate in the clinical trial as well as an informed consent document. Subsequently, the subject may decide to either take the document home to study it alone or with their family, or review it at the study site. In both cases, the process continues with offering explanations and providing appropriate answers to the subjects’ possible questions, so that an educated and informed decision can be made. We hypothesized that the likelihood of potential participants in a clinical trial of whether to ask questions or not, as well as their quality, is a reflection of the degree of their interest, awareness, and understanding of the information presented in the informed consent form. Consequently, we studied the subjects' behaviour before they signed the informed consent form.Following an outpatient medical consult, the indication to participate to an ongoing clinical trial was established by the physician. On this occasion, the patients were invited to participate in that trial. If interested, they received an information brochure related to the trial. Preliminary discussions with the physician and questions & answers sessions before reading the brochure did not occur in the case of patients included in this prospective transverse observational study. The patients read the brochure without being offered any explanations at this point. The informed consent process allowed the patient to take the document home and study it alone or with the family, but this analysis included only patients who went through the informed consent process at the medical center in order to have the same conditions for all participants. Informing sessions consisted in reading the brochure, asking questions, providing answers, then explanations and discussions with the investigator, followed by another questions & answers session, if needed. These informing sessions lasted between an hour and an hour and a half, depending on the clinical trial described and the participant's reading skills. The decision to participate was made at that moment, or later.Our study did not involve direct research on subjects. The observation protocol requested the members of the research team to write down on a form all the questions asked by the patients. All the patients' questions, as well as the multiple questions asked by the same patient, were grouped according to the inquiry moment , as follSpontaneous questions (after reading the brochure and before the discussion with the investigator)Facilitated questions (asked during or after the discussion with the investigator)In order not to influence the participants' behaviour in this observational study, they were informed only at the end of the informed consent process that the investigator watched certain aspects of their attitude: whether the subjects asked questions, what kind and how many. The data were analyzed anonymously. Demographical data were collected, but not personally identifiable. We have not evaluated clinical data for our analysis. 68 patients with indication to participate in one of the 4 clinical trials  that took place at the Neomed Medical Center from Brasov between March and July 2013 gave their verbal consent to have these data processed. The local ethics committee of Neomed Medical Center specifically waived the need for written informed consent and also gave their approval for our analyses.Therefore, as far as the number of questions addressed is concerned, out of the 68 forms submitted by the investigators who wrote down the received questions, most of the patients (24) did not ask any questions while the rest asked between 1 and 4 .Regarding the type of asked questions, all 92 questions were grouped in 3 categories :General aspects of clinical trials (19 questions) Specific questions related to the clinical trial (67 questions) Questions unrelated to the clinical trial, regarding the disorder and its prognosis (6 questions) Following the descriptive statistical analysis of the data, we notice that out of all the subjects’ questions, addressed before and after the discussion with the investigator, the number of questions related to specific aspects of the clinical trial is predominant. 2χ test for association. As it can be noticed in We wanted to find out if this predominance was statistically significant. To test if the awareness level of the patients’ informed consent varies according to the number and type of asked questions, the moment of being addressed (before and after the discussion with the investigator), as well as the educational level, we applied the 2 (2) = 6,928, p = 0,003. This relation shows that besides the specific questions, the subjects with general studies tend to ask questions unrelated to the clinical trial after the discussion with the investigator. The effect size indicator, Phi = 0,589, p = 0,003, shows that the relation between the two variables is strong.Specific to the general studies group is the existence of a significant difference in the number of questions ratio, χWe have observed that many patients do not ask any questions (35.3%). From those who do, part of the questions  referred to general aspects (addressed the form but not the gist) of the clinical trial, some  referred to specific aspects of the clinical trial they will attend and others  unrelated to the clinical trial. This attitude can reflect the lack of or insufficient awareness of the informed consent process.There are a few possible explanations for our findings. First, presumably “a misunderstanding of the terms in the consent document may leave patients incapable of asking questions” Another possible explanation is that subjects might have felt overwhelmed and intimidated by the quantity of information presented during the informed consent process. While the document is being read by a physician, the subject might lose attention; alternatively, when the subjects read the consent on their own, they might do so superficially, skimming the text as they would do with a utility contract. After the interviews with the research teams, we observed another situation that is unfortunately common: some of our patients did not appear motivated to understand the informed consent; they adhered to the study schedule and procedures, but they were not interested in the clinical trial or its objectives. For these patients, what mattered was the end result of the trial (an improvement in their condition), even if they were informed that this was not guaranteed. Lack of interest or reactivity (the fact that they don't ask questions) could also be due to cultural and educational conditions, which could be specific to Romania, as, historically, questioning was seen as a sign of insubordination and challenging authority. This attitude discouraged an open attitude and feedback from the individual due to the collective system that put people in their place, patronized them, and discouraged questioning. These factors may have prevented subjects from asking questions even if they were motivated and interested in learning more. As a result, subjects may be less informed than desired at the completion of the informed consent process.From an administrative perspective, signing the informed consent document is proof that the subject understood the information presented and that they had authorized the investigator to proceed with the proposed study. Signing this document, however, does not demonstrate that the subjects actually read, understood, and retained this information. Over the last 10 years of practice in clinical research, the informed consent document has evolved from a short document to a longer one, sometimes exceeding 20 pages, containing a large quantity of complex information that describes many procedures that may be difficult to understand. Many pieces of information might be required to be presented from a liability perspective, but are irrelevant to the patient's consent. It is then the investigator’s responsibility to translate this complex information into simpler explanations and support the subjects in discussing the medical and research procedures.From an ethical perspective, it is essential that the subjects understand what their participation in clinical research entails. The goal of the consent process is achieved only when patients understand the information disclosed in the informed consent and by the medical team. In current research practice, a number of patients who are less educated or older find that reviewing the informed consent document is quite tiring. In a culture where the patient-physician relationship is often dominated by deferring to the physician’s authority, subjects who do not clearly grasp the difference between a treating physician  and a clinical investigator  often make statements such as, “doctor, you know better what I should do,” which is indicative of the expectation that the investigator (often times their physician) should function as a substitute decision maker. In this respect, patients may give their consent to participate in research because their physician tells them that they should. Also, “the real power scale in the patient-physician is out of balance, always tilted towards the physician” From a legal perspective, there may be situations in which “both the investigators and the patients' lawyers need to unquestionably prove that a person has been informed and has understood what his voluntary participation in a clinical trial involves”.


We also studied the electrical transport properties of Au/MoS2 junction devices for systematic comparison. A previous work has demonstrated the existence of a positive Schottky barrier height (SBH) in the metal/MoS2 system. However, analysis of the SBH indicates that the contacts of the multi-layered graphene/MoS2 have tunable negative barriers in the range of 300 to −46 meV as a function of gate voltage. It is hypothesized that this tunable SBH is responsible for the modulation of the work function of the thick graphene in these devices. Despite the large number of graphene layers, it is possible to form ohmic contacts, which will provide new opportunities for the engineering of highly efficient contacts in flexible electronics and photonics.We fabricated multi-layered graphene/MoS Here, an alternative approach was engineered by inserting a thick graphene layer instead of single-layer graphene. To compare the characteristics of the devices, reference samples without MGr contacts were also fabricated. The heterostructured FETs exhibited a dramatic reduction in SBH based on the framework of the thermionic emission theory. It appeared that the observed negative SBH gave rise to true ohmic contact.In this study, we present heterostructured MoS2 heterostructured devices, we deposited mechanically exfoliated graphite and single-crystalline 2H-MoS2 onto a SiO2/Si substrate on which Au metal pads (110 nm thick) had been pre-patterned, as described in the Methods section. The dry transfer technique used for both 2D materials began with the introduction of an intermediate polydimethylsiloxane (PDMS) layer, which acted as a contamination-free viscoelastic material to avoid wet chemistry  in 2 FETs (denoted below as Au/MoS2) without inserting an MGr layer; for these devices, the transfer procedure was reduced to only one step. The MoS2 channel was verified using Raman spectroscopy and atomic force microscopy (AFM) to confirm the thickness of the MoS2 flakes (see −1 between the in-plane mode (E12g) and the out-of-plane mode (A1g) corresponds to the signal of bi-layer MoS2, which is consistent with our MoS2 reference sample  and other reports2 stack. An HRTEM image of a clear and compact interface, without other impurities or significant gaps between the MoS2 and MGr, is shown in 2 heterostructure, and the corresponding energy-dispersive X-ray spectroscopy (EDX) mapping is shown in A 3-dimensional (3D) schematic representation of our heterostructured device architecture is depicted in akes see . The fre2 devices and its influence on the Schottky barrier, the devices were electrically characterized at room temperature. DS-VBG transfer characteristics of the back-gated heterostructured FETs, which are presented as a normalized sheet conductivity (defined as σ = IDS/VDS(L/W)) as a function of VBG-VT, where VBG is the gate voltage and VT is the threshold voltage. The devices displayed obvious n-type behavior and produced a high on/off ratio (~106) with a maximum normalized transconductance gM/W of approximately 30.4 nS/μm at VDS = 500 mV. I-V hysteresis behavior is also shown in Figure S6. After in situ thermal heating, the devices exhibited nearly “hysteresis-free” behavior, indicating that the amount of adsorbates was negligibleDS = 500 mV, which is 2 orders of magnitude lower than that of the MGr/MoS2 FETs, as shown in Figure S7. The field-effect mobility, μFE, was estimated from the maximum value of the transconductance using the expression μFE = gML/WCOXVDS. Here, COX = εrε0/dOX ~ 1.23 × 10−8 F/cm2 , which is the back-gate capacitance per unit area, and gM = dIDS/dVBG is the transconductance. Thus, the field-effect mobility for our bi-layer MGr/MoS2 device appeared to be approximately 17.9 cm2/Vs in the bi-layer MoS2 channel, as presented in Figure S8. This measured mobility is comparable to those of mono- or bi-layer MoS2 in high-κ gate dielectric capping devices222) with an Au metal contact had a mobility of 0.2 cm2/Vs, which is consistent with other reports3242 interface that hinders efficient carrier injection. To extract an accurate gate capacitance for the mobility calculation, a 100-kHz capacitance-voltage (C-V) profile was obtained by measuring between the source and back-gate electrodes . The floor value of the capacitance (the minimum capacitance in the inversion regime) represented the actual electrode capacitance−8 F/cm2) was very close to the value of 1.23 × 10−8 F/cm2 obtained in the above calculation for a 4.52-mm2 contact area.To investigate the presence of the intermediate MGr layer in our MGr/MoSDS-VDS output characteristics of the MGr/MoS2 devices at various gate voltages. The saturation current could be reached at high drain voltages under certain gate biases, opening a wide drain voltage window. Moreover, the drain current, IDS, exhibited symmetrical and linear behavior [exp(eVDS/kBT) − 1], where A*, A, e, VDS, and kB are the Richardson constant, the area of the contact, the elemental charge, the drain voltage, and the Boltzmann constant, respectively. Temperature-dependent I-V data were collected in the high-temperature regime (above room temperature) to avoid weak conductivity variations (see the DS/T2) fit curve was plotted for various gate voltages at the Au/MoS2  and becomes more positive with the formation of a highly conductive MoS2 channel at VBG = −1.6 V, as depicted in 2 contact and is contrary to the results of several studies that have investigated this phenomenon in the 0–300 meV range161718e.g., MoS2 with ferromagnetic permalloy contacts2 with MoOX2 contact considered in this study, under a negative gate bias, the current through the Schottky barrier via thermionic emission resulted in a linearly decreasing trend (from 765.9 to 118.8 meV) as the gate bias became forward directed.To better understand how the carriers travel in the presence of a Schottky barrier, we employed a well-known model,  see the . Such va see the . To dete Au/MoS2  and MGr//MoS2 /S0 ≈ 7.9 × 1013 states/eV cm2, where the slope is S0 = dΦSB/dΦM ≈ 0.12 and ΦM is the metal work function. However, when MGr and MoS2 are brought into contact, the Schottky-Mott model does not hold, because DIT ≠ 0. A modified model can be written as followsGenerally, the semiconductor surface states and the work functions of the metal play a decisive role in the determination of the SBH, but the details of Schottky barrier formation in metal/semiconductor systems are not well understood. Various previous studies on metal/MoSMGr = 4.5 eV and χ = 4.0 eV are the work function of the graphene and the electron affinity of the MoS235G = 1.6 eV is the band gap of the bi-layer MoS2; and ΦN = 1.53 eV is the neutral level of the interface states. In practice, the Fermi level is pinned at a certain energy, and then, a depletion contact with a positive Schottky barrier is created at the MGr/MoS2 interface. Considering this behavior, the first term of equation MGR depends on the back-gate electric field, the carrier density in the MGr shifts the Fermi level by ΔEF,MGr = ħνF√νr|COX/e(VBG − VT)|), where νF is the Fermi velocity. The tunability of the work function of graphene has been demonstrated by Yu, Y.-J. et al.MGr, is less than that of the MoS2, i.e., ΦB(VBG) = ΦMGr − ΦMoS2(VBG), then it is possible for a negative contact potential, ΦB(VBG), to arise as shown in et al.2 is estimated to be ΦMoS2(VBG) = e(χ + ϕS) − ħπ(COX/e)(VBG − VT)/2 m*, where ϕS, ħ, and m* are the surface potential, Planck’s constant, and the effective mass of bi-layer MoS2, respectively. For the values given in ref. F,MoS2, of the bi-layer at the turning point voltage (VBG = −3.8 V) is only approximately 31 meV, which is quite small. Therefore, we suggest that the tunable Schottky barrier is primarily responsible for the modulation of the work function of the thick graphene. Despite the large number of graphene layers, ohmic contacts can be formed, which will provide new opportunities for the engineering of highly efficient contacts in flexible electronics and photonics applications.where Φ2 FETs with multi-layered graphene contacts and systematically investigated their electronic properties at the MGr/MoS2 interface. Negligible hysteresis was observed because of the optimized fabrication procedure. Upon the insertion of an intermediate tunnel layer, the devices displayed highly improved performance in terms of carrier mobility. An interesting negative Schottky barrier behavior was observed between the MoS2 and the multi-layered graphene. We also qualitatively discussed the formation of ohmic contacts, which likely contributed to the controllability of the graphene work function by gate bias modulation. Thus, we were able to improve the carrier injection behavior and reduce the contact resistance, thereby developing a new possible approach for future electronics applications.In summary, we fabricated heterostructured MoSMulti-layered graphene was mechanically exfoliated from single-crystalline graphite (from Graphene Supermarket) using the standard Scotch-tape-based cleavage method. The thick graphene flakes were transferred onto bottom gold electrodes, which served as the source and drain, using techniques described previously2. We intentionally chose two similar thicknesses of graphene flakes to perform the above techniques twice. Subsequently, MoS2 (2D semiconductor) flakes were precisely deposited on top of the graphene contacts to serve as a channel material. The fabrication process described above avoids the use of any chemical solutions to minimize contamination. The devices were subsequently annealed at 399 K for 12 hours in a cryostat  at a base pressure of ~2 × 10−2 Torr for the removal of adsorbates. Electrical measurements were performed using a semiconductor parameter analyzer  after in situ vacuum annealing. C-V measurements were performed using a Precision LCR meter  with the samples in the same chamber.Before the dry transfer was performed, the gold electrodes were pre-patterned on a degenerately doped Si substrate covered with a 280-nm layer of SiOA focused ion beam  was employed to prepare cross-sectional TEM samples after the deposition of a carbon/Pt coating. HRTEM imaging was performed using a spherical-aberration-corrected TEM  operating at 200 kV. Chemical composition mapping was performed with an EDX apparatus attached to the TEM instrument.2 were determined based on the color contrast in the optical microscope images and were further confirmed by AFM . Raman spectroscopy  with laser excitation at λ = 532 nm was employed to analyze the spectra of the MoS2 reference flakes and the FET channels.The numbers of layers of graphene and MoSHow to cite this article: Qiu, D. and Kim, E. K. Electrically Tunable and Negative Schottky Barriers in Multi-layered Graphene/MoS2 Heterostructured Transistors. Sci. Rep.5, 13743; doi: 10.1038/srep13743 (2015).


Enhanced proliferation of pulmonary arterial vascular smooth muscle cells (PASMCs) is a key pathological component of vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Mammalian targeting of rapamycin (mTOR) signaling has been shown to play a role in protein translation and participate in the progression of pulmonary hypertension. Eukaryotic translation initiation factor-2α (eIF2α) is a key factor in regulation of cell growth and cell cycle, but its role in mTOR signaling and PASMCs proliferation remains unknown. Pulmonary hypertension (PH) rat model was established by hypoxia. Rapamycin was used to treat rats as an mTOR inhibitor. Proliferation of primarily cultured rat PASMCs was induced by hypoxia, rapamycin and siRNA of mTOR and eIF2α were used in loss-of-function studies. The expression and activation of eIF2α, mTOR and c-myc were analyzed. Results showed that mTOR/eIF2α signaling was significantly activated in pulmonary arteries from hypoxia exposed rats and PASMCs cultured under hypoxia condition. Treatment with mTOR inhibitor for 21 days attenuated vascular remodeling, suppressed mTOR and eIF2α activation, inhibited c-myc expression in HPH rats. In hypoxia-induced PASMCs, rapamycin and knockdown of mTOR and eIF2α by siRNA significantly abolished proliferation and increased c-myc expression. These results suggest a critical role of the mTOR/eIF2αpathway in hypoxic vascular remodeling and PASMCs proliferation of HPH. Pulmonary hypertension (PH) is a multi-factorial disease with poor prognosis . The disHypoxia–induced PH (HPH) is one of the most common classes of PH. A number of intracellular signaling mechanisms are observed to be involved in PASMCs proliferation and vascular remodeling in HPH, among which hypoxia-inducible factor 1α (HIF-1α), transforming growth factor (TGF), Wnt and mitogen-activated protein kinases (MAPKs) are widely studied –8. UnforRecent research has indicated that the mammalian target of rapamycin (mTOR) in PH is potentially involved in pathogenesis of PAH , 10. EvimTOR is a highly conserved member of the serine/threonine protein kinase family activated by phosphorylation. There is also evidence shows that mTOR plays an important role in regulation of translation initiation , 15. S6KIn summary, the purpose of this study was to explore the contribution of mTOR and eIF2α in vascular remodeling in HPH rat model, and the role of the mTOR/eIF2α pathway in hypoxia induced PASMCs proliferation and vascular remodeling in PH.Male Sprague–Dawley rats (weighing 180–220 g) were obtained from the Laboratory Animal Center, Xiangya School of Medicine, Central South University . All surviving animals were handled in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the medicine animal welfare committee of Xiangya Medical School, Central South University .Rats were randomly divided into three groups (n = 10/group): (i) normoxia group; (ii) hypoxia group, and (iii) hypoxia plus rapamycin group. Rats in the hypoxia or hypoxia plus rapamycin groups were placed in a chamber and exposed to 10% O2 continuously for 21 days, and rats in the hypoxia plus rapamycin group were administrated with rapamycin  from the first day of their exposure to hypoxia for 21 days. On day 22, animals were anesthetized with sodium pentobarbital , right ventricle systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) were monitored. After sacrificing the animals, the right and left ventricle  and the interventricular septum (S) were dissected. The tibia length was measured and weighed for calculating the ratio of RV to (LV+S) and tibia length/RV, which are the key indexes used for evaluating RV hyperplasia. The pulmonary arterial samples were isolated for determination of mRNA expression. Portions of excised lungs were fixed in 4% paraformaldehyde for hematoxylin–eosin and immunohistochemical staining. The remainder of the excised lungs was frozen in liquid nitrogen for immunofluorescent staining.ext−areaint/ areaext×100, where areaext and areaint are the area bounded by external and internal elastic lamina, respectively. For immunohistochemistry and immunofluorescent staining, sections were stained with anti-eIF2α antibody , anti-p-eIF2α antibody , anti-mTOR antibody , anti-p-mTOR antibody , anti-c-myc antibody , anti-α-SMA, developed with DAB, and counterstained with hematoxylin. The immunohistochemistry samples were treated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies  for 1 h at 37°C. Images were acquired using a Nikon Olympus BX51. The immunofluorescence samples were treated with cy3-conjugated  and/or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies  for 1 h at 37°C. Analysis of the intensity in smooth muscle actin (SMA)-positive areas of small muscular PAs was performed using an Olympus BX51.Vascular remodeling of lung tissues was studied. Lung hematoxylin–eosin staining was performed as previously described . Slices 2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 20% fetal bovine serum (FBS). PASMCs were identified by immunohistochemical staining using an antibody against smooth muscle α-actin . PASMCs between passages 3 and 8 were used for the experiments.PASMCs were isolated from the pulmonary artery of 10-week-old male Sprague-Dawley rats using the method described previously . The cel2, 5%CO2 and 92%N2) for 48 h. Cell proliferation assays were performed. eIF2α and c-myc expression were analyzed. The 48 h hypoxia duration was determined based on previous pilot study. The three series of experiments were designed to explore the effect of rapamycin (RAPA) on PASMCs proliferation and involvement of the eIF2α/c-myc pathway. PASMCs were treated with rapamycin , then subjected to hypoxia  for 48 h. Cell proliferation assays were performed and the expression of eIF2α, mTOR and c-myc was analyzed.Three series of experiments were designed. The first series of experiments investigated the role of hypoxia on PASMCs proliferation. PASMCs were subjected to hypoxia for 12, 24, 48, 60 and 72 h to determine the optimal duration of exposure to hypoxia. The second series of experiments were designed to explore the effect of eIF2α siRNA on PASMCs proliferation and the involvement of c-myc pathway. PASMCs were treated with eIF2α siRNA  under normal culture conditions for 4 h, then subjected to hypoxia . TurboFect transfection reagent  were used for cell transfection. PASMCs grown to 60% to 70% confluence were starved with 2% fetal bovine serum in Dulbecco’s modified Eagle’s medium (DMEM) for 24 h and then transfected with Turbofect transfection reagent for 4–6 h, and then fresh medium were replaced according to the manufacturer’s instruction. Cells were harvested 4h after for the experiments as mentioned above. Transfection efficiency was evaluated by eIF2α mRNA and protein expression using real-time PCR and Western blot analysis, respectively. Each experiment was repeated using primarily cultured cells (3–8 passage). The experiments were repeated three times.ueous One Solution cell Proliferation Assay  according to the manufacturer’s instruction. CellTiter 96 AQueousOne Solution Cell Proliferation Assay is a colorimetric method for determination of cell viability in proliferation or cytotoxicity assays. PASMCs were plated at a dense of 6,000 cells/well in 96-well plates. After the cells were attached to the 96-well plates, the medium was replaced with DMEM culture supplemented with 2% fetal bovine serum and incubated for 24 h. The first experiment was designed to determine the effect of hypoxia on the proliferation of PASMCs. Cells were subjected to hypoxia for 12, 24, 48, 60, or 72 h, cultured, and then incubated for 2 h with 20 μl/well MTS. Absorbance was measured at 490 nm . To determine the effect of eIF2α siRNA on proliferation of PASMCs, cells were exposed to hypoxia for 48 h in the presence or absence of eIF2α siRNA  for 4 h, 20 μl/well MTS was added followed by incubation for 2 h. Absorbance was measured at 490 nm. All experiments were performed in triplicate and repeated at least three times.DNA synthesis analysis was performed using CellTiter 96 AQTotal RNA was extracted by TRIzol . For detection of mRNAs and miRNAs expression, RNA (0.2–0.5 μg) was subjected to reverse transcription reaction using the PrimeScript reverse transcription reagent Kit  according to the manufacturer’s instructions. Quantitative analysis of mRNAs and miRNA expression was performed using SYBR Premix Ex Taq  at ABI 7300 system. PCR cycling conditions were as follows: an initial incubation at 95°C for 15 s, followed by 40 cycles of denaturation at 95°C for 5 s, and annealing at 60°C for 31 s. For detection of mature miRNAs, Bulge-Loop miRNA Primers (Ribobio) were used. The sequences of oligonucleotides used were as follows: Primers for eIF2α were: 5′- GGACAAATGGAAGTATGGGATG-3′ (forward), 5′-CAAGAGAGAGCCAGTGTAATGC-3′ (reverse). Primers for mTOR were: 5′- ATCCAGACCCTGACCCAAAC-3′(forward), 5′- TCCACCCACTTCCTCATCTC -3′(reverse). Primers for c-myc were: 5′- TGTCCGTTCAAGCAGATGAG -3′(forward), 5′- CGGTCAGTTTATGCACCAGA -3′(reverse). Primers for β-actin were: 5′-TGTCACC-AACTGGGACGATA-3′(forward), 5′-ACCCTCATAGATGGGCACAG-3′(reverse). Data analysis was performed by comparative Ct method using the ABI software. U6 and β-actin were used to normalize the expression of mRNA and miRNAs, respectively. The results were repeated at least three times independently using three different pools of templates.Protein was extracted from PASMCs with RIPA buffer (contain 0.1% PMSF), and equal amounts of protein from each sample (20 or 40 μg) were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidine fluoride membranes. The membranes were then incubated with primary antibodies overnight at 4°C and horseradish peroxidase-coupled goat anti-rat secondary antibody . The chemiluminescence signals were detected with the EasySee Western Blot Kit . The densitometric analysis was conducted with Image J 1.43 . Primary antibodies against eIF2α  were purchased from abcam . Primary antibodies against p-eIF2α , c-myc , mTOR , and p-mTOR  were obtained from Cell Signaling Technology. Primary antibody against β-actin  was purchased from Boster.t-test for two groups or analysis of variance followed by Newman–Student–Keuls test for multiple groups. Statistical significance was defined as P<0.05.Data were presented as the mean ± S.E.M (standard errors). Statistical analysis was performed by unpaired Student’s Immunofluorescence analysis of lung tissues from hypoxia-induced PH rats showed a significant increase in p-S2481-mTOR expression level, a marker for mTOR catalytic activity. The expression of mTOR was located in SMA-positive areas in small remodeled pulmonary arteries in lungs of hypoxia-induced PH rats, suggesting a role of mTOR in medial hyperplasia of pulmonary arteries . PASMCs eIF2α is a critical protein for cell growth and cell cycle regulation. A recent study demonstrated that eIF2α is involved in the regulation of airway smooth muscle cell proliferation, hyperplasia and migration . Thus, wc-myc is an important member of the myc family and is involved in the regulation of cell proliferation and differentiation . Our preIn keeping with a previous study , three wAs observed in our study, mTOR and eIF2α were up-regulated both in the media of pulmonary arteries and in hypoxia-induced cultured PASMCs. We next determined whether mTOR contributes to PASMCs proliferation and pulmonary vascular remolding via eIF2α. We performed immunofluorescence staining and immunohistochemical analysis of lung tissues from rats with chronic hypoxia-induced pulmonary vascular remodeling. Rapamycin markedly decreased mTOR, p-eIF2α and eIF2α levels . At the in vivo and in vitro , endothelin 1 receptor (ET1R) antagonist, prostacyclin and sildenafil are focused on improving vasodilatation. However, preventing vascular remodeling is supposed to have a greater impact on improving prognosis , 5, 7. RIn vitro experiments showed that chronic hypoxia induced mTOR activation in primarily cultured rat PASMCs. These results suggest that mTOR is activated in PASMCs proliferation and pulmonary vascular remodeling in HPH. To further investigate whether activated mTOR is involved in the progression of PH, the mTOR inhibitor rapamycin was used in vivo and in vitro. Our findings showed that rats treated with rapamycin were resistant to chronic hypoxia, as evidenced by lower right ventricle systolic pressure (RVSP) and mean pulmonary arterial pressure (mPAP) and thinner pulmonary vessel walls. Pretreatment with rapamacin or knockdown of mTOR by siRNA also abolished hypoxia-induced proliferation of PASMCs. Collectively, our results demonstrate that mTOR plays an important role in PASMCs proliferation and vascular remodeling in HPH.A critical role of mTOR signaling in cellular function has been reported mainly in cancer cells . EmerginResults from our lab and other scientists –38 proviThe present study suggested that eIF2α acts as a new mediator of mTOR and an important regulator of PASMCs proliferation in HPH. Firstly, under hypoxic conditions, mTOR activation was accompanied by up-regulation of eIF2α expression and activity (as determined by phosphorylation) in pulmonary arteries and PASMCs. Secondly, rapamycin treatment significantly reversed the activation of eIF2α in vitro and in vivo. Thirdly, knockdown of mTOR with siRNA reversed the increase in eIF2α expression and phosphorylation in hypoxia-induced PASMCs. In addition to eIF2α, other translation initiation factors are also involved in mTOR signaling. For example, mTOR can bind to eIF3 and lead to release and activation of S6K. 4E-BP1 is one of the main downstream proteins of mTOR , 17. AllAnyway, a critical role of eIF2α in PASMCs proliferation and vascular remodeling in HPH enlarged our understanding on the role of eIFs in PH. In this study, we observed a marked increase in p-eIF2α, a marker for eIF2α catalytic activity, in SMA-positive areas in small muscular remodeled vessels of lungs of HPH rats. Importantly, we found that eIF2α siRNA prevented hypoxia-induced PASMCs proliferation. It suggested that eIF2α might be crucial for the media hyperplasia in HPH rat through stimulation of PASMCs proliferation. We think it is only the first step to reveal the role of eIFs in PH, investigating the effect of other eIFs including eIF3, eIF4 on PASMCs proliferation and vascular remodeling would be helpful to decipher the role of eIFs in PH and provide more evidence for potential therapeutical strategy targeting eIFs.In this study, we found that eIF2α siRNA can reverse hypoxia-induced PASMCs proliferation and c-myc expression. However, much work remains to be done to elucidate the mechanism underlying eIF2α regulation of PASMCs proliferation. Previous studies showed that activation of c-myc is considered to be one of the initiating steps in vascular smooth muscle cell proliferation. An aberrant expression of c-myc is observed in injured vessels after angioplasty . ConsistOur findings suggest a critical role of the mTOR/eIF2α pathway in hypoxic vascular remodeling and PASMCs proliferation in HPH. Inhibition of mTOR/eIF2α pathway could serve as a new solution for the treatment of PH.S1 FigP<0.05 vs. con; **P<0.01 vs. con; ***P<0.001 vs. con; #P<0.05 vs. hypoxia; ##P<0.01 vs. hypoxia. The above experiments were repeated three times with similar results.Cellular proliferation stimulated by 20% FBS (48 hours) was assayed in PASMCs treated with siRNA against mTOR or eIF2α, or in the presence of rapamycin. As shown in MTS experiments, cell proliferation was inhibited by either rapamycin  (A), eIF2α siRNA  (B), or mTOR siRNA  (C). These results are in agreement with cell proliferation data acquired with hypoxia. Data represent the means ±S.E.M. n = 3. *(TIF)Click here for additional data file.S2 FigP<0.05 vs. control; **P<0.01 vs. control; #P<0.05 vs. hypoxia; ##P<0.01 vs. hypoxia. The above experiments were repeated three times with similar results.(A) The expression of Ki-67 mRNA was determined by real-time PCR. (B) The expression of PCNA mRNA was determined by real-time PCR. Data represent the means ±S.E.M. n = 3. *(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.


Charge transport measurements form an essential tool in condensed matter physics. The usual approach is to contact a sample by two or four probes, measure the resistance and derive the resistivity, assuming homogeneity within the sample. A more thorough understanding, however, requires knowledge of local resistivity variations. Spatially resolved information is particularly important when studying novel materials like topological insulators, where the current is localized at the edges, or quasi-two-dimensional (2D) systems, where small-scale variations can determine global properties. Here, we demonstrate a new method to determine spatially-resolved voltage maps of current-carrying samples. This technique is based on low-energy electron microscopy (LEEM) and is therefore quick and non-invasive. It makes use of resonance-induced contrast, which strongly depends on the local potential. We demonstrate our method using single to triple layer graphene. However, it is straightforwardly extendable to other quasi-2D systems, most prominently to the upcoming class of layered van der Waals materials. The past years have seen a tremendous increase in available quasi-2D materials, extending from graphene3461VE , is considered a fingerprint of a specific surface structure18Vbias is applied over a sample. This causes the local electron landing energy (wavelength) to become position-dependent, as depicted in The basic idea behind LEEP is to determine the local potential at each position on a sample from LEEM images, i.e., from the intensity of specularly reflected low-energy electrons (0–100 eV) that are projected onto a pixelated detector141516VE . Importan non-degenerate states for materials with n + 1 layers. When the electron landing energy in a LEEM experiment is equal to the energy of one of these states, resonant coupling between the electron wave and the interlayer state suppresses electron reflection. This yields minima in the IV-curve, with the number of such minima corresponding to the number of interlayer states n. For graphene on silicon carbide (SiC), for example, the number of minima corresponds to the number of conducting graphene layers, because the bottommost carbon layer is a buffer layer that is insulating, but does contribute to the formation of interlayer statesIn principle, any clean material has its characteristic IV-curve and can thus be studied by potentiometry. Still, LEEP is expected to work best if the intensity exhibits a strong and well-defined energy dependence. The layered structure of van der Waals heterostructures, for example, has interesting consequences for LEEM IV-curves. The properties of these stacks of 2D systems like hexagonal boron nitride, graphene or transition metal dichalcogenides, held together by van der Waals forces, are tunable by choosing a specific stackingVE = 2.7 V (VE = 3.5 V) causes contrast inversion. We have taken IV-curves at every point of this field of view by recording LEEM images while sweeping VE . Note that the distinct shape of the IV-curves due to the resonant coupling to interlayer graphene states allows us to deduce this shift particularly precisely and thus enhances the resolution of the LEEP technique. Similar minima are expected for many layered quasi-2D crystals. In fact, most materials show clear structure in LEEM-IVWe next use the rich structure of the graphene IV-curves to precisely measure local potential values. In such a LEEP experiment, we apply an in-plane bias voltage n VE see . Figure sed case  and the sed case  are immeV, derived using the LEEP technique. The grainy structure of the image is mainly caused by residues of the resist used and is considered noise in the following. As expected for an Ohmic material like graphene, a voltage drop from left to right is apparent.A complete potential map of the sample can now be produced by comparing the IV-curve at every pixel with a reference IV-curve taken at zero bias .


By using one stage amplifier in the anomalous dispersion regime, the laser can be amplified up to 450 mW and the narrowest pulse duration of 1.4 ps can be obtained simultaneously. Our work paves the way to graphene Tm/Ho co-doped mode-locked all-fiber master oscillator power amplifiers as potentially efficient and economic laser sources for high-power laser applications, such as special material processing and nonlinear optical studies.Mid-infrared ultrafast fiber lasers are valuable for various applications, including chemical and biomedical sensing, material processing and military applications. Here, we report all-fiber high-power graphene mode-locked Tm/Ho co-doped fiber laser at long wavelength with evanescent field interaction. Ultrafast pulses up to 7.8 MHz are generated at a center wavelength of 1879.4 nm, with a pulse width of 4.7 ps. A graphene absorber integrated with a side-polished fiber can increase the damage threshold significantly. Harmonics mode-locking can be obtained till to the 21 Long-wavelength >1.55 μm) ultrafast lasers can potentially be applied in various applications, such as free-space optical communication due to atmospheric transparency window, molecular spectroscopy, medical diagnostics, laser surgery, long-range radar, and gas detection.55 μm ul23Mode locking is the most typical approach to generate ultrashort pulses. Various methods have been reported to achieve mode locking: Nonlinear polarization rotation (NPR) is the one of the most commonly used methods for fiber laser mode locking, which have been studied for many years6791112131415171820192527In order to generate ultrashort pulse at wavelength above 1.55 μm and even up to 3.9 μm3234353637In this paper, we utilize the side-polished fiber together with graphene grown by CVD method to achieve mode locking in a Tm/Ho co-doped ring fiber laser. It is demonstrated that mode locking can be obtained at a center wavelength of 1879.4 nm, spectral bandwidth of 1.8 nm, and pulse width of about 4.7 ps. High-order harmonic mode locking (up to 164.6 MHz) can be obtained with the increase of pump power. After we use a Tm-doped fiber amplifier (TDFA) to boost the output power, the maximum output power is ~450 mW, corresponding to the peak power of about 9.7 kW. Our results shows graphene all-fiber Tm/Ho co-doped mode-locked fiber laser with single mode output and high peak power. These unique properties enable the advantages of compactness, good beam quality and high power output, which make them potential candidates in the molecular spectroscopy, nonlinear frequency conversion, and laser surgery applications.2, Graphene Supermarket) is transferred onto a side-polished fiber embedded in a glass plate by a modified transfer process. W~996000 by GPC, Sigma Aldrich, dissolved in chlorobenzene with a concentration of 50 mg/mL), which is then cured at 110 °C for 10 min. The underneath Cu foil is then dissolved in an aqueous (NH4)2S2O3 solution (10%). The remaining PMMA/graphene film is cleaned by repeatedly dipping it in deionized water, and then a 0.5 M HCl bath for 5–10 min. Then, the PMMA/graphene film is picked up through the target side-polished fiber. After drying in the ambient condition, some PMMA solution is dropped onto the side-polished fiber to dissolve the pre-coated PMMA and flatten out winkles on the graphene. The PMMA is then dissolved in acetone, leaving SLG on glass with side polished fiber.CVD grown single layer graphene (SLG) (~20 × 20 mmG/I2D) is 0.54, which is recognized as intrinsic single layer grapheneAfter transfer, the graphene on the side-polished fiber was characterized by Raman spectroscopy (under 532 nm excitation) and absorption microscopy. A representative Raman spectrum of the transferred graphene on side-polished fiber is shown in When the pump power reaches 371 mW, mode-locking can be self-started. When we decrease the pump power, pulse train can be maintained till 326 mW, which is due to the hysteresis effect2 profile with a pulse width of 4.67 ps. The corresponding 3-dB spectral width is 1.8 nm, corresponding to a time-bandwidth product (TBP) of about 0.714. The TBP is larger than transform-limited sech2 pulse, which indicates the pulses are chirped. Compared with Ref. 2, is comparable. The pulse width can be further reduced by controlling the dispersion of the cavity.The typical output of the fiber laser  is shown in th harmonics mode locking, which gives an output of 10.1 mW with a repetition rate of 164.6 MHz. Because of the limitation of the damage threshold of the fiber components (500 mW), the pump power are not further increased. th, 8th, 11th, 15th, and 21th harmonic mode locking versus the different pump power. With further increasing the pump power, our fiber laser can operate from the fundamental mode locking regime to the high-order harmonic mode-locking regime. th harmonic, which is limited by the damaged threshold of the WDM coupler rather than the absorber. The absorber with a sandwich structure, however, cannot bear such high pump power. Thus, it is difficult to observe higher-order harmonics mode locking based on the sandwich-structure absorber.The absorber structure in our work is quite different from those with the sandwich structure. In our work, higher-order harmonic mode locking is observed till to the 21To increase the output power of our graphene based fiber laser, we use a home-made TDFA to amplify the seed laser. The maximum output power after amplification is ~450 mW by removing the residual pump power, when the passively mode-locked fiber oscillator operates at the fundamental mode locking (output power of the oscillator is 3 mW). The amplification coefficient is more than 21 dB. The spectral evolution at different output power is shown in Conventionally, chirped pulse amplification (CPA) technique can overcome the pulse break-up, which is widely used in the normal-dispersion regime. After being amplified, the pulse can be further compressed due to the linear chirp of the pulse. Different from the CPA technique, the pulse can be amplified and compressed simultaneously in the anomalous dispersion regime. This can help to get shorter pulse than the transform-limited pulse due to the nonlinearity and high order soliton effect. Our approach provides a scheme to further compress the transform-limited pulse.In order to demonstrate that the mode locking is solely mode locked by the SLG SA with graphene, we remove the side polished fiber with graphene or just remove the graphene on the side-polished fiber. No clear mode-locked pulse train or typical mode-locked spectrum can be observed. Only the CW regime was obtained from the laser cavity, which confirms that the mode locking is solely contributed by the side-polished fiber with graphene. The stability of our laser has been tested for a few days, no performance degradation was observed.th order, which is limited by the damaged threshold of fiber components. The output power of the proposed fiber laser can be further amplified to more than 450 mW, corresponding to a peak power of about 9.7 kW. The high peak-power Tm/Ho co-doped mode-locked fiber laser, with single mode output and high beam quality, can be potentially applied with further development in applications such as laser surgery, medical diagnostics, and gas detection, etc.In conclusion, we demonstrate passively mode-locked high power Tm/Ho Co-doped fiber laser based on a side-polished fiber incorporated with CVD grown graphene. The obtained pulse width is about 4.7 ps, the spectral width is about 1.8 nm with Kerr side band, indicating that the fiber laser operates in the conventional soliton regimes. With further increase of the pump power, the Tm/Ho co-doped mode-locked fiber laser can operate in high-order harmonic mode locked states till to the 21Output spectra of our fiber lasers are measured through an optical spectrum analyzer . Mode-locked pulse train and radio frequency (RF) spectra are detected by using a 33-GHz mixed signal oscilloscope , one signal source analyzer  coupled with 12-GHz photodetector (Newport 818-BB-51F), respectively. A wide-range autocorrelator (From Femtochrome FR-103HP) is used to detect output pulse width.How to cite this article: Li, X. et al. High-power graphene mode-locked Tm/Ho co-doped fiber laser with evanescent field interaction. Sci. Rep.5, 16624; doi: 10.1038/srep16624 (2015).


The simple treatment of dispersed graphenides suspended on silica substrates with benzonitrile leads to the clean conversion to graphene. This unprecedented procedure represents a rather mild, scalable and inexpensive method for graphene production surpassing previous wet-chemical approaches.Herein, we report on a significant discovery, namely, the quantitative discharging of reduced graphite forms, such as graphite intercalation compounds, graphenide dispersions and graphenides deposited on surfaces with the simple solvent benzonitrile. Because of its comparatively low reduction potential, benzonitrile is reduced during this process to the radical anion, which exhibits a red colour and serves as a reporter molecule for the quantitative determination of negative charges on the carbon sheets. Moreover, this discovery reveals a very fundamental physical–chemical phenomenon, namely a quantitative solvent reduction induced and electrostatically driven mass transport of K Graphite intercalation compounds are promising precursors for the reductive exfoliation of graphene. Here, the authors unveil the discharging mechanism of reduced graphitic compounds in a solid/liquid phase reaction, and further demonstrate its practical use for graphene production. The fundamental finding is that the treatment of GICs with benzonitrile (PhCN), leads to a quantitative discharging of the individual graphenide sheets upon the formation of the coloured radical anion PhCN˙−, which is easy to monitor quantitatively, the accompanying exhaustive and Coulomb force-driven migration of the potassium counterions from GICs into the surrounding benzonitrile phase, the suppression of any reactions of dispersed graphenides with moisture and air that is shown to take place when no treatment with benzonitrile is provided and the successful generation of defect-free single-layer graphene suspended on silica substrates. This latter discovery represents a rather mild, scalable, and inexpensive method for the wet-chemical graphene production.We report here on a significant discovery, which gives answer to these questions and solves the associated challenges. Moreover, this discovery reveals a very fundamental physical–chemical phenomenon, namely a quantitative solvent reduction induced and electrostatically driven mass transport of KN,N-dimethylformamide (DMF). Generally, typical strong oxidizing agents show much higher reduction potentials, for instance tetracyanoquinodimethane with −0.2 V versus Ag in DMF2122+ cations and PhCN˙− radical anions2425˙− displays a broad absorption pattern with a peak at ∼390 nm and another feature of bands at 500 nm. For the determination of the extinction coefficient of PhCN˙−, which has not been reported in literature so far, we performed a dilution series of potassium in benzonitrile. The linear correlation between amount of potassium and measured absorption reveals quantitative reactions  L mol−1 cm−1 (in situ Raman spectroscopy under inert conditions (8 in PhCN (−1) by brief ultrasonication  under argon atmosphere. The resulting formation of PhCN˙− was investigated by UV/Vis absorption spectroscopy in sealed cuvettes to exclude any air exposure  measurements under inert gas atmosphere thus inhibiting any reoxidation processes that could take place under ambient conditions. In An important question that arose from these investigations was what happens with the GICs after the quantitative discharging process. For addressing this point we applied Raman spectroscopy. To suitably handle the highly reactive intercalation compounds we carried out 8 is verified by the characteristic broad Fano-line-shape spectrum of KC8  of graphite after the treatment of the GIC with benzonitrile (8 and recovered graphite with the interlayer distances of 3.35 Å (graphite) and 5.35 Å (KC8) are exactly matching the literature values.After melting of potassium in the presence of graphite, the formation of a stage I GIC corresponding to KCm of KC8 2728. Besonitrile . The XRD˙− anions is accompanied by mass transport of interlayer K+ counterions from the interior of the solid GIC to the benzonitrile solution. This can be explained by an initial heterogeneous electron transfer reaction from the solid GIC to the liquid benzonitrile phase. As a consequence for a possible mechanism, it can be assumed that electrostatic forces are build up between the liquid phase, enriched with proximate, negatively charged PhCN˙− anions and the solid graphitic phase, enriched with positive charges . At this step, the solvent-driven electron trapping induces an intermediately built-up Coulomb attraction between the liquid and solid phase, while the subsequent migration of the K+ leads to energy minimization of the entire system which is indicated by the intermediate step iii) in − as a reporter molecule could find beneficial applications in battery technology. The current method for the state of charge determination by measuring voltage is frequently associated with errors for some materials.Obviously, the reoxidation of the negatively charged graphenide sheets and the formation of PhCNID/IG ratios  of the generated bulk solid sample after workup under ambient conditions . The reoG ratios  that matG ratios . Only a 8 were dispersed in 200 ml absolute tetrahydrofuran (THF) by tip ultrasonication  as depicted in GA and GB of 100 ml, each. In contrast to GA, the fraction GB was treated with an excess of 10.0 ml PhCN resulting in a colour change to red, which is indicative for the formation of the respective PhCN˙− anion in THF. Both fractions were subjected to aqueous workup under ambient conditions. The dispersions were filtrated and the resulting black powders were dried at 70 °C before the final characterization by TG-MS and SRS . Mass spectrometry analysis of the cleavage products reveals fragments with m/z 2 and 18 demonstrating the detachment of -H and -OH moieties in GA. On the other hand, for GB these characteristic mass traces are almost absent and only a very small signal for m/z 18 can be detected. These experiments provide the first experimental proof of the expected reaction of graphenides with water and oxygen—previously monitored for carbon nanotubesSignificantly, sample ormation . As a diphite GP . Obviousigations  provides8 in 100 ml dimethyl sulfoxide (DMSO). After 2 h of sedimentation, monolayer graphene films (1LG) on Si/SiO2 wafers were formed via drop casting of one drop in the glovebox.A direct consequence of these studies is that wet-chemical graphene production should be possible on the basis of this novel protocol, as long as reaggregation of the individualized graphene sheets to graphite can be suppressed. To prove this we have prepared graphenide solutions by dissolving 12.0 mg KC(ac)) or subjected to treatment with PhCN (1LG(PhCN)). The so obtained graphene flakes exhibit single-layer nature bearing a mean height of 1.5 nm over a lateral size of 10–18 μm grain size in pristine graphite SGN18—as depicted in the atomic force microscopy (AFM) image in (PhCN) by SRS  by SRS .(ac) display a mean ID/IG ratio of 2.5 which correlates with an average degree of defects of θ=0.5%. This shows that also on a substrate the deposited graphenides undergo chemical reactions leading to sp3-defects. In the case of 1LG(PhCN), however, the characteristic Raman features of high-quality single-layer graphene are observed. In particular, the D-mode intensity is almost vanishing, indicative for a very low density of sp3 carbon atoms (θ=0.003%). Moreover, the 2D-mode exhibits the typical narrow Lorentzian line shape and a drastically increased intensity with respect to the G-mode. In fact the spectroscopic features of 1LG(PhCN) reveal the formation of defect-free graphene, with even superior quality with respect to chemical vapour deposition grown samplesI2D/IG ratio in relation to the ID/IG ratio is presented for each data point within the investigated area of the Raman map. The sample quality of the graphene prepared in this way is extraordinary and can easily compete with bottom-up synthetic routesThe samples 1LG˙−) serves as an ideal reporter molecule for the quantification of the amount of negative charges on GICs or graphenides using absorption spectroscopy. Associated with the discharging of the GICs and the documented conversion to the parent graphite is the quantitative migration of the interlayer potassium cations into the liquid phase. The mass transport is driven by the electrostatic potential caused by adlayers of initially formed PhCN˙− anions on the surface of the GIC and their diffusion into the liquid phase. The opportunities of this unprecedented and simple solvent-driven mass transport (which is related to that in lithium-ion batteries) for applications in energy release technologies did not escape our attention. We have also demonstrated direct practical use of our discovery, namely, the straightforward formation of graphene from graphenides deposited on surfaces. The advantage of this simple and inexpensive graphene production surpasses previous wet-chemical methods such as exfoliation of neutral graphite in solvents and the reduction of graphene oxide, since both features are combined in our method namely, the complete exfoliation down to single-layer sheets and the application of rather mild reaction conditions avoiding a priori any formation of σ-defects (holes) in the carbon plane.The discovery of a facile and quantitative electron trapping from reduced graphite forms such as graphite interaction compounds (GICs), graphenide dispersions, and graphenides deposited on surfaces by simple exposure to benzonitrile opens unprecedented perspectives in graphene chemistry and engineering. Benzonitrile is a common and cheap organic solvent, which at the same time has a rather low reduction potential. This allows for a quantitative discharging of reduced graphites in a heterogeneous solid/liquid phase reaction—a quantitative process that has been overlooked so far. At the same time the red coloured benzonitrile radical anion (PhCNx–y table in a continuous linescan mode (SWIFT-module) in order to obtain faster measurements. In situ Raman measurements were carried out in a quartz tube sealed by an ISO-KF flange in order to keep inert conditions. The laser power was kept below 0.01 mW to avoid laser induced deintercalation of intermediate species.Raman spectroscopic characterization was carried out on a Horiba Jobin Yvon LabRAM Aramis confocal Raman microscope (excitation wavelength: 532 nm) with a laser spot size of ∼1 μm . Raman measurements were carried out using a micro-Raman setup in backscattering geometry. A charge-coupled device is used to detect the signal after analysing the signal via a monochromator. The spectrometer was calibrated in frequency using a HOPG crystal. Statistical Raman measurements were obtained through a motorized Atomic force microscopy was carried out on a Solver Pro scanning probe microscope equipped with NSG-10 cantilevers (NT-MDT) and a Sony Exwave HAD camera optical zoom (6.5) in tapping mode.m/z>45, the thermogravimetric analysis of pure PhCN solutions was carried out on a Perkin Elmer Pyris 1 TGA instrument. For the analysis of m/z<45  in GA and GB , equipped with a gas filter to remove solvents and an argon cooling systems, with an oxygen content <0.1 p.p.m. and a water content <0.1 p.p.m.λ=1.542 Å) equipped with a pinhole camera  and an image plate system . All two-dimensional X-ray images were radially averaged and background corrected to obtain the scattering intensities in dependence on the scattering angle 2θ.X-ray diffraction was performed by placing the material in a glove box into glass capillaries with 1.5–2 mm diameter and 10 μm wall thickness  and subsequent sealing. X-ray patterns were measured using a microfocus X-ray source with a copper target , a synthetic graphite  with a comparatively small mean grain size of 18 μm , a high material .Potassium chunks were bought from Sigma-Aldrich Co. and used as received.2O, <1 p.p.m. O2) over molecular sieves (3 Å). Finally, pump–freeze technique was used to completely degas the solvent before the reaction.Benzonitrile (PhCN) was received anhydrous from Sigma-Aldrich Co. and dried to absolute quality . Finally, pump–freeze technique was used to completely degas the solvent prior to use.THF was received anhydrous from Sigma-Aldrich Co. and dried over molecular sieves (3 Å). Subsequently, it was distilled over Na/K alloy to remove inhibitor and achieve absolute quality (<1 p.p.m. HDMSO was received anhydrous and purified (99.99%) from Sigma-Aldrich Co. and pump–freeze technique was used to completely degas the solvents before the reaction.The data supporting the findings of this study are available within the article and its How to cite this article: Vecera, P. et al. Solvent-driven electron trapping and mass transport in reduced graphites to access perfect graphene. Nat. Commun. 7:12411 doi: 10.1038/ncomms12411 (2016).Supplementary Figures 1-6, Supplementary Table 1, Supplementary Methods and Supplementary References