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


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


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


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