This paper reports a highly reliable transparent conductive electrode (TCE) that integrates silver nanowires (AgNWs) and high-quality graphene as a protecting layer. Graphene with minimized defects and large graphene domains has been successfully obtained through a facile two-step growth approach. Ultraviolet light emitting diodes (UV-LEDs) were fabricated with AgNWs or hybrid electrodes where AgNWs were combined with two-step grown graphene (A-2GE) or conventional one-step grown graphene (A-1GE). The device performance and reliability of the UV-LEDs with three different electrodes were compared. The A-2GE offered high figure of merit owing to the excellent UV transmittance and reduced sheet resistance. As a consequence, the UV-LEDs made with A-2GE demonstrated reduced forward voltage, enhanced electroluminescence (EL) intensity, and alleviated efficiency droop. The effects of joule heating and UV light illumination on the electrode stability were also studied. The present findings prove superior performance of the A-2GE under high current injection and continuous operation of UV LED, compared to other electrodes. From our observation, the A-2GE would be a reliable TCE for high power UV-LEDs. The two-step growth is highly effective in reducing the point defects and increasing the graphene domain size, both factors benefit the graphene to be used as conformal layer to protect the AgNWs from oxidation. Herein, one-dimensional AgNWs network is integrated to graphene synthesized by two-step growth method without any considerable loss in optical transmittance to fabricate exceptionally stable and conductive TCEs. As a proof of concept, we demonstrate operation of a 380 nm UV-LED using this hybrid electrode and compare the performance and long-term reliability with AgNWs only electrode and similar hybrid electrode comprising of AgNWs and conventional one-step grown graphene.2, respectively. The corresponding nucleation densities are calculated to be 2.77 × 106/cm2 and 5.21 × 105/cm2, respectively. Raman spectra were acquired on each graphene films to examine the crystal quality, as shown in −1 and 2685 cm−1, respectively. Raman spectra recorded from both samples reveal typical characteristic of monolayer graphene, i.e. a single Lorentzian peak with a full width at half maximum of 28 cm−1 and the 2D to G intensity ratio greater than 2. Moreover, the so-called defects or disorder-provoked D-band peak around 1350 cm−1 is negligible, implying that both graphene layers are high quality with minimized defects. Though Raman analysis evaluate the defect distribution and quality of graphene by the D band intensity, it is difficult to detect accurately the low-density defects that unroll across over large area. To give a further insight into structural quality of graphene on large scale, the film-induced frustrated etching (FIFE) test is performed. Commercial copper etchant, ammonium persulfate [(NH4)2S2O8], is dropped on to graphene grown on Cu foils. After 10 sec, the dropped etchant is rinsed with deionized water and then the surfaces of one-step and two-step graphene on Cu foils are probed by atomic force microscope (AFM), as shown in 7 cm−2 and 2 × 106 cm−2, signifying that the etch-pit density in two-step graphene is about one order less due to the decrease of the amount of graphene grain boundaries. The reduction of grain boundaries is a result of minimization of nucleation density of graphene during the first growth step and associated decrease in defects caused by better stitching between domain boundaries by fast lateral growth during the second step. To confirm the barrier properties of graphene, the oxygen transmission rate (OTR) measurement is carried out after the graphene films being transferred to poly ethylene terephthalate (PET). The values of OTR for bare PET and PET substrates cover by one-step graphene (1-G/PET) and two-step graphene (2-G/PET) are measured to be 20.89 ± 0.4, 12.52 ± 1.2, 5.39 ± 0.9 cc/m2-day, respectively. The 2-G/PET exhibits the lowest OTR value, which is 74% less than that of bare PEF owing to the impermeability of graphene with the minimized defect density and large graphene domain. It is evident that graphene plays an important role as gas barrier against oxygen.In the growth of graphene by CVD, the initial stage of growth is a key to obtain large domain size and good crystal quality. TC) of the three different TCEs are depicted in TC = T10/Rshsh) and transmittance measured at 380 nm. The transmittance of the AgNWs, A-1GE, and A-2GE at 380 nm is measured to be 91.5%, 93%, and 93%, respectively. Generally, some loss in optical transmittance could occur in hybrid TCEs due to the additional absorption by graphene in the visible wavelength region. Conversely, the transmittances of A-1GE and A-2GE at 380 nm are increased about 1.5% compared to the transmittance of AgNWs (91.5%), which is a likely result of partial screening of plasmon absorption of AgNWs. This is because the dielectric environment of the AgNWs is interrupted by the graphene, which inhibits the collective oscillation of free electrons in AgNWs arising due to interacting electromagnetic field26,28TC due to the reduced sheet resistance and increase in transmittance at 380 nm, as shown in Optical transparency and sheet resistance are the two important criterias that must be considered for the use of TCEs in LEDs, because the efficiency of the LEDs largely associated with these two parameters. The transmittance, sheet resistance, and figure of merit  intensity is enhanced for the LED with A-2GE in comparison to LEDs made with AgNWs or A-1GE, as shown in p-electrode for the LED with AgNWs due to insufficient current spreading by the formation of loosely bound random networks of AgNWs. Of particular point of interest is that a blue LED fabricated with similar AgNWs only electrode offered well-distributed current over the whole emission area, as shown in p-GaN/p-AlGaN layers in the former, and hence it will have an additional influence on current spreading and light output. However, recent studies demonstrated uniform light emission over the entire area of an UV-LED with the application of AgNWs TCE12max-EQE)/EQEmax is found to be 72%, 55.8%, and 47% for UV-LEDs with AgNWs, A-1GE, and A-2GE, respectively, at an injection current of 100 mA. The primary reasons for the reduced efficiency droop associated with the use of A-2GE are decrease in the heating effect caused by the reduced series resistance and improved current spreading30The performances of AgNWs, A-1GE, and A-2GE as TCEs in UV LEDs of 380 nm emission wavelength are evaluated. For practical application of TCE, it is important to take into account the long-term stability and reliability of TCE material. Herein, we intend to compare the performance of all the three devices after one month. Meanwhile, studies have shown that moderate UV exposure could lead to degradation of the performance of AgNWs32In summary, the impact of the graphene quality on the performance of a hybrid electrode of graphene on Ag NWs in GaN-based UV light-emitting diodes has been studied. Two dissimilar graphene films grown by one-step and two-step approaches were evaluated as a protective transparent conductive coating to Ag NWs towards the fabrication of extremely stable TCEs for UV-LEDs. The hybrid electrode using two-step graphene showed good ambient stability with stable sheet resistance over time. The UV LED using this TCE offered a low forward voltage, an increase in the EL intensity, and a reduction of efficiency droop. Furthermore, the device exhibited stable light emission even at high injection currents while the Ag NWs only electrode degraded over time. Our findings suggest that high-quality graphene on Ag NWs as hybrid TCE has great potential in high-power devices owing to its stability and reliability.The AgNWs used in this work was purchased from NANOPYXIS Corp. and diluted to a concentration of 5 mg/ml before being used. The diluted aqueous dispersion was then spin-coated on to desired substrates at 1000 rpm for 40 seconds to form the transparent electrodes.2 flow rate of 15 standard cubic centimeters per minute (sccm) by split-tube furnace. Concurrently, the CVD chamber was pumped down to 0.072 Torr. Then, the Cu foil was annealed for 50 min. In the one-step growth, graphene synthesis was carried out under flowing CH4 of 13 sccm and H2 of 15 sccm at 1030 °C for 23 min. In the case of two-step growth, large size graphene domains were initially achieved on Cu foils by reducing the nucleation sites in a mixed CH4/H2 (5 sccm/100 sccm) ambient for 1 min at 1030 °C. In the second step, continuous graphene was obtained when the flow rate of CH4 and growth time were increased to 13 sccm and 8 min, respectively, while maintaining the H2 flow rate and temperature constant. Finally, the chamber was cooled down to room temperature by injecting 500 sccm of Ar. Polymethyl methacrylate (PMMA) was spin-coated onto graphene surface at 4200 rpm for 50 s to make a supporting layer for the graphene during the transfer to other substrates of interest. Thereafter, the Cu foil with PMMA-spun graphene was immersed in 0.1M Ammonium Persulfate [(NH4)2S2O8] solution for 4 h to remove the Cu foil. PMMA/graphene layer was transferred to AgNWs-coated substrates in order to form the hybrid electrode. Finally, the PMMA was removed by using acetone. More details on the growth of graphene and transfer can be found in ref. We prepared three different TCEs as follows: (1) a bare AgNWs electrode, (2) a hybrid electrode of AgNWs-graphene synthesized by conventional one-step growth (A-1GE) and (3) a hybrid electrode of AgNWs-graphene synthesized by two-step growth (A-2GE). Firstly, an aqueous solution containing AgNWs was spin-coated on substrates of interest at 1000 rpm for 40 s, which is the optimum condition in terms of transmittance-sheet resistance tradeoff and more details can be found in ref. n-type GaN layer, five-pairs of In0.04Ga0.96N/Al0.08Ga0.02N MQWs, a Mg-doped p-Al0.25Ga0.75N electron blocking layer, and a p-type GaN cladding layer. Briefly, the Si-doped n-GaN layer of 2-μm was grown on un-doped GaN layer at 1100 °C under a pressure of 400 mbar for 60 min by metal organic chemical vapor deposition (MOCVD). Subsequently, five pairs of InGaN quantum wells and GaN barrier layers of thickness 3 nm and 12 nm, respectively, were grown at 790 °C and 810 °C, to form the active region. Then, a 25 nm-thick Mg-doped p-Al0.25Ga0.75N electron blocking layer and a 100 nm-thick p-GaN contact layer were grown at 1040 °C. Following the growth of LED epi-wafer, discrete LED devices were fabricated with a chip size of 350 × 350 μm2 in which the mesa region was defined by an inductively coupled plasma reactive ion etching system using Cl2/BCl3 gases until n-GaN layer was revealed for n-electrode contact. Subsequently, different TCEs were formed on p-GaN layer as explained in the previous section. As a final step, Cr (50 nm)/Au (250 nm) metals for the n- and the p-electrode were deposited onto both the transparent electrode and the n-GaN layer using electron beam evaporator.The UV LED structure used in this study is composed of an un-doped GaN layer, a Si-doped Field emission scanning electron microscopy  was used to probe the surface morphology of the samples investigated in this work. Optical transmittance and sheet resistance measurements were performed on a UV/VIS spectrometer (V-670EX) and a four-point probe system (CMT-SR1000N), respectively. The surface topography of graphene on Cu after the film-induced frustrated etching (FIFE) test was probed by atomic force microscope  in tapping mode. The quality of graphene was characterized by Raman spectroscopy using 514 nm-line of an Ar ion laser as an excitation source. Current-Voltage (I–V) and electroluminescence (EL) measurements on the LED devices were performed using a probe station system. The oxygen transmission rate (OTR) was analyzed by a commercial MOCON instrument.How to cite this article: Seo, T. H. et al. The role of graphene formed on silver nanowire transparent conductive electrode in ultra-violet light emitting diodes. Sci. Rep.6, 29464; doi: 10.1038/srep29464 (2016).


Understanding the processes that influence the structure of biotic communities is one of the major ecological topics, and both stochastic and deterministic processes are expected to be at work simultaneously in most communities. Here, we investigated the vertical distribution patterns of bacterial communities in a 10-m-long soil core taken within permafrost of the Qinghai-Tibet Plateau. To get a better understanding of the forces that govern these patterns, we examined the diversity and structure of bacterial communities, and the change in community composition along the vertical distance  from both taxonomic and phylogenetic perspectives. Measures of taxonomic and phylogenetic beta diversity revealed that bacterial community composition changed continuously along the soil core, and showed a vertical distance-decay relationship. Multiple stepwise regression analysis suggested that bacterial alpha diversity and phylogenetic structure were strongly correlated with soil conductivity and pH but weakly correlated with depth. There was evidence that deterministic and stochastic processes collectively drived bacterial vertically-structured pattern. Bacterial communities in five soil horizons (two originated from the active layer and three from permafrost) of the permafrost core were phylogenetically random, indicator of stochastic processes. However, we found a stronger effect of deterministic processes related to soil pH, conductivity, and organic carbon content that were structuring the bacterial communities. We therefore conclude that the vertical distribution of bacterial communities was governed primarily by deterministic ecological selection, although stochastic processes were also at work. Furthermore, the strong impact of environmental conditions  on these communities underlines the sensitivity of permafrost microorganisms to climate change and potentially subsequent permafrost thaw. Furthermore, Faith’s PD [Chao1-richness was computed to estimate the community diversity using the MOTHUR program . Chao1-rith’s PD  was calcP < 0.05 or P > 0.95). For a single community, a significantly positive or negative NTI value indicated that co-occurring species were more closely or distantly related than expected by chance. Based on the hypothesis that closely related taxa were more ecologically or functionally similar (phylogenetic niche conservatism), the obtained NTI measure can be used to infer ecologically similar (phylogenetic clustering) or ecologically dissimilar (phylogenetic overdispersion) taxa within a given community [To characterize phylogenetic community structure within each sample, we calculated the mean nearest taxon distance (MNTD) . A randoommunity . A mean ommunity . To corrTo examine the beta diversity patterns of bacterial communities, the Bray-Curtis metric was computed to describe the dissimilarity in species community composition (taxonomic beta diversity) between all pairwise comparisons of bacterial communities . PhylogeTo estimate the relative importance of deterministic and stochastic processes on bacterial distributions, variation partitioning was performed for taxonomic and phylogenetic beta diversity. We partitioned beta diversity into spatial and environmental components using both the distance-based approach and the raw data approach as previously described ,15,24, ar = 0.769, P < 0.001) and conductivity  increase significantly with depth. No observable correlations were found between the other variables and depth.Our soil samples were collected from different depth intervals within a 10-m-long permafrost core, which progresses from surface active layer soil into a layer of permafrost soil. The stratigraphic profile and soil physicochemical characteristics across depths are shown in The soil characteristics at the very bottom of the permafrost core (9.75 m) were completely different from those of the other soil horizons . This hoNothing was recovered from the negative parallel controls, suggesting that contamination was not introduced during extraction and purification processes. To yield enough DNA for PCR amplification and minimize individual extraction biases, DNA extractions from three replicate subsamples were combined for each sampling depth and used as one sample for the construction of clone library also see . A totalAlphaproteobacteria and Actinobacteria, which showed the highest diversity, consisting of 46 phylotypes, respectively. Sequences related to Betaproteobacteria, Gammaproteobacteria, Firmicutes, Acidobacteria and Bacteroidetes were also frequently detected. In addition, phylotypes of Deltaproteobacteria, Gemmatimonadetes, Planctomycetes, Verrucomicrobia, Saccharibacteria (former candidate division TM7), Armatimonadetes (former candidate division OP10), Cyanobacteria, Chloroflexi, and Nitrospirae were identified at relatively low diversity, as well as members of an unclassified phylum , while the pure effect of environmental distance was significant for all metrics after controlling for spatial distance . These results suggest that bacterial communities in 11 soil horizons were phylogenetically clustered and the other five communities were phylogenetically random, whereas there was an average trend towards clustering across all communities.The analysis of phylogenetic structure revealed the phylogenetic patterns of bacterial communities through the permafrost core profile. The MNTD of 11 bacterial communities was significantly different from the null communities , whereas it was not significantly different from the null communities  in the other five communities, of which two (at depths of 150 and 225 cm) originated from the active layer and three  from permafrost . All theP = 0.006) than the pure effect of space (depth) . When the distance-based method was employed, variation partitioning showed that the pure environmental distance explained 9.53% and 22.91% of the variation in taxonomic dissimilarity and betaMNTD, respectively, while the pure spatial distances explained less than 0.32% for the both metrics ; of which, the soil conductivity  was the most significant variable  was the most relevant variable, followed by the soil conductivity .The dissimilarity in species community composition of bacteria was significantly related with conductivity, C/N ratio, SOC, pH and TN  [Upon observing significant vertical structure we evaluated whether the vertical patterns of the sampled bacterial communities were governed primarily by deterministic or stochastic processes. Analyses of phylogenetic structure can complement analyses of taxonomic structure, potentially providing additional insights into the factors that shape local communities ,47. Our clusion)  as well clusion) . HoweverOn the other hand, we observed that 5 of 16 bacterial communities were phylogenetically random, which suggested that the bacterial community assembly in these soil horizons of the permafrost core was mainly determined by stochastic processes. According to neutral theory , these rAlthough the main aim of this study was not to examine the specific environmental factors that determine the biodiversity patterns, our results do suggest that they show potential in affecting the bacterial alpha diversity, phylogenetic structure, and spatial turnover across the permafrost core. Multiple stepwise regression analysis revealed that soil conductivity and pH were the most important explanatory variables for bacterial alpha diversity and phylogenetic structure, respectively. The change in taxonomic and phylogenetic community composition was significantly correlated with most measured soil variables, especially the soil conductivity and organic carbon content. These results accord with many previous findings, emphasizing the importance of pH in explaining phylogenetic structures of lacustrine bacterioplankton communities , as wellIn conclusion, our study reveals the vertically-structured patterns of bacterial communities and infers the ecological processes driving these patterns in a permafrost core. We observed that the bacterial communities were not randomly distributed along the soil core, but rather showed a vertical distance-decay relationship. The vertical distribution of bacterial communities was mainly driven by deterministic processes such that the observed distance-decay relationship was most likely the result of physicochemical environmental conditions , although stochastic processes were also involved. Our findings highlight the importance of considering information on both the taxonomic and phylogenetic structure of microbial communities and of using multiple lines of evidence to carefully evaluate whether distance-decay relationships indicate a strong influence of deterministic or stochastic processes. Our study further contributes to the emerging body of literature aimed at understanding the microbial ecology of permafrost , a key rS1 FigBootstrap values above 50% are shown as a percentage of 1000 replicates. The scale represents the number of mutations per nucleotide position. Numbers in parenthesis represent the number of phylotypes and orders assigned to each division respectively.(TIF)Click here for additional data file.S2 FigSolid lines represent linear regressions and the significance levels are determined by Mantel test (9999 permutations).(TIF)Click here for additional data file.


Graphene grown by high temperature molecular beam epitaxy on hexagonal boron nitride (hBN) forms continuous domains with dimensions of order 20 μm, and exhibits moiré patterns with large periodicities, up to ~30 nm, indicating that the layers are highly strained. Topological defects in the moiré patterns are observed and attributed to the relaxation of graphene islands which nucleate at different sites and subsequently coalesce. In addition, cracks are formed leading to strain relaxation, highly anisotropic strain fields, and abrupt boundaries between regions with different moiré periods. These cracks can also be formed by modification of the layers with a local probe resulting in the contraction and physical displacement of graphene layers. The Raman spectra of regions with a large moiré period reveal split and shifted G and 2D peaks confirming the presence of strain. Our work demonstrates a new approach to the growth of epitaxial graphene and a means of generating and modifying strain in graphene. The introduction of strain provides a route to modify both the electronic properties and phonon spectrum of graphene monolayers1234678910111231516et al.31333637Growth by MBE is realised through the direct sublimation of one or more source materials, in this case carbon, onto a substrate surface and allows independent control of growth temperature and deposition rate. MBE is widely used to grow conventional semiconductor device-grade materials, particularly those which incorporate heterojunctions, but has so far had little impact on the growth of graphene. For this relatively new material chemical vapour deposition (CVD) is much more widely used19212312272829We have grown graphene by MBE on hBN flakes which are exfoliated from high-temperature high-pressure grown bulk hBN crystals31et al.The regions between these aggregates, which have typical separation of ~1 μm, have a uniform contrast in et al.120, is reported to be in the range 13.9–15 nm which is consistent with the value, 13.9 nm, expected for unstrained graphene  (Δλ/λs). For the values observed in The period of the hexagonal array in In other areas of the flake . This peak is present in all spectra with a near-constant energy but strong variation in amplitude, reflecting the inhomogeneous distribution of aggregates on the surface. The position of the red-shifted peaks shows a monotonic variation with λs, strongly supporting the hypothesis that the graphene is strained; this has not been observed in previous reports of exfoliated or CVD graphene on hBN1229The presence of strain is also expected to influence the Raman spectrum of the graphene and we have acquired a full two-dimensional Raman map of the flake in −1 and 1615 cm−1 close to the expected values for graphene. However, we also observe a shoulder on the low energy side of the G peak which evolves into a more distinct feature which can be decomposed into two broad peaks centred on 1553 and 1527 cm−1 for λs = 26.3 nm. Other peaks present in the spectrum but not shown in −1. These peaks are therefore associated, at least partially, with the presence of carbon aggregates.As shown in s ~ 14 nm (green) and the red-shifted components for λs ~ 26 nm (red)); the false colour map in To generate a Raman map we fit the 2D region of the measured spectrum at each point to the red, green and blue spectra shown in the inset to max/λmin−1) where λmax/min are the maximum/minimum periods extracted locally; note that g = 0 for a non-distorted moiré pattern). These data, plotted in In −1/% strain and −86 ± 6 cm−1/% strain. In addition, we observe a small shift even for Δλ = 0. It has previously been shown experimentally−1/%. The dependence of the red-shifted G peak is more difficult to determine from our data since the peaks are rather broad. Assuming a linear dependence on strain (see SI) we find a red-shift dependence of −37 cm −1/% and −28 cm−1/% for the two additional components. However, from symmetry considerations we would not expect either the G or the 2D peaks of a freestanding graphene layer to be split for isotropic strain. We speculate that the split peak is due to interactions with the hBN substrate, for example a difference in local environment of carbon atoms in analogy with the formation of 2D1 and 2D2 features in graphiteWe also extract the dependence of the 2D peak positions on percentage strain, determined from the moiré period as discussed above see . SpecifiIn As the carbon dosage is increased  we see aWe have confirmed that the cracks, such as in g, and hBN, ∝BN. A tensile strain of ~1%, as observed here, implies that (∝g–∝BN), averaged over the relevant temperature range (300–1770 K) is positive with a value 8 × 10−6/K. The coefficient for hBN  is −3 × 10−6/K at 300 K and remains negative, but with reduced magnitudeg; Bao et al.g is negative at 300 K, but then increases to a positive value for a temperature, T > 350 K, while Yoon et al.et al. argued that ∝g is positive over all measured temperatures in agreement with theoretical predictionsg–∝BN) ~3 × 10−6/K. This is lower than the average quoted above, but we emphasise that data are not available between 800 K and the growth temperature, 1770 K, and, given the uncertainty related to these quantities, thermally-induced strain is a plausible origin for the observed effects.We propose that the strain in the graphene layer is introduced during post-growth cooling and is maintained by a quasi-random distribution of nucleation sites which act to pin the material. The simplest explanation for the origin of the strain is a difference between the thermal coefficients of expansion of graphene, ∝o; in fact we would not expect a bright dislocation edge to be formed parallel, and close to, a free boundary since the mismatch in lattice constants which occurs in these regions must be stabilised by compressive stress arising from the lattice-matched graphene on each side of the dislocation. This implies a complex evolution of the dislocation network due to finite size effects as the island continues to grow and a possible activated process for the introduction of new dislocation lines. Note that the introduction/elimination of a dislocation parallel to an island edge generates an advancement/retraction of the edge and therefore modifies the overall strain in the island. It is possible that thermally activated fluctuating displacements of the islands edge occur until neighbouring islands coalesce thus stabilising a strained configuration.Graphene grown by CVD on hBN does not exhibit the large moiré pattern reported here and one possible reason for this difference is the pinning role of the nucleation sites in MBE growth; these aggregates are not observed in CVD growth. There are other differences, for example the growing edge of graphene is likely terminated by hydrogen in CVD growthOur results demonstrate, for the first time, that MBE can be used to grow large areas of aligned graphene and also, unexpectedly, that the resulting material can be highly strained. Moreover, the strain, and physical properties of the graphene, can be modified through the generation of cracks using the probe of an AFM. This also leads to a physical displacement of the edges of graphene domains so the AFM actuates a nanoscale relative displacement of the grown layer, and, furthermore, gives rise to highly parallel edges which appear regular on a nanometre scale. The strongly anisotropic deformation close to a crack indicates the possibility of better controlled routes towards the engineering of more complex strain fields in graphene, for example through the lithographic formation of arrays of etched holes, or the formation of artificial pinning sites on hBN through nanofabrication prior to growth. Our work motivates further exploration of MBE growth as a route to the controlled introduction of strain into graphene layers for the generation of pseudomagnetic fields of order 10 T, novel electronic structure and new device paradigms.−10  Torr) supplied by Veeco which is modified to reach growth temperatures of 1850 °C and is compatible with substrates up to 3 inches in diameter. In this system the substrates are mounted in a vertical configuration with the substrate heater mounted above the substrate. To deposit carbon we use a SUKO-63 sublimation source, essentially a high purity pyrolytic graphite filament which is Joule heated, supplied commercially by Dr. Eberl (MBE-Komponenten GmbH). As substrates we use exfoliated flakes of hBN from high-temperature high-pressure grown bulk hBN crystals2 double sided polished sapphire (0001) substrates (SurfaceNet GmbH) and cleaned by immersion in toluene  overnight and heating at 400 °C for eight hours in a flow of 0.15 sl/min of Ar/H2 (95:5). The substrates are loaded on a Ta holder and following entry to the MBE system are pre-annealed at 400 °C for 0.5 hour prior to growth. Due to the use of the transparent sapphire substrate, pyrometer measurements give a reading only of the heater temperature. Instead we estimate the substrate temperature during growth using a thermocouple mounted in the substrate heater. The carbon deposition rate is estimated to be in the range 18–22 nm/hour from AFM measurements of the thickness of adsorbed carbon on the exposed sapphire regions of the substrate; note that the fraction of adsorbed carbon incorporated into epitaxial graphene on the adsorbed hBN flake is small, ~1%, so a growth time of ~4 hours is required to form a partial monolayer. The lifetime of the carbon source under these conditions is 2–6 hours so we are limited in our current work to monolayer growth. All of the samples discussed below were grown at the same substrate temperature which we estimate to be 1500 °C.We use a custom-designed dual-chamber GENxplor MBE system (base pressure ~10AFM images were acquired using an Asylum Research Cypher-S instrument operating in AC mode under ambient conditions and using Multi75AI-G cantilevers (Budget Sensors) supplied commercially by Windsor Scientific.Raman spectra were recorded on a Horiba–Jobin–Yvon LabRAM Raman microscope, with a laser wavelength of 532 nm operating at low power (~4 mW) and a 600 lines/mm grating. The detector was a Synapse CCD detector. The Raman shift was calibrated using an Si(100) reference sample. The Raman CLS map was obtained by extracting the 2D region of each spectra in the map. The model spectra were selected from the dataset to best represent the different regions observed in the map. The presence of a small carbon aggregate peak (blue spectrum) in the red shifted spectra (green and red) was removed by subtracting the blue spectrum after applying a multiplier to it to ensure complete subtraction of the unwanted feature. CLS fitting to each spectrum in the map was performed using the Horiba Labspec 6 software.How to cite this article: Summerfield, A. et al. Strain-Engineered Graphene Grown on Hexagonal Boron Nitride by Molecular Beam Epitaxy. Sci. Rep.6, 22440; doi: 10.1038/srep22440 (2016).


Self-heating induced failure of graphene devices synthesized from both chemical vapor deposition (CVD) and epitaxial means is compared using a combination of infrared thermography and Raman imaging. Despite a larger thermal resistance, CVD devices dissipate >3x the amount of power before failure than their epitaxial counterparts. The discrepancy arises due to morphological irregularities implicit to the graphene synthesis method that induce localized heating. Morphology, rather than thermal resistance, therefore dictates power handling limits in graphene devices. Epitaxial (Epi) graphene realized via the sublimation of silicon (Si) from silicon carbide (SiC) is limited only by the size of the supporting substrateBeyond the curiosity of an atomic dimension, it is the transport properties of graphene that differentiates it from traditional materials. Exhibiting a mobility that can exceed 200,000 cmThe same syntheses processes that provide scale, however, also induce morphological imperfections that limit device performance. Both epitaxial and CVD syntheses result in small regions of non-uniform layer number71012Self-heating of exfoliated graphene devices has been examined previously using infrared-thermography (IR)14151714141517e.g., SiO2) separating the graphene from the more thermally conductive substrate. While transfer of CVD graphene to SiC can capitalize upon the high thermal conductivity of the substrate, weak van der Waals bonding will result in a comparatively large thermal resistance at the interface 232425Solely from the perspective of thermal resistance then, epitaxial graphene provides an avenue to minimize self-heating and maximize power dissipation. Silicon carbide has a thermal conductivity ~3x that of silicon at room temperature21Self-heating is not determined exclusively from the thermal resistance, however, but also the area over which heat is generated. Factors localizing heat generation will exacerbate its effect. Here, we demonstrate that morphological imperfections localizing heat generation in a graphene device ultimately determine its power handling capability. Specifically, the breakdown power of epitaxial graphene on SiC devices is shown to be <3x that of comparable devices made up of CVD graphene atop a similar substrate. Correlations between the temperature distribution obtained with infrared–(IR) thermography and the morphology acquired using Raman imaging indicate that the difference arises from heat localization stemming from morphological features characteristic of the synthesis process. Altogether, the results demonstrate that the morphology of the graphene, rather than the system’s thermal resistance, dictates the amount of power that it can sustain.2 deposited via plasma enhanced chemical vapor deposition. This is termed— epitaxial/covered (Epi/Cov). The final architecture is composed of commercially synthesized CVD graphene  that is transferred to a SiC substrate and then covered with 50 nm of HfO2 realized via atomic layer deposition (ALD). This is termed—CVD/covered (CVD/Cov). Device geometries ranged in size from from 5 × 15 μm to 20 × 60 μm for each architecture and were contacted utilizing a Ti/Au stack (10/100 nm). Resistivity varied by ≤2x between the differing architectures and had comparable Raman responses largely absent of any defect signature (I(D)/I(G) ≤ 0.15, see Three graphene device architectures were fabricated at chip scale using standard photo-lithographic processes as shown schematically in 28For each architecture, six different devices of varying size were subject to increasing levels of Joule-heating until breakdown occurred. Breakdown was defined when channel resistance suddenly increased by several orders of magnitude. Only devices in which failure occurred in the channel—as opposed to the contacts—were included in the analysis. Practically, heating was induced by ramping current across the outer two leads to a target value held constant for 30s to facilitate thermography. Powers were measured by monitoring voltage across the inner contacts see . UtiliziBreakdown power differs significantly between the architectures see . Quantit2 carbon bonds limits the susceptibility of the graphene to electromigration2030Heating is of particular salience as the breakdown of graphene devices is thermal in origin203031et al.T > 1400 °C) than during the self-heatingExposed to the environment, graphene reacts with oxygen upon reaching temperatures of ~450 °C202/Si stack of an exfoliated device exhibits an out of plane thermal resistance of at least Ri = Li/Ki assuming thicknesses of 300 nm and 400 μm for the SiO2 and Si, respectively. Thermal conductivities of the two layers are presumed to be: KSi = 148 W/mKμm of SiC (KSiC = 450 W/mK) induces a resistance of RSiC = 0.9 m2K/MW —3x less than the exfoliated condition. Second, the exfoliated device possesses two thermally resistive interfaces—graphene to SiO2 and SiO2 to Si—to the epitaxial devices one—graphene to SiC. Third, the exfoliated devices have an area that is at least 8x less than the epitaxial devices. Under the assumption of uniform heating, these effects together will cause the exfoliated devices to heat up much faster and, by extension, fail at significantly lower powers. This is contrary to observation as, in reality, the epitaxial devices do not heat uniformly.Bare epitaxial devices failed at similar powers compared to previous reports of exfoliated devices see 20. This Epitaxial graphene emerges from the sublimation of Si from SiC resulting in concentration gradients of carbon that drive layer formation. The morphology of the underlying SiC affects the carbon concentration and thus the resulting graphene layerFor example, graphene multilayers form near atomic steps of the SiC leading to monolayer multilayer boundaries9Similarly, electrical resistance, and by extension heating, will increase locally in regions of lower mobility. Lower mobility has been correlated with regions of high variation in the 2D-mode peak positionLocalized heating and failure was also observed in epitaxial devices covered in an oxide implying that hot spot formation observed in 2. SiO2 degradation initiates at temperatures of 900–1000 °C3839402 as shown by the inset of Relative to the Epi/Bare device, the Epi/Cov architecture is capable of withstanding much larger powers see . The hig2, ALD layers of hafnia seeded with alumina begin to degrade near 900 °C43442 and HfO2 is ~900 °C. These oxides have negligible influence on the heat dissipation in either case since they are thermally insulative and extremely thin. Taken together, the difference in oxide species cannot be the cause for the 3x larger power handling capability of the CVD devices as compared to those epitaxially derived  Detector. A 20X objective having a lateral resolution of ~2 μm in size using standard photolithographic processes. Analogous CVD devices were fabricated by transferring ACS “Trivial Transfer” graphene onto 6H-SiC substrates using a procedure specified by the vendor. Conventional photolithographic techniques were employed to define and contact the devices2 was deposited using an established plasma enhanced chemical vapor deposition process.Quasi-free standing epitaxial graphene monolayer was synthesized using established methodologies292 was deposited using atomic layer deposition. To prevent graphene decomposition during the ALD process, a thin alumina layer is first grown onto the graphene surface. This protective alumina layer is formed by the controlled oxidization of a 10 Å, 0.2 Å/s electron beam evaporated aluminum thin film in pure oxygen at a partial pressure of 10 Torr. After a twenty minute exposure of the aluminum to this oxygen enriched environment, the high quality protective alumina layer is completely formed. This alumina layer not only serves to protect the graphene from potential reactants, it also appears to enhance the ALD hafnia nucleation process. Hafnia ALD was grown using a Picosun Sunale R150 hot-wall reactor operating at 250 °C using tetrakis(dimethylamido)hafnium(IV) (TDMAH) and deionized H2O precursors. At a source temperature of 75 °C, the TDMAH is introduced into the reaction space for a period of 1.5 s followed by a 15 s, N2 purge. To complete the formation of a single monolayer of hafnia, deionized water is then introduced for a period of 0.1 s followed by a 40 s, N2 purge. Both the TDMAH and H2O reaction cycles are repeated 550 times to grow a 50 nm hafnia gate dielectric.For the CVD/Cov devices, HfOHow to cite this article: Beechem, T. E. et al. Self-Heating and Failure in Scalable Graphene Devices. Sci. Rep.6, 26457; doi: 10.1038/srep26457 (2016).


V600E cells showed significantly higher baseline expression of PD-L1 at mRNA and protein levels compared to BRAFWT cells. MEK inhibitor treatment resulted in a decrease of PD-L1 expression across all cell lines. BRAFi treatment decreased PD-L1 expression in BRAFV600E cells, but paradoxically increased its expression in BRAFWT cells. BRAFV600E mutated patients samples had a higher level of PD-L1 mRNA compared to BRAFWT (p=0.015). Immunocompetent mice (B6129SF1/J) implanted with syngeneic 3747 BRAFV600E/WT P53−/− murine tumor cells were randomized to control, PLX4720, anti PD-L1 antibody and their combination. In this model of aggressive thyroid cancer, control tumor volume reached 782.3±174.6mm3 at two weeks. The combination dramatically reduced tumor volume to 147.3±60.8, compared to PLX4720  or PD-L1 antibody  alone. Immunohistochemistry analysis revealed intense CD8+ CTL infiltration and cytotoxicity and favorable CD8+:Treg ratio compared to each individual treatment. Our results show anti PD-L1 treatment potentiates the effect of BRAFi on tumor regression and intensifies anti tumor immune response in an immunocompetent model of ATC. Clinical trials of this therapeutic combination may be of benefit in patients with ATC.The interaction of programmed cell death-1 and its ligand is widely studied in cancer. Monoclonal antibodies blocking these molecules have had great success but little is known about them in thyroid cancer. We investigated the role of PD-L1 in thyroid cancer with respect to BRAF mutation and MAP kinase pathway activity and the effect of anti PD-L1 antibody therapy on tumor regression and intra-tumoral immune response alone or in combination with BRAF inhibitor (BRAFi). BRAF Thyroid cancer is the most common endocrine malignancy . ClinicaIn the current era of personalized medicine, strategies that exploit the mutational profile of individual tumors have come to the fore. Recent work in both preclinical studies and clinical trials has focused on the known driver mutations in thyroid cancer. Altered MAPK signaling pathway in many thyroid cancers leads to ERK phosphorylation, proliferation, and invasion hence BRAF inhibitors (BRAFi) and MEK inhibitors (MEKi) have been the subject of intense investigation. Various inhibitory molecules have been tested, including vemurafenib (PLX4032), which demonstrated a reasonable response in patients with progressive metastatic, radio-iodine refractory BRAF-positive PTC , but theAn alternative approach involving immune therapy, which previously had been rejected by most physician scientists as ineffective and cumbersome, has regained favor as a result of new knowledge about the immune checkpoint receptor, PD-1 and its ligand, PD-L1, which regulate T-cell activity in the tumor microenvironment. A role of the immune checkpoint regulators is to prevent autoimmunity and to reduce collateral tissue damage by modulating the immune response in chronic infection. Cancer cells capable of up regulating the expression of PD-L1 have the potential to become immune resistant , thereby+ T cells or NK cells in the tumor microenvironment. Indeed, human tumors show a correlation between PD-L1 levels and T cell infiltration reflecting an ongoing but unsuccessful anti-tumor immune response. PD-L1 has been investigated as a potential marker for tumor aggressiveness [ex vivo experiments. [PD-L1, also known as CD274, is a cell-surface glycoprotein of the B7 family. It is expressed on various tissues including solid tumors and can facilitate immune evasion and T cells exhaustion . The intsiveness , 16, andsiveness , 18. Theriments. –24in vivo immunocompetent murine model of anaplastic thyroid cancer. In undertaking this study, we hypothesized that PD-L1 expression in non-medullary thyroid cancer would correlate with MAP kinase signaling pathway activity, and as a result, targeted therapies that reduce MAP kinase activity, such as BRAFi and MEKi, would be found to regulate PD-L1 expression in BRAF-mutated tumors. We further proposed that blocking the interaction between PD-L1 and PD-1 with an anti-PD-L1 antibody, would have the added effect of increasing the anti-tumor activity of BRAFi-induced infiltrating T cells. In the first phase of our investigation, we studied 5 human and 4 murine thyroid cancer cell lines to determine baseline expression of PD-L1. Next, we investigated the effect of manipulating MAP kinase activity on PD-L1 expression in vitro and in vivo. Finally, we tested the effect of combining BRAFi and anti-PD-L1 antibody on tumor regression and intra-tumoral immune response in an orthotopic immunocompetent mouse model of ATC.Our study was designed to advance current understanding of the role of PD-L1 in thyroid cancer cells, thereby paving a path for future testing of PD-L1-based therapies in thyroid cancer patients. It is the first study to look at the expression profile of PD-L1 in a panel of nonmedullary thyroid cancer cells at baseline, after IFN-γ stimulation, and after treatment with MAP kinase inhibitors. It also represents the first attempt to determine the impact of PD-L1 antibodies, alone or in combination with BRAF inhibitor, on tumor volume in an V600E melanoma cell lines  and one BRAFWT (MelJuso) . In fact, the normal HTORi cell line had the lowest expression of PD-L1. Western blot analysis pointed toward higher PD-L1 protein expression in the mutant BRAF cells compared with wild type across all cell lines investigated.Given the known therapeutic implications of PD-L1 expression in melanoma, PD-L1 mRNA levels in 6 human thyroid cancer cell lines was quantified against 3 BRAFV600E-mutated tumors also expressed higher levels of PD-L1, PD-L1 mRNA expression levels were analyzed in randomly selected 28 fresh frozen PTC tumors and their matched normal thyroid tissue samples. Fifty-seven percent had BRAFV600E mutations on routine sequencing. None of the demographic or tumor characteristics were significantly different between the BRAFV600E (n=16) and the BRAFWT (n=12) groups (Table V600E-mutated tumors showed significantly higher expression of PD-L1 mRNA compared to BRAFWT tumors (log2 of fold changes: median (inter quartile range): 0.51 (−0.05 to 1.04) vs. −0.70 (−2.24 to 0.28) (P = 0.015) , whereas in the SW1736, only decreases in PD-L1 protein expression were observed. In BRAFWT cells treated with PLX4720 in the presence of IFN-γ, consistent increases were only observed in the protein levels of PD-L1. Significant increases in PD-L1 mRNA were observed only in the HTh-7 (ATC) cell line  at baseline and in response to PLX720, IFN-γ, or both. Although IFN-γ caused a dramatic increase of PD-L1 expression; 3601R (93.7-fold), 3868 (13.9- fold), 3743 (74.8-fold), 3403 (20.2-fold) as compared to untreated control, none of the four cell lines showed significant (>2-fold) change in PD-L1 expression in the presence of BRAFi Figure , regardl5 genetically engineered murine ATC; TBP 3743 BRAFV600E/WT P53−/− cells are implanted into an immunocompetent mouse thyroid, resulting in a very rapidly enlarging tumor with marked infiltration of CD8+ T lymphocytes when treated with PLX4720 [3. Treatment with anti-PD-L1 Ab alone did not significantly reduce tumor volume , whereas PLX4720 treatment resulted in significant tumor volume reduction . Using the Wald chi-square for statistical analysis showed that the combination of PLX4720 and anti-PD-L1 antibody acted synergistically, and was the most effective in reducing tumor volume compared to all other treatment groups  and impressively over 66% better than those treated with PLX4720 alone (P=0.023)  Figure .+ cell exhaustion; and Granzyme B, a marker of T-cell cytotoxicity. Tumors from animals treated with PD-L1 blockade or PLX4720 alone had visibly higher infiltration of CD8+ T cells compared to controls; in the tumors treated with the BRAF inhibitor most of the T cells appeared to have infiltrated into the peripheral aspects of the tumor. Combining both drugs, however, dramatically increased CD8+ T-cell infiltration throughout the tumor  have been shown to selectively interfere with the release of cytolytic granules by cytotoxic T lymphocytes (CTLs) in a reversible and TGFβ-dependent manner, thereby attenuating CTL-mediated cytotoxicity. Previous studies have shown that the CD8:Treg ratio is an important indicator of therapeutic benefit in melanomas, where potent synergistic effects of combination therapy using BRAFi and PD1 blockade cause increased CD8d humans . Indeed,l Figure .The baseline expression of mouse PD-L1 (mPD-L1), as seen in the IHC staining of the control group, was observed to increase dramatically upon treatment with PLX4720 alone; while treatment with anti-PD-L1 antibody alone or in combination with BRAFi reduced the IHC staining expression of mPD-L1 as compared with control and BRAFi alone, respectively.V600E, in an effort to alter thyroid cancer progression. Unfortunately, the response to these efforts to inhibit the MAP kinase pathway has been relatively short-lived as a consequence of the development of resistance to treatment. Coincident with this work, immune therapy, has regained favor as a result of new knowledge about the immune checkpoint receptor, PD-1 and its ligand, PD-L1, which regulate T-cell activity in the tumor microenvironment. In particular, studies involving the use of monoclonal antibodies directed against the PD-1 receptor and its ligand PD-L1 have shown promising response rates, leading to FDA approval of PD-1 drugs for advanced melanoma, lung and renal cancer [During the last decade, most thyroid cancer therapeutics have targeted known oncogenic mutations, such as BRAFl cancer , 32. OneIn melanoma patients, the initiation of BRAFi therapy causes rapid T-cell infiltration into tumor, but the T cells are rendered ineffective after two weeks, probably resulting from a surge of expression of PD-L1 on the tumor cells . This fiThis approach might also be the logical next step in thyroid cancer, given the deregulation of the MAP kinase pathway in many aggressive thyroid cancers, but little is known about the baseline characteristics of these immune regulators in thyroid malignancy. Furthermore, as clinical trials in other malignancies mature, it is becoming increasingly clear that toxicity is a major barrier to treatment. Additional preclinical data providing mechanistic insight into the possible beneficial synergistic and toxic effects of these combinations in the various tumor components is essential.+, CD4+, FoxP3+ lymphocytes and tumor associated macrophages [+ T lymphocytes to be enriched in metastatic lymph nodes especially in recurrent disease and were markedly associated with extra nodal invasion [V600E and high level of PD-L1 in 33 PTC samples was recently reported [V600E mutations, and 3 had high expression of PD-L1, there was one patient with both [The presence of PD-L1 in thyroid tumors was first described in 2003 by Brown et al. . Subsequrophages , 20. Freinvasion . Interesinvasion . Given treported . Both BRith both .V600E -mutated tumors have higher levels of PD-L1 compared with either BRAFWT tumors or matched normal tissue. Additionally, we have validated the combination of PD-L1 blockade and BRAF-targeted therapy in a novel immunocompetent model of ATC using rapidly growing genetically engineered BRAFV600E-mutated tumors. Our model is clinically relevant on multiple fronts. First, the genetically engineered cell lines have both BRAF and p53 mutations allowing for treatment to be initiated when the tumors are large and still rapidly growing. Second, the immune competent nature of our model permits us to study the immune milieu inside the tumor. Our studies did show that PD-L1 blockade has very minimal effects on the tumor when given alone, despite fairly impressive infiltration of CD8+ T cells and high activity of Granzyme B. This finding is not entirely unexpected, however, given the aggressiveness of this specific ATC model in which more conventional agents such as BRAF or Src inhibitors were less effective [In this study, we have established that thyroid cancer cell lines and tumor samples from patients with BRAFffective In contrast, the combination of PD-L1 antibody and BRAF inhibitor produced a powerful synergistic response. The synergistic improvement in tumor shrinkage in those animals treated with a combination of BRAFi and PD-L1 antibody was also associated with an increase in tumor-infiltrating lymphocytes (TIL) and TIL function. The mechanisms underlying TIL infiltration were not studied here. It could be the result of clonal expansion or increased trafficking of T cells into the tumor. Previous studies in melanoma animal models and patient trials indicate that both mechanisms are likely at play . Clearlyin vivo studies. PD-L1 expression in lymphoid organs may suppress the initial activation of T cells or perhaps even induce Tregs. PD-L1, expressed on both tumor and non-tumor cells has the potential to suppress antitumor immunity by contributing to T-cell exhaustion.Little is known about the effects of immune checkpoint regulators in thyroid cancer. The therapeutic effect of blocking either PD-1 or PD-L1 previously has not been tested. PD-L1 is expressed by multiple cell types in tumors, thus underscoring the importance of these in vitro. Both thyroid cancer cell lines and tumor samples from patients with BRAFV600E-mutated tumors had higher levels of PD-L1 compared with either BRAFWT tumors or matched normal tissue.Treatment with MAP kinase inhibitors, such as BRAFi and MEKi, can result in complex interactions given the varied effects of these inhibitors on immune cells and tumor cells Figure . In thisV600E cells, whereas in BRAFWT tumors, BRAFi activates the RAF-MEK-ERK pathway and could enhance tumor growth in xenograft models [WT) cells were treated with PLX4720 [V600E cells with BRAFi and MEKi and we activated and blocked the same pathway in BRAFWT cells with BRAFi and MEKi, respectively. We demonstrated tight correlation between MAP kinase activity (as measured by ERK phosphorylation) and the expression of PD-L1 both in the mRNA and protein levels. IFNγ, a known and potent upregulator of PD-L1, abrogated the suppression that BRAFi imposed on PD-L1, but it was MAP kinase activity that consistently correlated with fine differences of expression as exhibited in our Western blot results.Accumulating evidence points to a relationship between MAP kinase signaling activation and the induction of PD-L1 expression , 38, 39.t models , 41. Mor PLX4720 . We bloc+ T cell infiltration in melanoma patient samples [The combination of BRAFi and immunotherapy is gaining acceptance as a rational approach to cancer treatment, and is undergoing clinical investigation in melanoma , where B samples , 43 and  samples . Atefi e samples . These i+ T cell staining for cytotoxic marker. Moreover, the blockade of PD-L1 mitigated the effect of CD8+ T cells on the number of FoxP3 T regulatory cells in the tumor microenvironment. Multiple previous studies have shown that tumor-specific CD4+FoxP3 Tregs express multiple inhibitory receptors, including PD-1 and TIM3, which likely reduce the capacity of T cells in the tumor microenvironment for proliferation and cytokine production [Our previously established mouse model served as an ideal platform to explore the effect of PD-L1 blockade in the presence of BRAFi. On the basis of the above rationale, we hypothesized that a synergistic clinical and immunologic effect would be observed by combining both approaches. Indeed, our results showed dramatic tumor shrinkage when anti-PD-L1 Ab and BRAFi were combined compared to BRAFi alone. IHC analyses demonstrated an intense and dense infiltration of CD8oduction .+ cells to over express TIM3 is not clear, but its co-expression with PD-1 on tumor specific CD8+ T cells makes it an important future candidate for combined immunotherapy [It is noteworthy that PD-L1 blockade and not BRAFi resulted in higher expression of the exhaustion marker, TIM3. This observation was not consistent with Fredrick et al., who reported increased expression of TIM3 with BRAFi treatment . Ascribiotherapy –48. NeveOur study does have a number of important limitations. Although the toxicity and tolerability profile of anti-PD-1/PD-L1 in clinical trials is promising , 49, ourAdverse events (AE) related to anti PD-1/PD-L1 antibody as a single therapy and to a greater extent when combined with anti CTLA-4 or BRAFi are well documented in patients with melanoma and lung cancer , 51. MosSince previous studies have not established whether tumors that have been growing slowly over time versus those that grow more aggressively, such as in our model, alter the native immune response to the tumor, it is not entirely clear where our model fits in the spectrum. In addition, genetically engineered tumor cell lines (such as ours) may have a smaller mutation burden compared to the equivalent native tumor. It is not entirely clear whether the immune response in the genetically engineered models is a true representation of the immune response in human tumors. Since thyroid cancers generally lie on the low end of the mutation burden, this might be less of an issue in thyroid cancer than other genetically engineered models used for preclinical studies . Future in vivo model that permits exploration of the various players of antitumor immunity in the growing tumor. More than the exciting and robust immune infiltration that occurs when BRAFi and anti-PD-L1 work synergistically; it is obvious from the IHC results of our control group that aggressive thyroid tumors potentially grow without propagating anti tumor immune response. This observation, although hardly new, is striking given the robust immune infiltration when treatment with BRAFi and combinatorial therapies are implemented. Evidence from other malignancies, along with this preclinical study, suggests promise for strategies that combine immunotherapy with targeted therapy in thyroid cancer patients. The scarce number of aggressive thyroid cancer patients treated with BRAFi and the lack of ex vivo samples from these patients is a missing piece in the puzzle. We hope that in the near future, as clinical trials with targeted therapies and immune checkpoint regulators are developed for thyroid cancer, that pre and on treatment biopsy samples will be obtained from these patients to allow detailed analysis of the immune environment inside the tumors. This will allow a better understanding of the immune landscape of those who respond versus those who do not.The fact that the study of tumor immunity has gone from a fringe effort to a viable and creative option in a wide range of tumors led us to take the first exploration in aggressive thyroid cancers. Our study is unique for its utilization of an immunocompetent 2 incubator. Cells were plated at 70-80% confluence and treated for 24 hours with either 10μM PLX4720 , 10μM PD03250901 , or 500U/ml hIFNγ for human cells and 200ng/ml mIFNγ for murine cells  either as single agents or in combination as specified in the results section. At the end of the treatment, cells were collected for RNA or protein isolation. Cell characteristics and mutational status are presented in Table Six human thyroid cancer cell lines; 8505c (Deutsche Sammlung von Mikroorganismen und Zellkulturen), BCPAP , SW1736, TPC-1 , HTh-7 and HTORi; four murine thyroid cancer cell lines; 3601R, 3868, 3743, 3403 , and fouIn preparation for these studies, a dose-response curve for hIFN-γ treatment was generated and a working concentration of 500U/ml, the minimum dose required to reach a saturated response for the 24-hour treatment, consistent with previously validated concentrations (data not shown), was selected .phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (phospho-ERK), p44/42 MAPK (ERK1/2)  and β-actin were purchased from Cell Signaling Technology.The anti-PD-L1 antibody (10F.9G2) has been described previously , had lesRNA isolation was performed using Trizol , and the cDNA was synthesized using the Superscript VILO cDNA synthesis kit (Invitrogen) according to the manufacturer's instructions. PD-L1 gene expression was measured using TaqMan Gene Expression Assays  by real-time reverse transcriptase polymerase chain reaction (RT-PCR) with technical triplicates and repeated at least twice. Gene expression levels were normalized to GAPDH expression.At the end of treatment, cells were washed twice with PBS and lysed rapidly in RIPA lysis buffer containing protease and phosphatase inhibitor cocktails  by incubating on ice for 10 minutes. Total lysates were centrifuged at 15,000 G for 15 minutes at 4°C, and supernatants collected and quantified using Bradford protein assay kit . Western blot analysis was performed using standard procedure, and the proteins were identified using specific antibodies; total ERK (tERK), phosphorylated p42/44 ERK (pERK), β-actin and PD-L1.6 cells injected into the left thyroid gland using a 27-gauge needle. The right side of the thyroid gland was not manipulated and was used as an internal control. Mice were randomized and started treatment at two weeks for the 8505 and 4 weeks for the BCPAP-implanted groups . After 14 days of treatment, mice were euthanized and both fresh and formalin-fixed tumors were collected for RNA isolation and IHC analysis, respectively.All animal experiments were conducted at the Massachusetts General Hospital in accordance with federal, local, and institutional guidelines. To test PD-L1 expression and response to BRAFi therapy using human thyroid cancer cells: 24 ten-week-old female SCID mutant mice were injected with human thyroid cancer cells (12 with BCPAP and 12 with 8505c) as previously described . Briefly5 of the TBP  3747 BRAFV600E/WT P53−/− murine tumor cell line in 24 immunocompetent syngeneic B6129SF1/J mice as previously described [To test tumor growth and anti-tumoral immune indices in response to PD-L1 – PD1 blockade, with or without BRAFi therapy, we used the genetically engineered murine ATC cell line in an immunocompetent setting. First we implanted 10escribed . In thisescribed .4-μm sections of the formalin-fixed orthotropic thyroid tumors were stained with Hematoxylin and Eosin stain and underwent IHC analysis for CD8 (Leica Microsystems), FoxP3 (eBioscience), TIM3 (Millipore), Granzyme B (abcam), mouse PD-L1 (clone 10F.9G2) (Provided by GF), and human PD-L1 (ProSci-Iinc Poway CA) primary antibodies. The primary antibody was detected using the respective biotin-free secondary antibody.V600E and 12 BRAFWT tumors with their matched normal thyroid tissue samples were analyzed for PD-L1 mRNA expression.Using the IRB-approved MGH Endocrine Surgery Tissue Bank and the Endocrine Surgery Data Registry, we obtained clinical data, fresh frozen tumors, and matched normal tissues from randomly selected patients with PTCs >1 cm. A total of 16 BRAFStatistical analysis was carried out using Microsoft Excel  and IBM SPSS Statistics . BRAF mutant and wild type groups were compared using the Student t-test for age, Fisher's exact test for sex, tumor size (T1-2 vs. T3-4) and lymph node status, and Pearson chi-square for TNM stage. All experiments were performed in technical triplicate and repeated twice unless specified otherwise. A fold change in mRNA expression of 2 or more was considered significant. A log power of 2 for fold changes in expression was used to generate box-and-whisker plots based on the median and inter quartile range, and the nonparametric Mann-Whitney U- test was used to compare independent groups. Two-way factorial analysis of variance (ANOVA) was applied to test the effects of treatments on reduction in tumor volume with the Wald chi-square test to assess the additive vs. supra-additive (synergistic) effect of two-drug combinations. Two-tailed values of P < 0.05 were considered significant for all analysis.


Comprehensive microscopic and spectroscopic characterizations confirm the resulting hybrid material possesses a 3D crumpled few-layered graphene network structure decorated with MoS2 nanoparticles. Electrochemical characterization analysis reveals that the resulting hybrid material exhibits efficient electrocatalytic activity toward HER under acidic conditions with a low onset potential of 112 mV and a small Tafel slope of 44 mV per decade. The enhanced mechanism of electrocatalytic activity has been investigated in detail by controlling the elemental composition, electrical conductance and surface morphology of the 3D hybrid as well as Density Functional Theory (DFT) calculations. This demonstrates that the abundance of exposed active sulfur edge sites in the MoS2 and nitrogen active functional moieties in N-RGO are synergistically responsible for the catalytic activity, whilst the distinguished and coherent interface in MoS2/N-RGO facilitates the electron transfer during electrocatalysis. Our study gives insights into the physical/chemical mechanism of enhanced HER performance in MoS2/N-RGO hybrids and illustrates how to design and construct a 3D hybrid to maximize the catalytic efficiency.An efficient three-dimensional (3D) hybrid material of nitrogen-doped graphene sheets (N-RGO) supporting molybdenum disulfide (MoS Monolayer metallic MoS2 with high conductivity have been explored for HER catalyst172 chemically exfoliated via lithium intercalation from semiconducting 2H-MoS2 nanostructures grown directly on graphite was reported as a highly competitive earth-abundant catalyst due to favorable kinetics, metallic conductivity, and proliferation of active sites2 materials with conductive templates or supports to form a multidimensional structure has been recently presented as an effective approach to improve the catalytic activity2 can be implemented by incorporating MoS2 with carbon nanotube (CNTs), graphene, graphite or other mesoporous carbon to form a MoS2-based hybrid or composite20212223et al. developed an HER advanced catalyst of MoS2 nanoparticles grown on graphene via a facile solvothermal approach, the resulted highly exposed edges and excellent electrical coupling to the underlying graphene sheets endowed the hybrid catalyst with excellent HER activity233N4 nanolayers@N-graphene films by integrating porous C3N4 nanolayers with nitrogen-doped graphene sheets, displaying an unbeatable HER performance which stems from highly exposed active sites, hierarchical porous structure and 3D conductive graphene network2 nanoplatelets grown on the n-type nitrogen-doped reduced graphene oxide (N-RGO) can form p−n junctions on nanoscale and act as an outstanding catalyst in photocatalytic HERHydrogen has emerged as an effective alternative to fossil fuels because it is environment-friendly energy with water as exhaust2 nanoparticles decorated nitrogen-doped reduced graphene oxide (N-RGO), a three-dimensional (3D) hybrid structure with macro-porosity by a hydrothermal route, which can efficiently catalyze HER under acidic conditions with low overpotential and small Tafel slope. The resulting MoS2/N-RGO hybrid materials display high-performance HER catalytic activity. By controlling the elemental composition, electrical conductance and morphology of the as-prepared 3D MoS2/N-RGO hybrid, the enhanced electrocatalytic activity mechanism toward hydrogen evolution was investigated in detail.Herein, we report MoS4)2MoS4 were sonicated in N, N-dimethylformamide (DMF) to generate a highly dispersed suspension. Hydrazine and polypyrrole (PPy) were then slowly added into the suspension, followed by hydrothermal assembly at 180 °C for 12 h. Besides as a reductant, hydrazine could work as a nitrogen source as PPy during the assembly process. Regarding to PPy, it acted not only as the nitrogen source, but also was used to fabricate 3D network structure which promoted the BET specific surface and conductivity of the final product. The hydrothermal environment plays a significant role in the nitrogen doping because in the absence of hydrothermal environment, the GO could be reduced directly without the incorporation of nitrogen into the graphene network. During the hydrothermal process, (NH4)2MoS4 and GO were reduced to MoS2 nanoparticles and RGO respectively by hydrazine; and MoS2 nanoparticles were grown on the RGO accompanied by incorporation of nitrogen species into the graphene lattice4)2MoS4 is removed from the starting materials; MoS2’/N-RGO where nitrogen-doped graphene sheets decorated with half quantity of MoS2; MoS2/N’-RGO where the addition order of hydrazine and PPy is exchanged; MoS2/N-RGO’ is the sample fabricated by GO of higher oxidation degree.Firstly, graphene oxide (GO) and (NH2/N-RGO synthesized was comprehensively characterized by a number of microscopic and spectroscopic tools. Scanning electron microscopic (SEM) images revealed a 3D network structure with crumpled few-layered graphene sheets with plenty of folded edges conglutinated by PPy and decorated by numerous MoS2 nanoparticles with a lateral dimension of about 35 nm (2nanoparticle growth and created an efficient template to restrain the aggregation of particles. The information presented by the transmission electron microscopic (TEM) images was consistent with the SEM in that there were laminar structures of graphene in the catalyst and MoS2nanoparticles were evenly distributed on N-RGO sheets, suggesting successful assembly between the nanoparticles and the graphene sheets  assigned to the N-RGO can also be observed. Notably, a distinguished and coherent interface was observed in MoS2/N-RGO, which is known to facilitate the electron transfer to enhance catalytic activityThe 3D MoSut 35 nm . The lare sheets . The higinset in . The two2/N-RGO hybrid. As shown in 2/N-RGO hybrid shows the characteristic peaks of N-RGO; including a prominent graphitic C 1 s peak at 284 eV, a strong O 1 s peak at 532 eV and an N 1 s peak located at 402 eV. The peaks assigned to Mo 3d and S 2p in MoS2 were also present in the survey spectrum, which confirmed the successful assembly between the nanoparticles and the graphene sheets. The detailed information of elemental composition is presented in 2. It is worthy mentioning that the N/C atomic ratio of the 3D MoS2/N-RGO hybrid was calculated up to be 5.9%, much higher than that of N-doped graphene from previous reports31322/N-RGO hybrid could lead to a more efficient catalytic activityX-ray photoelectron spectroscopy (XPS) measurements were performed to investigate the components of the prepared 3D MoS2/N-RGO hybrid. The binding energy of C-C/C=C (284.5 eV) in the 3D MoS2/N-RGO hybrid was downshifted by ∼0.5 eV compared to the graphitized carbon peak of pristine GO (285.0 eV)2, which can improve both the current density and catalytic activity of the catalyst. The N 1 s region shown in 352H4In the high resolution XPS of C 1 s , a promi4+ 3d5/2, Mo4+ 3d3/2, MoO3 or MoO42−, Mo5+ and S 2 s, respectively, are displayed in the high resolution XPS of Mo 3d5/2 and Mo 3d3/2 peaks are close to the meta-stable 1 T phase of MoS2 (associating with better HER performance)406+ results from the oxidation of the catalyst in air to form MoO3 or MoO42−6+ results in the formation of Mo5+3/2, S 2p1/2, S22− (or S2−) ligands and the S4+ of sulfate groups, respectively404322− and/or S2− is likely to be caused by the doping of S in the graphene sheet or formation of an S-rich MoS2 structure. This facilitates the development of high HER activity at the active sites for HER on the edge of MoS2where the unsaturated S atoms are exposed to adsorb H74043As shown in 2/N-RGO hybrid were characterized using the nitrogen isotherm (2 adsorption-desorption isotherm of 3D MoS2/N-RGO hybrid showed an intermediate shape between types II and IV (in the IUPAC classification) with a small H3 hysteresis loop extending from P/P0 = 0.45 to 0.95, demonstrating the presence of mesoporous structure and slit-shaped pores2 adsorption-desorption measurement revealed the as-prepared 3D MoS2/N-RGO hybrid had a Brunauer-Emmett-Teller (BET) specific surface area of 1066.6 m2 g−1, which was significantly larger than many reported porous carbon nanomaterials4647482/N-RGO hybrid materials and formation of numerous porous structures during the sintering and elimination process in the presence of PPy. The pore-size distribution of the 3D MoS2/N-RGO hybrid calculated by the DFT method exhibited one dominant peak at 5.30 nm and another two weak peaks at ∼1.56 and 3.79 nm, respectively , (103) and (110) of MoS2 could be observed in MoS2/N-RGO, indicating the MoS2 nanoparticles have been successfully grown on the N-RGO surface. The disappearance of the typical (002) peak of MoS2located at 14.1° resulted from the small size of the MoS2 nanoparticles preferring to attach to the surface of the silicon supports5051The porosity structures of the 3D MoSisotherm . The N2 Inset of , suggest2/N-RGO hybrid was further confirmed by Raman spectra analysis. As shown in −1 associated with the D and G bands of graphene can be observed in GO, N-RGO and MoS2/N-RGO. The reduction of GO induces the recovery of the graphene structure, which leads to the increase of 2D bands in N-RGO and MoS2/N-RGO. Notably, compared to the GO with a G band at 1599 cm−1, the G band of N-RGO (1586 cm−1) and MoS2/N-RGO (1593 cm−1) showed a downshift due to the incorporation of N heteroatoms2 located at 375.5 and 403.5 cm−1  were observed in the Raman spectrum of MoS2/N-RGO ranging from 300 to 450 cm−1  suggested that the interaction between the Mo precursors and graphene could efficiently avoid the aggregation of MoS2The successful fabrication of the 3D MoS450 cm−1 53. In co2/N-RGO hybrid toward the HER was investigated in 0.5 M H2SO4 solution with commercial Pt catalyst (10 wt % Pt on carbon black) as the reference. N-RGO, MoS2’/N-RGO, MoS2/N-RGO, MoS2/N’-RGO and MoS2/N-RGO’ were also prepared to investigate in detail the influence of composition and structure to the HER catalytic activity. XPS , MoS2/N’-RGO (155 mV) and MoS2/N-RGO’ (200 mV) showed more negative onset potential, indicating lower HER activity. The current intensity of the 3D MoS2/N-RGO hybrid was higher than the other catalysts along the whole potential region. To further investigate the HER activity, the linear portions of the Tafel plots were fitted to the Tafel equation (η = a + b log |j|), where j is the current density and b is the Tafel slope. In 2/N-RGO hybrid showed a Tafel slope of 44 mV per decade, which outperformed MoS2’/N-RGO of 114 mV per decade, MoS2/N’-RGO of 71 mV per decade and MoS2/N-RGO’ of 57 mV per decade. The slight higher Tafel slope compared to MoS2 nanoparticle on graphene with a value of 41 mV2 nanosheets on graphite with a value of 43 mV2/N-RGO hybrid. Electrochemical impedance spectroscopy (EIS) was measured to evaluate the HER catalytic activity of these catalysts. The MoS2/N-RGO displayed the lowest faradaic impedance among these prepared catalysts , which was larger than MoS2’/N-RGO and MoS2/N’-RGO, which lead to better reactant accessibility and superior efficiency of build of the triple-phase boundaries (gas–electrode–electrolyte)582 582  and MoS2ted MoS2  displaye2 and nitrogen impurities in RGO boost the activity of the catalyst. The relative content (RC) of S22− and/or apical S2−related to active HER catalytic activity, where RC is mean that sulfur elemental compositions (wt %)  calculations showed that graphite N shows the smallest Gibbs free-energy (|∆GH*|) value of 0.76 eV, followed by pyrrole N of 1.10 eV and pyridinic N of 1.73 eV, indicating graphite N was the best electrocatalytic activity from the viewpoint of thermodynamics and pyrrolic N also possessed the enhanced electrocatalytic activity than pyridinic N toward HER   multiplys (wt %) , in MoS22’/N-RGO , MoS2/N-2/N-RGO’ , while t2/N’-RGO  was not ydrazine . The MoS2/N-RGO’ , it was ward HER . Additioward HER , larger ward HER , which l2/N-RGO hybrid were characterized by comprehensively microscopic and spectroscopic methods including SEM, TEM, HRTEM, BET, XRD, XPS and Raman spectroscopy. Electrochemical characterization data including low onset potential, small Tafel slope, low charge transfer resistance and high electrochemical stability revealed the high performance of the MoS2/N-RGO hybrid toward HER catalytic activity compared to analogous composites. Detailed experimental analysis implies that the abundant active S22− and/or S2− ligand species and pyrrolic nitrogen and graphene N of the 3D MoS2/N-RGO hybrid and its good conductance were all beneficial to its enhanced HER catalytic activity. The exploration of the enhanced HER mechanism of the MoS2/N-RGO hybrid provides guidelines to design and construct 3D hybrids to maximize their catalytic efficiency.In summary, we have developed an efficient hydrothermal route to successfully synthesize a 3D hybrid material of nitrogen-doped graphene oxide sheets-supported molybdenum disulfide nanoparticles with high capability of hydrogen evolution. The morphology and element of the as-generated 3D MoS2H4·H2O, 80%) was purchased from Guangdong Guanghua Sci-Tech Co., Ltd (JHD). (NH4)2MoS4, Polypyrrole  and Pt/C (10% Pt) were obtained from Sigma-Aldrich. N, N-dimethylformamide  and KOH were purchased from Sinopharm Chemical Reagent Co., Ltd. Sulfuric acid  and ethanol (99.9%) was acquired from Beijing Chemical Works. All aqueous solutions were prepared with doubly distilled water.Graphene oxide  was obtained from XF NANO, INC and graphene supermarket, respectively. Hydrazine monohydrate (N4)2MoS4 and 10 mg of GO was dispersed in 10 ml of DMF by 10 min of sonication at room temperature. Then 0.1 ml of N2H4•H2O was added to the above solution followed by 30 min of sonication. 100 mg PPy was then added, followed by sonication for 10 min to form a stable complex solution. The mixture solution was transferred to a 40 mL Teflon-lined autoclave and heated in an oven at 180 °C for 12 h with no intentional control of ramping or cooling rate. The product was collected by centrifugation at 10000 rpm for 5 min and washed with DI water several times to remove most of the DMF. Subsequently, the product was redispersed in 3 ml of DI water and freeze-dried overnight, followed by thermal treatment at 600 °C for 3 h in N2 gas with 400 standard cubic centimeters per minute (sccm) to remove the organic species (DMF) and improving crystallinity. The obtained product was denoted as 3D MoS2/N-RGO. Other samples were also fabricated in the same way for comparison: N-RGO where (NH4)2MoS4 is removed from the starting materials; MoS2’/N-RGO where Nitrogen-doped graphene sheets decorated with half quantity of MoS2; MoS2/N’-RGO where the addition order of hydrazine and PPy is exchanged; MoS2/N-RGO’ is the sample fabricated by GO of higher oxidation degree.22 mg of (NHDLD instrument equipped with an Al Kα X-ray source. Raman spectrum of powder samples were recorded on an InVia-Reflex Raman microscope with a laser excitation wavelength of 532 nm.X-ray diffraction (XRD) was performed with a Rigaku X-ray diffractometer with Cu KR target. The porosity was measured with a nitrogen adsorption-desorption isotherm using a surface area analyzer . The structure of products was observed under a field-emission scanning electron microscope  and a transmission electron microscope . X-ray photoelectron spectroscopy (XPS) spectra were obtained using an AXIS ULTRA2). After the catalyst ink dried, 5 μl of 1 wt% Nafion solution was dropped onto the GCE and the working electrode was prepared. Liner sweep voltammetry (LSV) was conducted in nitrogen-purged 0.5 M H2SO4 at a scan rate of 3 mV s−1 at 1400 rpm. Electrochemical impedance spectroscopy (EIS) was carried out in the same configuration in nitrogen-purged 0.5 M H2SO4 from 10−2 to 106 Hz with a modulation amplitude of 5 mV. SCE was calibrated with respect to reversible hydrogen electrode (RHE). The calibration was performed in the high purity H2 saturated electrolyte with a Pt wire as the working electrode and the counter electrode. LSV were run at a scan rate of 0.1 mV s−1, and the potential at which the current crossed zero was taken to be the thermodynamic potential for the hydrogen electrode reactions. In 0.5 M H2SO4, E (RHE) = E (SCE) + 0.314 V. All the potentials reported in our manuscript are against RHE.All electrochemical studies were performed using a CHI 852C electrochemical workstation  in a standard three-electrode setup. A three-electrode configuration consisting of a saturated calomel electrode (SCE) as the reference electrode, a graphite rod as the counter electrode and a glass carbon RDE after loading the catalyst as the working electrode was employed. Typically, 0.5 mg of catalyst was dispersed in 500 μl of DI water by sonication to form a homogeneous ink. Then, 20 μl of the catalyst ink  was loaded onto a glassy carbon electrode (GCE) of 3 mm in diameter .


Resistive random access memory (ReRAM) devices have been extensively investigated resulting in significant enhancement of switching properties. However fluctuations in switching parameters are still critical weak points which cause serious failures during ‘reading’ and ‘writing’ operations of ReRAM devices. It is believed that such fluctuations may be originated by random creation and rupture of conducting filaments inside ReRAM oxides. Here, we introduce defective monolayer graphene between an oxide film and an electrode to induce confined current path distribution inside the oxide film, and thus control the creation and rupture of conducting filaments. The ReRAM device with an atomically thin interlayer of defective monolayer graphene reveals much reduced fluctuations in switching parameters compared to a conventional one. Our results demonstrate that defective monolayer graphene paves the way to reliable ReRAM devices operating under confined current path distribution. Nowadays one of the most widely used nonvolatile memories is flash memory. It has been employed to numerous mobile devices and becomes a representative product supplied by the Si based semiconductor industries. The flash memory is expected to reach limitations in operating speed, power consumption, and density of memory in near future because it is a charge-storage type memory based on a Si transistor. To overcome the limitations, many researchers have tried to develop next generation nonvolatile memories (NG-NVM) with high performances, which do not rely on stored charges and Si transistors123ReRAM whose resistive change is induced by applied external electrical stress is considered as one of these NG-NVMs. In addition to the advantageous properties of oxide-based ReRAM such as simple composition, facile fabrication process, and compatibility with conventional semiconductor processes, this resistance-change memory has presented promising nonvolatile memory effects including fast operation speed, low power consumption, and high scalability5678Resistive switching can be classified into two categories: Uni-polar and bi-polar resistive switching caused by conducting filament formation in bulk and oxygen migration at interface, respectively. Especially, uni-polar resistive switching, which is usually observed in binary oxide, has been explained by conducting filament (CF) mechanism in switching materials. The creation and rupture of CFs presumably result from Joule heating910111213161920+ ion-assisted reaction (IAR) system, which is monitored by Raman spectroscopy24In this paper, to reduce fluctuation in switching parameters, we suggest the insertion of a highly defective graphene (d-graphene) monolayer between an oxide film and an electrode. Because localized electronic structure of graphene can be modified by ionic treatments2223242/Si substrate using chemical vapor deposition (CVD) method . In Raman spectroscopy data from honeycomb lattice of graphene, three most considerable features are the G, 2D, and D peaks, which appear around 1580 cm−1, 2700 cm−1, and 1340 cm−1, respectively. Our Raman data only shows G and 2D peaks with G/2D peak ratio of about 0.5, which are very similar with those of intrinsic MLG2427282925+ ion beam was generated by a cold hollow cathode-type ion source. Generated number of Ar+ ions is measured by Faraday cup placed at a distance of 50 cm from the ion source. Working pressure during Ar+ ion bombardment is kept at 0.1 mTorr with Ar flow rate of 5 sccm.Before we fabricate a MGIM structure, we need to confirm quality of our graphene. Raman spectroscopy analysis in + ions at kinetic energies of 240 eV, 250 eV, 260 eV, and 270 eV, respectively, with number of bombarded Ar+ ions about 5 × 1014 per unit area (cm2) and time (second). The 240 eV of applied kinetic energie is minimum value of stably controllable area in our experimental system. Relative defect concentrations in the pristine and defective graphenes can be compared using Raman spectroscopy data, as shown in + ion bombardment induces the breaking of carbon-carbon bonds in graphene honeycomb structure31242633+ ions. To identify the location of defects, we obtained Raman scanning microscopy images of D/G peak ratio in 5 μm × 5 μm scanning areas of two d-graphenes irradiated with Ar+ ions at kinetic energies of 240 eV and 270 eV, as shown in the upper and lower panels of + ions at kinetic energies of 240 eV and 270 eV, respectively. The observed bright spots designate the positions of conducting paths where higher local current (~50 pA) passes through d-graphenes than that (~1 pA) of the other regions. + ion irradiation with lower kinetic energy induces less conducting paths on d-graphene than that with higher kinetic energy. As Jafri et al. mentioned, the higher concentration of conducting paths on d-graphene with more defects can be attributed to the defect induced mid-gap states, which create a region exhibiting metallic behavior around the vacancy defects on grapheneWith attempt to provide confined current path distribution, we introduce defects on graphene by bombarding it with Ar2/Si substrate using dc reactive sputtering method with the same condition of our previous works 3637383940414243To fabricate a MGIM structure, d-graphene introduced in forming), measured current abruptly increases and the structure reaches a low resistance state (LRS). During the following voltage sweep with step voltage of 0.05 V from 0 V, switching from LRS to HRS and switching from HRS to LRS occur at Vreset and at Vset, respectively. Compliance current of 1 mA is considered as a minimum value which can allow forming and reproducible set process in our NiO capacitor systems. The MGIM structure initially shows higher Vset value and lower current level of HRS than those of the MIM structure owing to the additional resistance from the inserted d-graphene interlayer. + ions at kinetic energies of 240 eV (MGIM240), 250 eV (MGIM250), 260 eV (MGIM260), and 270 eV (MGIM270) as well as a MIM structure. The cumulative probability for each structure was obtained after over 200 switching cycles. The distributions of the switching voltages, Vset and Vreset, are important parameters indicating memory device performances. According to the cumulative probability data, we can confirm that the switching voltage distributions of MGIM structures are narrower than that of a conventional MIM structure. Especially, MGIM240 shows much narrower distributions of Vset  of 0.32 V) and Vreset  than those of Vset  and Vreset  in the MIM structure. Due to such wider distribution, Vset of the MIM occasionally becomes higher than that of the MGIM. MGIM240 also shows the narrowest distributions in the cumulative probability plots of resistance values in HRS and LRS states  and after (less-residue MGIM240) transfer of the MLGs on NiO films. Less-residue MGIM240 shows narrower distribution of Vset  than that for MGIM240 due to the removal of residues on the MLG during Ar+ ion bombardment although distribution of Vreset  for less-residue MGIM240 is comparable to that for MGIM240. Each resistance state for less-residue MGIM240 is stable similarly to that for MGIM240, as shown in In previous reports, graphene devices have shown suppressed performances than those predicted by theory owing to residues, ripples, vacancies, etc4546+ ions bombarded on MLG/NiO may induce defects in the NiO layer as well as on the MLG. According to a previous study about ReRAM treated using IAR+ bombardment. It was found that the forming voltage could be controlled by modification of surface roughness and oxygen vacancy concentration of an oxide layer, which depended on kinetic energy of the bombarded Ar+ ion. However, in our case, the existence of MLG interlayer might reduce the effect of Ar+ bombardment on the underlying NiO layer. To demonstrate the protection role of graphene, we fabricated MIM structures with defective NiO layers directly bombarded by Ar+ ions at kinetic energies of 240 eV (DMIM240), 250 eV (DMIM250), and 260 eV (DMIM260).During the modified fabrication process, Ar+ ion increases, they are comparable to those of MIM. In contrast, DMIM240 reveals larger fluctuations in switching voltages and currents than those of less-residue MGIM240 with d-graphene, as shown in + ion bombardment.+ ions at kinetic energy of 240 eV has lower conducting path concentration than that of 270 eV leading to more reduced randomness during formation and rupture of CFs in an oxide layer underneath a d-graphene. To vary the number of defects on graphene, we fabricated two MGIM240 structures in which graphenes are bombareded with different numbers of Ar+ ions: 3 × 1014/cm2 and 8 × 1014/cm2  simply modified by insertion of a defective graphene layer between a top electrode and on oxide layer. This atomically thin interlayer inserted into an MIM device can reduce randomness during formation and rupture of conducting filaments by confined current path distribution in the modified system. MGIM structures show much narrower range of V2/Si substrate. During the growth process, the substrate is heated to 650 °C within 10 min under ~10−7 torr and then treated with H2 plasma. After purging with Ar gas for a couple of minutes, C2H2 is added (C2H2:Ar = 1:40) for graphene growth at the same temperature. A 35 nm thick polycrystalline NiO thin film was prepared by dc reactive sputtering method on Pt/Ti/SiO2/Si substrate .Monolayer graphene was synthesized by inductively coupled plasma enhanced chemical vapor deposition (ICP-CVD) on a Cu/Ni/SiO2/Si was spin-coated with polymethyl methacrylate (PMMA) 950 C4 on which we attached a pressure sensitive adhesive ultraviolet tape. Peeling the tape against the Si wafer physically separated the tape/PMMA/graphene/Cu/Ni layer due to poor adhesion of the metal films to SiO2. After etching of the underlying Cu/Ni by soaking in FeCl3 and cleaning in water, the tape/PMMA/graphene layer was pressed onto the NiO/Pt/Ti/SiO2/Si. The successive removal of the tape and PMMA in ethanol and acetone, respectively, left only the graphene layer on the NiO/Pt/Ti/SiO2/Si.For the monolayer graphene transfer, graphene/Cu/Ni/SiOMost electrical properties of the MGIM device (such as resistive switching characteristics and fluctuations of the switching parameters) were measured using a Keithly 2400 sourcemeter at room temperature and atmospheric pressure, but all retention measurements were performed additionally at 85 °C and 1 mTorr. The electrical data were obtained on a single device for each kind of structure, which showed the best performance among several ten devices. The Raman spectroscopy measurements were performed at room temperature with a 532 nm diode-pumped solid state laser and a microscope setup with a laser spot diameter of 200 nm.How to cite this article: Lee, K. et al. Enhancement of resistive switching under confined current path distribution enabled by insertion of atomically thin defective monolayer graphene. Sci. Rep.5, 11279; doi: 10.1038/srep11279 (2015).


Cultured cells were characterized by immunophenotyping. Before and after administration of the agents, the cultures were observed by inverted and environmental scanning electron microscopy (ESEM). The number of live cells and the proliferation rate were monitored by MTT assay.Rituximab and adalimumab were the least toxic agents to chondrocytes, whereas adalimumab and etanercept were to osteocytes.During periods of intense active inflammation, the concentration of the preferred BAs after inhibition of inflammation needs to be emphasized when their effects on cartilage and bone tissue are considered at the cellular level if the clinical practice is to continue. Rheumatic diseases are systemic inflammatory conditions that may cause severe joint degeneration and are associated with high morbidity and mortality rates . In receDisease-modifying anti-rheumatic drugs are declared as the traditional treatment of rheumatic diseases and, even if these drugs reduce the symptoms of the disease in a long-term, they have limited effects to terminate the disease completely . Involvein vitro). Using these methods, we aim to evaluate the toxicity and effects of the BAs on the viability and proliferation of the cultured cells.In the present study, we aim to observe the effects of three TNF inhibitors, etanercept (ETA) (soluble TNF receptor fusion protein (p75-IgG fusion protein)), infliximab (INF) , and adalimumab (ADA) , and two IL-1 antagonists, abatacept (ABA) (CTLA4-IgG1 fusion protein) and rituximab (RIT)  was approved by the Local Scientific Research Ethics Committee of Gulhane Military Medical Academy (GMMA) on February 27, 2014, with the authorization number 1491–29–14/1539. To perform molecular analyses on cellular material, written informed consent was obtained from patients. Specific BAs were numbered and researchers were blinded to each specific BA added to each culture medium. All experiments were carried out at least three times.n = 6, mean age 65 years; stage 4 gonarthrosis according to the Kellgren-Lawrence protocol ). RIT allowed chondrocyte proliferation up to 36 h (p < 0.01 [p = 0.0000]). In the same period of time, the viability rate of chondrocytes in the ADA group was similar to the RIT group (p < 0.01 [p = 0.0000]). In the ETA, ABA, and INF groups, the number of live chondrocytes was lower than that of the RIT, ADA, and control groups, suggesting that ETA, ABA, and INF inhibited chondrocyte proliferation (p < 0.01 [p = 0.0000]). Proliferation analysis performed 48 h after BA application showed no living cells or proliferation in all groups, other than the control group (p < 0.01 [p = 0.0000]).At 24 h, in the RIT group, more viable chondrocyte cells were present, compared to the other groups (p < 0.01 [p = 0.0000]). From the results of the proliferation test, which was made at the 24th hour, it was clear that proliferation occurred in ADA and ETA groups at 36th hour (p < 0.01 [p = 0.0000]).In the osteocyte cultures, the number of live cells was the greatest in the ADA group, followed by the ETA group, 24 h after BA application, compared to the control and all other groups (p < 0.01 [p = 0.0000]).The viability rate of osteocytes in the ABA, INF, and RIT groups was found to be very low compared to that in the control, ADA, and ETA groups, suggesting that these BAs inhibited osteocyte proliferation (p < 0.01 [p = 0.0000]).The results of proliferation test at the 60th hour showed no proliferation in all BA groups, while proliferation in the control group was continued , INF , and ADA , and two IL-1 antagonists, ABA (CTLA4-IgG1 fusion protein) and RIT  on human primary chondrocyte and osteocyte cultures. An additional aim was to evaluate the toxicity and effects of these BAs on the viability and proliferation of the cells in question.Owing to difficulty in isolating osteocytes from the bone tissue, characterization of osteocytes and chondrocytes via immunoflow cytometry was performed before their use in the experiments. Immunophenotypic characterization of these cells is usually performed on expanded cells rather than primary cell cultures. Although no perfect marker has been defined for cells grown in culture, it is known that hematopoietic markers, such as CD34, CD14, and CD45, are not expressed. CD44, a receptor for ligands such as hyaluronan and osteopontin, is a marker of osteocytes, but there is no such a specific receptor for chondrocytes , 34. ForPharmacological alternatives of BAs used in this study, including bevacizumab, ranibizumab, aflibercept, and Ziv-aflibercept were studied on different tissues, and mild mitochondrial toxicity was reported . FurtherThe cases included in the study were those which confirm that the toxicity in osteocytes and chondrocytes was a result of BA therapy and not a patient-specific cellular response; we ensured that the patients from whom the tissue samples were provided were not exposed to methotrexate, fludarabine, cyclophosphamide, high-dose steroids, and/or an anti-pneumococcal vaccination, and that they did not have protein allergies or a history of RA.p < 0.01 [p = 0.0000]).In the present study, the data obtained at the 24 h showed that the number of live cells and the rate of chondrocyte proliferation were highest in the RIT group, and that ABA and INF were the most toxic to chondrocytes. However, at the 48 h, neither viable cells nor proliferation were observed in all groups, except the control (p < 0.01 [p = 0.0000]).At the 24 h, the highest number of viable osteocytes was in the ADA group, followed by the ETA group. But, viability rates in the ABA, INF, and RIT groups were lower. Based on the proliferation analyses at the 48 h, similar to the chondrocyte cultures, there were no viable osteocytes and proliferation in all groups, except for the control. Osteotoxicity was highest in the RIT group, followed by the INF group . Tested doses are determined based on the previous studies and systemic peak levels of patients. Our data indicate that chondrotoxicity and osteotoxicity of BAs should be taken into consideration on the treatment regimen during active inflammation periods.RIT and ADA were found to be the least toxic biologics for cartilage, whereas ADA and ETA were the least toxic for bone cells (