In immunocompetent mouse models, blocking PD-L1 induced a longer tumour growth delay following irradiation. The inhibition of T cell functions including proliferation and cytotoxicity against tumor cells was responsible to the effects of PD-L1 on radiation response. In conclusion, PD-L1 could be a significant clinical predictor for clinical stage and treatment response of bladder cancer.Identification of potential factors that can stratify a tumor’s response to specific therapies will aid in the selection of cancer therapy. The aim was to highlight the role of programmed cell death 1 ligand 1 (PD-L1) in bladder cancer. In this study, 92 of muscle-invasive bladder cancers and 28 of non- muscle invasive bladder cancers were selected for immunohistochemical staining analysis. Furthermore, human and murine bladder cancer cell lines were used to examine the correlation between PD-L1 and radiation response. Our data revealed that PD-L1 was overexpressed in the bladder tumor specimens compared with adjacent non-malignant specimens. Furthermore, the staining of PD-L1 was significantly linked to higher clinical stage, lower complete response rates and reduced disease-free survival rates. By  Bladder cancer is a significant public health issue worldwide and manifests itself in two distinct forms with different clinical and biological behaviors3Tumor-induced immune suppression in cancer patients is a major issue that not only promotes tumor progression but also inhibits the efficiency of anti-cancer treatment579111213141617Bladder tissue specimens retrospectively collected from 65 patients with MIBC  were constructed into TMA. IHC staining of TMA slides demonstrated that PD-L1 was overexpressed in the tumour tissues of 40 patients (61%) compared with adjacent non-malignant epithelial tissues . Figure in vitro. For human bladder cancer cell line HT1197, RT increased the expression of PD-L1 in tumour cells compared with nontreated cells, especially at 48h, and the increased levels of this protein were positively associated with the irradiation dose  with or without anti–PD-L1. The results shown in As shown in We analysed the recruitment of CD8+ T cells in tumours using irradiated mice treated with or without anti-PD-L1. The data in Malignant tumors possess mechanisms for evading host immune responses. A novel mechanism that tumor may evade host immune response through the expression of PD-L1. PD-L1 expression has been observed in a wide variety of solid malignancies, and might be correlated with the higher malignant grade of tumours, increased tumour growth, and the patients’ prognoses101920in vitro. The upregulation of PD-L1 was also observed in irradiated tumours in mice, suggesting that the alteration of PD-L1 levels in tumour microenvironments might play a role in the radiation response of bladder cancer. To address this issue, we combined irradiation with anti-PD-L1 antibody therapy to treat murine MB49 bladder cancer in immunocompetent animal models. The data revealed that the anti-PD-L1 antibody sensitized bladder cancer to radiation, as demonstrated by a longer tumour growth delay for ectopic tumors. Furthermore, by mouse orthotopic models, blocking PD-L1 augmented the RT-induced tumour cell death associated with decreased tumor cell proliferation compared with tumours after radiation therapy alone.Emerging evidence suggests that the generation of antitumor immune responses might play an important role in the effectiveness of radiotherapy22911in vitro. The data revealed that tumor cells’ PD-L1 expression was triggered by radiation. Blockade of PD-L1 effectively inhibit CD8+ T cells cytotoxicity against irradiated bladder cancer cells. Furthermore, we demonstrated that the increase in PD-L1 expression on irradiated bladder cancer cells was associated with a decreased T cell proliferation upon co-culture experiments, and anti-PD-L1 had reduced the suppressive ability for T cell proliferation. Thus, these results showed that PD-L1 expression negatively correlated with the numbers and function of cytotoxic T cells, which linked to tumor irradiation response demonstrated by the immunocompetent animal model. Therefore, we suggested that PD-L1 affects the radiation response of bladder cancers mediated by regulating the tumor microenvironment in the immunocompetent host.Radiation increased the T cell recognition of irradiated tumour cells, making the cells vulnerable to cytotoxic T lymphocyte-mediated clearance242628For the interpretation of our results, different limitations have to be taken into account. First, we examined PD-L1 expression mainly in biopsy tissue specimen from TURBT to evaluate its prognostic value. Second, the patient population enrolled in the retrospective study are heterogeneous with relatively small group sizes. Thus, further investigations that included more patients in a prospective trial are needed.The ultimate cancer treatment would leverage different cytotoxic pathways for the complete eradication of tumours. The data indicated that PD-L1is a predictor of treatment response and loco-regional recurrence for bladder cancer. Therefore, measured PD-L1 expression levels in bladder cancer excision tissues provides further data stratifying patients to higher risk disease and allow selection for personalized treatment.This study was approved by the Institutional Review Board of Chang Gung Memorial hospital, and performed in accordance with the approved guidelines. Informed written consent for the acquisition and storage of medical information and tissue specimens was obtained from all of the patients. There were 72 patients with histopathological proven muscle invasive urothelial cell carcinoma who completed definite chemoradiotherapy (CCRT) treatment enrolled in the study. On completion of definite CCRT, patients underwent abdomen CT, cystoscopy and urine cytology to assess the treatment response and disease status every 3 to 4 months for the first 2 years, and then every 6 months during follow-up. The median follow-up time was 3.8 years. Clinical CR was defined as no tumor visible on cystoscopy, negative tumor site biopsy, and negative urine cytology after the completion of CCRT. The identification of clinical lymph node (LN) involvement with CT was based on measurements of node size, with greater than 1 cm being the criterion. Specimens were retrospectively collected from the 72 patients for immunochemical analysis. The end points were overall survival (OS), disease-free survival (DFS) and the response to definite CCRT. The loco-regional recurrence was defined by tumor progression in bladder or pelvic nodes assessed with CT and cystoscopy. Survival probabilities were analyzed using the Kaplan-Meier method.Formalin-fixed, paraffin-embedded bladder tissues collected from TURBT at diagnosis and/or radical cystectomy for bladder cancer were subjected to immunochemical analyses. These 120 urothelial cell carcinoma patients in HT1197, a human bladder cancer cell line, was obtained from the American Type Culture Collection (ATCC). We maintained the bladder cancer cells in DMEM supplemented with 10% foetal bovine serum. Mouse bladder carcinoma MB49 cells were kindly gifted from Dr Yi-Wen Liuin vivo. In the ectopic tumour implantation model, 1 × 106 tumour cells were subcutaneously implanted by injection into the dorsal gluteal region . In the orthotopic tumour implantation model, we performed intravesical instillation of cancer cells as previously describedin vivo, local irradiation to 12 Gy was performed when the ectopic tumours reached 0.5 cm3 or at 2 weeks after orthotopic tumour implantation. Radiosensitivity was indicated as growth delay . Duplicate experiments were performed for growth delay analyses. To evaluate the effect of PD-L1 blockade on the radiation response and immune cells in vivo, the intraperitoneal (i.p.) injection of a PD-L1 neutralization antibody  was initiated at 1 day prior to irradiation and continued for 2 weeks.This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals as promulgated by the Institutes of Laboratory Animal Resources, National Research Council, USA. The protocol was approved by the Committee on the Ethics of Animal Experiments of Chang Gung Memorial Hospital. Eight-week-old female C57 mice were used as the tumour implantation model. To investigate the link between PD-L1 expression and the radiation response in immunocompetent hostTo obtain single-cell suspensions, the tumour tissues were digested using 1 mg/ml collagenase IV (Sigma-Aldrich) and 0.2 mg/ml DNase I (Sigma-Aldrich) for 45 minutes at 37 °C. The cells were blocked and subsequently stained with antibodies against CD8, CD3, and PD-L1. For cytotoxicity assays, tumor cells were stained with PI after the removal of CD8+ cells. The murine cytotoxic T cells from fresh tumour specimens were characterized and sorted as CD3+CD8+.5 cells/well), and tumors were previously isolated at a 2:1 ratio. T cell proliferation was induced using anti-CD3/CD28 stimulation beads . After three days, the suppression assay was analysed for T cell proliferation using flow cytometry.The negative regulation of tumour-infiltrating T cells through PD-L1/PD-1 might be an important host-mediated mechanism of acquired radioresistance in tumours33The significance of the differences between the samples was determined using Student’s t-tests. The data are presented as the means ± standard error of the mean (SEM). All experiments, comprising three replicates, were performed at least twice independently. A probability level of p < 0.05 was considered statistically significant, unless otherwise stated.How to cite this article: Wu, C.-T. et al. The role of PD-L1 in the radiation response and clinical outcome for bladder cancer. Sci. Rep.6, 19740; doi: 10.1038/srep19740 (2016).


Fourier transformed infrared spectroscopy analysis indicates that graphene oxide is reduced into graphene. Transmission electron microscope observation shows that SrTiO3 nanoparticles are well assembled onto graphene sheets. The photocatalytic activity of as-prepared SrTiO3-graphene composites was evaluated by the degradation of acid orange 7 (AO7) under a 254-nm UV irradiation, revealing that the composites exhibit significantly enhanced photocatalytic activity compared to the bare SrTiO3 nanoparticles. This can be explained by the fact that photogenerated electrons are captured by graphene, leading to an increased separation and availability of electrons and holes for the photocatalytic reaction. Hydroxyl (·OH) radicals were detected by the photoluminescence technique using terephthalic acid as a probe molecule and were found to be produced over the irradiated SrTiO3 nanoparticles and SrTiO3-graphene composites; especially, an enhanced yield is observed for the latter. The influence of ethanol, KI, and N2 on the photocatalytic efficiency was also investigated. Based on the experimental results, ·OH, h+, and H2O2 are suggested to be the main active species in the photocatalytic degradation of AO7 by SrTiO3-graphene composites.SrTiO61.46. + w; 78.67.Bf; 78.66.Sq In these processes, the high recombination rate of the photogenerated carries greatly limits the photocatalytic activity of catalysts. Therefore, the effective separation of photogenerated electron–hole pairs is very important in improving the photocatalytic efficiency.Semiconductor photocatalysts have attracted considerable attention over the past decades due to their potential applications in solar energy conversion and environmental purification ,2. Amongllutants . Furthernductors -10. It isp2-hybridized carbon atoms, possesses unique properties including high electrical conductivity, electron mobility, thermal conductivity, mechanical strength, and chemical stability [3-graphene nanocomposites has been rarely reported.Graphene, being a two-dimensional (2D) sheet of tability -13. On atability -21, supetability , field-etability , and fuetability . Particutability -21. In t4[Eg of the photocatalyst, during which graphene oxide receives electrons from the excited photocatalyst and is thus reduced to graphene. During the photocatalytic reduction process, photocatalyst nanoparticles are assembled onto graphene sheets to form photocatalyst-graphene composites. Herein, we report the synthesis of SrTiO3-graphene nanocomposites via the photocatalytic reduction method. The photocatalytic activity of the composites was evaluated by the degradation of acid orange 7 (AO7) under ultraviolet (UV) light irradiation, and the photocatalytic mechanism involved was discussed.Up to now, semiconductor-graphene nanocomposites have been generally prepared using graphene oxide as the precursor, followed by its reduction to graphene. To reduce the graphene oxide, several methods have been employed including chemical reduction using hydrazine or NaBH4, high-te4, hydroth4, green c4, and pho4-21. Amon3 nanoparticles were synthesized via a polyacrylamide gel route as described in the literature [3-graphene composites were prepared via a photocatalytic reduction route. A certain amount of graphene oxide was dispersed in 50 mL distilled water, followed by ultrasonic treatment of the suspension for 30 min. Then, 0.1 g SrTiO3 nanoparticles and 0.0125 g ammonium oxalate (AO) were added to the suspension under magnetic stirring. After stirring for 10 min, the mixture was purged with nitrogen and exposed to UV light irradiation from a 15-W low-pressure mercury lamp for 5 h under mild stirring. During the irradiation, the color of the mixture changed from brown to black, indicating the reduction of the graphene oxide. After that, the product was separated from the reaction solution by centrifugation at 4,000 rpm for 10 min, washed several times with distilled water and absolute ethanol, and then dried in a thermostat drying oven at 60°C for 4 h to obtain SrTiO3-graphene composites. A series of samples were prepared by varying the weight fraction of graphene oxide from 2.5% to 10%.SrTiOterature . The graλ = 254 nm). The initial AO7 concentration was 5 mg L-1 with a photocatalyst loading of 0.5 g L-1. Prior to irradiation, the mixed solution was ultrasonically treated in the dark to make the photocatalyst uniformly dispersed. The concentration of AO7 after the photocatalytic degradation was determined by measuring the absorbance of the solution at a fixed wavelength of 484 nm. Before the absorbance measurements, the reaction solution was centrifuged for 10 min at 4,000 rpm to remove the photocatalyst. The degradation percentage is defined as (C0 - Ct) / C0 × 100%, where C0 and Ct are the AO7 concentrations before and after irradiation, respectively. To investigate the photocatalytic stability of the SrTiO3-graphene composites, the recycling tests for the degradation of AO7 using the composite were carried out. After the first cycle, the photocatalyst was collected by centrifugation, washed with water, and dried. The recovered photocatalyst was introduced to the fresh AO7 solution for the next cycle of the photocatalysis experiment under the same conditions. The process was repeated four times.The photocatalytic activity of the samples was evaluated by the degradation of AO7 under UV light irradiation of a 15-W low-pressure mercury lamp . By measuring the photoluminescence (PL) intensity of TAOH that is pronounced around 429 nm, the information about · OH can be obtained. TA was dissolved in a NaOH solution (1.0 mmol L-1) to make a 0.25-mmol L-1 TA solution and then to the solution was added 0.5 g L-1 SrTiO3-graphene composites. The mixed solution, after several minutes of ultrasound treatment in the dark, was illuminated under a 15-W low-pressure mercury lamp. The reacted solution was centrifuged for 10 min at 4,000 rpm to remove the photocatalyst and was then used for the PL measurements through a fluorescence spectrophotometer with the excitation wavelength of 315 nm.Terephthalic acid (TA) was used as a probe molecule to examine hydroxyl (·OH) radicals produced over the irradiated SrTiOThe phase purity of the samples was examined by means of X-ray powder diffraction (XRD) with Cu Kα radiation. Fourier transform infrared spectroscopy (FTIR) measurements were carried out on a Bruker IFS 66v/S spectrometer . The morphology of the samples was observed by a field emission transmission electron microscope (TEM). The UV-visible diffuse reflectance spectra were measured using a UV-visible spectrophotometer with an integrating sphere attachment.3 nanoparticles. It is noted that the SrTiO3 particles have an isoelectric point at pH 8.5 [3 particles are added to the graphene oxide suspension, the pH value of the mixture is measured to be approximately 6.5, implying that the SrTiO3 particle surface is positively charged. On the other hand, the oxygen-containing functional groups of graphene oxide (such as carboxylic acid -COOH and hydroxyl -OH) are deprotonated when it immersed in water, which leads to negative charges created on graphene oxide [3 particles are expected to be adsorbed onto the graphene oxide sheets through electrostatic interactions. Upon UV-light irradiation, electrons and holes are produced on the conduction band (CB) and valence band (VB) of the SrTiO3 particles, respectively. The photogenerated holes are captured by ammonium oxalate that is a hole scavenger [3 particles. The electrons are injected from the SrTiO3 particles into the graphene oxide and react with its oxygen-containing functional groups to reduce graphene oxide.Figure t pH 8.5 ; that isne oxide . As a recavenger , leaving3 particles, and SrTiO3-graphene(10%) composites. In the spectrum of graphene oxide, the absorption peak at 1,726 cm-1 is caused by the C = O stretching vibration of the COOH group. The peak at 1,620 cm-1 is attributed to the C = C skeletal vibration of the graphene sheets. The absorption peak of O-H deformation vibrations in C-OH can be seen at 1,396 cm-1. The absorption bands at around 1,224 and 1,050 cm-1 are assigned to the C-O stretching vibration. For the SrTiO3 particles, the broad absorption bands at around 447 and 625 cm-1 correspond to TiO6 octahedron bending and stretching vibration, respectively [-1 is due to the bending vibration of H-O-H from the adsorbed H2O. In the spectrum of the SrTiO3-graphene composites, the characteristic peaks of SrTiO3 are detected. The absorption peak at 1,630 cm-1 is the overlay of the vibration peak of H-O-H from H2O and C = C skeletal vibration peak in the graphene sheets. However, the absorption peaks of oxygen-containing functional groups, being characteristic for graphene oxide, disappear. The results demonstrate that graphene oxide is completely reduced to graphene during the photocatalytic reduction process.Figure ectively . The abs3 particles and the SrTiO3-graphene (10%) composites. It is seen that all the diffraction peaks for the bare SrTiO3 particles and the composites can be index to the cubic structure of SrTiO3, and no traces of impurity phases are detected. This indicates that the SrTiO3 particles undergo no structural change after the photocatalytic reduction of graphene oxide. In addition, no apparent diffraction peaks of graphene in the composites are observed, which is due to the low content and relatively weak diffraction intensity of the graphene.Figure 3 particles, revealing that the particles are nearly spherical in shape with an average size of about 55 nm. The TEM image of the SrTiO3-graphene(10%) composites is presented in Figure 3 particles are well assembled onto the graphene sheet.Figure 3 particles and SrTiO3-graphene composites. The composites display continuously enhanced light absorbance over the whole wavelength range with increasing graphene content. This can be attributed to the strong light absorption of graphene in the UV-visible light region [R) with respect to wavelength λ , where the peak wavelength is characterized to be the absorption edge of the samples. It is seen that the SrTiO3 particles and composites present two absorption peaks in the derivative spectra. The strong and sharp absorption edge at approximately 370 nm is suggested to be attributed to the electron transition from valence band to conduction band. In comparison to the SrTiO3 particles, the SrTiO3-graphene composites show almost no shift in this absorption edge, indicating that the effect of graphene on the band structure of SrTiO3 can be neglected. From this absorption edge, the Eg of the samples is obtained to be approximately 3.35 eV. In addition, the relatively weak absorption edge at approximately 335 nm may be ascribed to the surface effects.Figure t region . Figure 3-graphene composites was evaluated by the degradation of AO7 under UV light irradiation. Figure 3-graphene composites as a function of irradiation time (t). The blank experiment result is also shown in Figure 3 particles, about 51% of AO7 is observed to be degraded. When the SrTiO3 particles assembled on the graphene sheets, the obtained samples exhibit higher photocatalytic activity than the bare SrTiO3 particles. In these composites, the photocatalytic activity increases gradually with increasing graphene content and achieves the highest value when the content of graphene reaches 7.5%, where the degradation of AO7 is about 88% after irradiation for 6 h. Further increase in graphene content leads to the decrease of the photocatalytic activity.The photocatalytic activity of the SrTiO3 particles and SrTiO3-graphene(7.5%) composites. The blank experiment result indicates almost no PL signal at 429 nm after irradiation without photocatalyst. On irradiation in the presence of the SrTiO3 particles, the PL signal centered around 429 nm is obviously detected, revealing the generation of · OH radicals. When the SrTiO3-graphene composites are used as the photocatalyst, the PL signal becomes more intense, suggesting that the yield of the · OH radicals is enhanced over the irradiated composites.Figure +, ·OH, ·O2, and H2O2 are thought to be the main active species responsible for the dye degradation [+[+ and · OH in the photocatalysis. The role of · O2 and H2O2, which are derived from the reaction between dissolved O2 and photogenerated e-, on the dye degradation can be examined by investigating the effect of N2 on the photocatalytic efficiency since the dissolved O2 can be removed from the solution by the N2-purging procedure. Figure 2 (bubbled at a rate of 0.1 L min-1), ethanol (10% by volume), and KI (2 × 10-3 mol L-1) on the degradation percentage of AO7 after 6 h of photocatalysis. It is demonstrated that when adding ethanol to the reaction solution, the photocatalytic degradation of AO7 undergoes a substantial decrease, from approximately 88% under normal condition to approximately 40% on addition of ethanol. This suggests that · OH radical is an important active species responsible for the dye degradation. Figure 3-graphene composites. The addition of KI to the reaction solution results in a higher suppression of the photocatalytic efficiency compared to the addition of ethanol, where only 16% of AO7 is caused to be degraded, indicating that the photogenerated h+ also plays a role in the degradation of AO7. In addition, the photocatalytic efficiency decreases slightly under N2-purging condition, implying comparatively minor role of · O2 and/or H2O2 for the dye degradation.Generally, hradation . It is kdation [+,33. By i3 can be calculated using the following relation [In order to understand the photocatalytic mechanism of semiconductor-based photocatalysts, it is essential to determine their energy-band potentials since the redox ability of photogenerated carriers is associated with energy-band potentials of photocatalysts. The conduction band and valence band potentials of SrTiOrelation :X is the absolute electronegativity of SrTiO3 (defined as the arithmetic mean of the electron affinity and the first ionization of the constituent atoms) and estimated to be 5.34 eV according to the data reported in the literature [Ee is the energy of free electrons on the hydrogen scale (4.5 eV), and Eg is the bandgap energy of SrTiO3 (3.35 eV). The conduction band and valence band potentials of SrTiO3 vs. normal hydrogen electrode (NHE) are therefore calculated to be ECB = -0.84 V and EVB = +2.51 V, respectively.where terature ,36, Ee i3-graphene composites toward the degradation of AO7 is schematically shown in Figure 3 is irradiated with light of energy greater than its bandgap energy, electrons are excited to the conduction band from the valence band, thus creating electron–hole pairs (Equation 2). Generally, most of the photogenerated electrons and holes recombine rapidly, and only a few of them participate in redox reactions. It is noted that graphene, which is an excellent electron acceptor and conductor, has a Fermi level (-0.08 V vs. NHE [3 (-0.84 V). When SrTiO3 particles are assembled onto graphene sheets, the photogenerated electrons can readily transfer from the conduction band of SrTiO3 to graphene (Equation 3). Thus, the recombination of electron–hole pairs can be effectively suppressed in the composites, which leads to an increased availability of electrons and holes for the photocatalytic reactions. The Fermi level of graphene is positive to the redox potential of O2/·O2 (-0.13 V vs. NHE) but negative to that of O2/H2O2 (+0.695 vs. NHE) [- which transferred onto the graphene cannot thermodynamically react with O2 to produce · O2, but can react with O2 and H+ to produce H2O2 (Equation 4). H2O2 is an active species that can cause dye degradation, and moreover, H2O2 can also participate in the reactions as described in Equations 5 and 6 to form another active species · OH. The valence band potential of SrTiO3 (+2.51 V) is positive to the redox potential of OH-/·OH (+1.89 V vs. NHE) [+ can react with OH- to produce · OH (Equation 7). As a consequence, the active species · OH, h+, and H2O2 work together to degrade AO7 (Equation 8).Based on the obtained experimental results, a possible photocatalytic mechanism of SrTiO vs. NHE ) positivvs. NHE) ,38. Thisvs. NHE) , indicat3 particles decorated on the surface of graphene is expected to increase, thus providing more photogenerated carriers for the photocatalytic reaction. When the graphene content in the composites reaches 7.5%, the SrTiO3 particles are decorated sufficiently, consequently leading to the achievement of the highest photocatalytic activity. However, with further increasing graphene content above 7.5%, the photocatalytic efficiency begins to exhibit a decreasing trend. The possible reason is that the excessive graphene may shield the light and decrease the photon absorption by the SrTiO3 particles, and moreover, the amount of available surface active sites tends to be reduced due to an increasing coverage of graphene onto the surface of the SrTiO3 particles.From Figure 3-graphene(7.5%) composites is examined by the recycling photocatalytic experiment, as shown in Figure 3-graphene composites. Figure 3 particles are still well decorated on the graphene sheets.Besides the photocatalytic activity, the reusability of photocatalysts is another crucial factor for their practical applications. The stability of the SrTiO3-graphene nanocomposites were prepared by irradiating the mixture solution of SrTiO3 nanoparticles and graphene oxide sheets, during which graphene oxide receives electrons from the excited SrTiO3 nanoparticles to be reduced to graphene, simultaneously leading to the assembly of SrTiO3 nanoparticles onto graphene sheets. Compared to the bare SrTiO3 nanoparticles, the as-prepared SrTiO3-graphene composites exhibit an enhanced photocatalytic activity for the degradation of AO7 under irradiation of UV light. This can be attributed to the effective separation of photogenerated electron–hole pairs due to the electron transfer from SrTiO3 to graphene and, hence, increased availability of electrons and holes for the photocatalytic reaction. The enhanced generation of · OH radicals is observed over the irradiated SrTiO3-graphene composites compared to the bare SrTiO3 nanoparticles. The photocatalytic efficiency is slightly deceased by purging with N2 but is significantly suppressed by the addition of ethanol and KI . Based on the experimental results, ·OH, h+, and H2O2 are suggested to be the main active species causing the dye degradation.SrTiO-: photogeneration of electron; Eg: bandgap energy; FTIR: Fourier transform infrared spectroscopy; h+: photogeneration of hole; H2O2: hydrogen peroxide; NHE: normal hydrogen electrode; OH: hydroxyl radicals; PL: photoluminescence; TA: terephthalic acid; TAOH: 2-hydroxyterephthalic acid; TEM: transmission electron microscope; UV: ultraviolet; VB: valence band; XRD: X-ray powder diffraction.AO: ammonium oxalate; AO7: acid orange 7; CB: conduction band; eThe authors declare that they have no competing interests.HY and TX conceived the idea of experiments. TX, LD, JM, and HZ carried out the preparation and characterization of the samples. HY, TX, and JD analyzed and discussed the results of the experiments. TX drafted the manuscript. HY improved the manuscript. All authors read and approved the final manuscript.HY is a professor and a Ph.D. degree holder specializing in the investigation of photocatalytic and nanometer materials. JD is a professor and a Ph.D. degree holder specializing in the investigation of nanometer materials. JM and HZ are instructors and M.Sc. degree holders specializing in the research of nanometer materials. TX is a doctoral candidate major in the study of photocatalytic materials. LD is a graduate student major in the preparation of photocatalytic materials.


The PD-1/PD-L1 axis plays a paramount role in the immune escape of tumor cells by negative regulation of T-cell functions. The aim of the present study was to characterize the PD-L1 expression pattern and its clinical implication in soft-tissue sarcomas (STS).We analyzed PD-L1 expression in 82 STS patients with 5 subtypes: rhabdomyosarcoma, synovial sarcoma, Ewing sarcoma, epithelioid sarcoma, and mesenchymal chondrosarcoma.P = 0.004); highest in epithelioid sarcoma , followed by synovial sarcoma , rhabdomyosarcoma , and Ewing sarcoma , while it was not expressed in mesenchymal chondrosarcoma . STS patients with PD-L1 expression had worse overall survival compared with those without PD-L1 expression . The Cox proportional hazard model adjusted for histologic subtype, initial metastasis, and PD-L1 expression showed that PD-L1 expression was significantly associated with shorter overall survival .The median age at diagnosis was 26 (range: 1–78) and the male to female ratio was 1.6. The majority (80 %) of patients showed locoregional disease rather than metastatic disease at diagnosis. Thirty-five cases (43 %) showed PD-L1 expression and the proportion of PD-L1 expression was significantly different according to histologic subtypes  contains supplementary material, which is available to authorized users. Sarcomas are a rare and highly heterogeneous group of neoplasms originating from the bone and soft tissue, which account for <1 % of all human malignancies . With stIt is well known that the prognosis of a malignant tumor is closely related to host immune responses. During the immune response, the priming and activation of T-cells are critical processes in the induction of adaptive immunity, and the ultimate amplitude of the immune response is regulated by a balance between co-stimulatory and inhibitory signals. In this T-cell-mediated process, cytotoxic T-lymphocyte antigen 4 (CTLA-4) provides inhibitory signals in the priming phase of the T-cell response within the lymph node. The programmed death 1 (PD-1) receptor is one of the key inhibitory signals that is induced during the chronic antigen exposure in peripheral tumor microenvironments. The interaction between PD-L1 in tumor cells and PD-1 in T-lymphocytes negatively regulates the effector function of tumor-specific T-lymphocytes and allows tumor cells to evade the host immune system. Recent studies have indicated that high expression of PD-L1 is associated with poor prognosis in non-small cell lung cancer (NSCLC), ovarian cancer, and kidney cancer . FurtherIn this study, we investigated PD-L1 expression in soft tissue sarcoma and evaluated its clinical relevance according to different subtypes of sarcoma. We thereafter analyzed the prognostic potential of PD-L1 to provide a practical guide as a diagnostic and therapeutic strategy.This study was conducted in a retrospective cohort of patients who were pathologically diagnosed with CD99 positive sarcomas such as rhabdomyosarcoma, synovial sarcoma, Ewing sarcoma, epithelioid sarcoma, and mesenchymal chondrosarcoma, between 1994 and 2013 at Yonsei Cancer Center. A total of 82 formalin-fixed, paraffin-embedded tissue blocks were available for examination of PD-L1 expression. All hematoxylin and eosin (H&E) slides were independently reviewed by two experienced pathologists (E.K.K. and S.H.K).The clinicopathologic variables such as sex, age, maximal tumor size, tumor histology and grade, tumor location, tumor stage, initial presentation of disease, and the status of the resection margin were reviewed retrospectively based on electronic medical records. Tumors were graded according to the French Federation Nationale des Centre de Lutte Contre le Cancer (FNCLCC) criteria . StagingSimple, inexpensive, and precise paraffin TMAs were constructed with a conventional microcompound table and a drill grinder. The original H&E slides were reviewed by two pathologists (E.K.K and S.H.K). One or two different tumor areas per case were selected for tissue microarray construction. Core tissue biopsies (3 mm in diameter) were taken from the individual paraffin blocks (donor blocks) and arranged in recipient paraffin blocks (tissue array blocks) using a trephine apparatus. All TMA blocks were confirmed by H&E staining.Immunohistochemistry (IHC) staining of PD-L1 was performed on the TMA blocks using a mouse monoclonal antibody for PD-L1 . Four-micrometer-thick sections were prepared and stained using a Ventana automatic immunostainer  . After dt-test for the continuous variables and the chi-square test for the discrete variables. For survival analysis, recurrence-free survival (RFS) was defined as the time interval between surgery and tumor recurrence or last follow-up. OS was defined as the time interval between the diagnosis of metastatic/recurrent disease and death or last follow-up. Survival analysis was performed using the Kaplan-Meier method with the log-rank test. Multivariate analyses for OS were performed with Cox’s regression. The accepted level of statistical significance was P < 0.05.The Statistical Package for the Social Sciences (SPSS) version 18.0  was used for statistical analyses. The correlations between PD-L1 expression and clinicopathologic variables were analyzed using the independent sample n = 32, 39 %), followed by synovial sarcoma , Ewing sarcoma , epithelioid sarcoma , and mesenchymal chondrosarcoma . One third of the tumors were located in the extremities . Most patients had no distant metastasis at the time of diagnosis (82 %) and underwent surgical resection (93 %). Adjuvant chemotherapy was administered to 47 patients (57 %), and 37 of these patients (79 %) also received adjuvant radiotherapy.The baseline clinicopathologic characteristics of the patients are summarized in Table P = 0.004). The proportion of PD-L1-expressing tumors was highest in epithelioid sarcoma , followed by synovial sarcoma , rhabdomyosarcoma , and Ewing sarcoma , whereas it was not expressed in mesenchymal chondrosarcoma . Patients with PD-L1 expression had a more negative resection margin than the PD-L1-negative group . There was no significant difference in PD-L1 expression regarding age, sex, tumor size, location, histologic grade, surgical resection, and adjuvant treatment.Among the 82 sarcoma patients, PD-L1 was expressed in 35 cases (43 %). Representative images of PD-L1-positive and -negative staining for each histologic type are shown in Fig. P = 0.015, Fig. P = 0.037, HR = 2.57, 95 % CI = 1.06–6.23).After a median follow-up duration of 33.8 months, 27 patients (33 %) had died at the time of survival analysis. The 5-year OS for all patients was 58.8 % and the 5-year OS rates of the subgroups were as follows: 64 % for rhabdomyosarcoma, 77 % for synovial sarcoma, 39 % for Ewing sarcoma, 18 % for epithelioid sarcoma, and 100 % for chondrosarcoma. There was a trend toward a worse RFS for the positive PD-L1 expression group compared to the negative group Fig. . ExpressIn this study, we evaluated the clinical relevance of PD-L1 expression in various sarcoma subtypes. PD-L1 was differently expressed according to the histologic subtypes of sarcoma and it was found to be an independent prognostic factor for OS.William Coley, a bone surgeon at New York Memorial Hospital, depicted the spontaneous tumor regression of sarcoma patients after severe bacterial infection more than 100 years ago . SubsequBased on this theoretical background, numerous clinical trials were attempted in sarcoma patients with various immunomodulatory agents such as macrophage-colony stimulating factor (GM-CSF), peptide vaccines, and anti-CTLA-4 antibody –16. InhaRecently, with the impressive and outstanding success of pembrolizumab and nivolumab in melanoma, NSCLC, and other malignancies , 18, immThe PD-1/PD-L1 expression level has been reported to be related to poor survival in other solid tumors. In a previous report of renal cell carcinoma, PD-1 expression in tumor-infiltrating lymphocytes was observed in half of the cases and was associated with poor survival . MoreoveMoreover, besides the prognostic value of PD-L1 expression in human cancers, it is becoming increasingly recognized as an important biomarker for predicting the treatment efficacy of PD-1/PD-L1 blockade. In the KEYNOTE-012 phase IB trial  , patientUnfortunately, the IHC criteria for PD-L1 expression have not yet been standardized. Indeed, different definitions of positive PD-L1 expression have been used in the clinical trials conducted to date. In the CheckMate 017 study with NSCLC patients, OS and response rate were significantly better with nivolumab than with docetaxel, regardless of the PD-L1 expression level . ConsideThe main limitations of our study include its patient selection, small sample size, and imbalance of histologic type. Because we initially intended to analyze the PD-L1 expression in CD99 positive tumors, this study mainly enrolled pediatric STS, which could result in relatively younger median age of patients and longer survival outcome. Moreover, due to the small sample size and imbalance of histologic type between PD-L1 positive and negative groups, especially epithelioid sarcoma and mesenchymal chondrosarcoma, there could be a possibility of type I error. Therefore, our findings should be validated in an independent STS cohort and according to the response to PD-1/PD-L1 inhibitors in future clinical trials.In conclusion, we have revealed PD-L1 expression in various STS of young population and demonstrated its independent negative prognostic role, thereby suggesting the PD-1/PD-L1 axis as a potential therapeutic target for the treatment of young STS patients.CTLA-4, cytotoxic T-lymphocyte antigen 4; IHC, immunohistochemistry; OS, overall survival; PD1, programmed death 1; PD-L1, programmed death ligand 1; RFS, recurrence-free survival; TMA, tissue microarray


TDP-43 is a DNA/RNA binding protein associated with TDP-43 proteinopathies. Many mutations have been identified in the flexible C-terminal region, which is implicated in the disease pathology. We investigated four point mutations in the amyloidogenic core region (residues 311–360) of TDP-43 by biochemical and spectroscopic methods. We found that the G335D mutation enhances the aggregation and inclusion formation of TDP-43 and this mutant in TDP-35  exaggerates the antagonist effect on RNA processing by endogenous TDP-43; whereas Q343R gives an opposite effect. As a comparison, M337V and Q331K have very little impact on the aggregation and inclusion formation of TDP-43 or TDP-35. NMR structural analysis showed that the G335D mutant in the core region forms a loop linker between the two α-helices and promotes α-to-β transition, but Q343R loses the second helix and consequently the structural transformation. Thus, the propensity of structural transformation in the amyloidogenic core of TDP-43 determines its aggregation and inclusion formation. This study may provide a molecular mechanism of the TDP-43 proteinopathies caused by genetic mutations. TDP-43 (TAR DNA-binding protein of 43 kDa) is a DNA/RNA binding protein containing two RNA recognition motifs (RRMs) that participate in RNA binding and a long C-terminal glycine-rich region (GRR) that is involved in protein-protein interactions1426TDP-43 is mainly localized in nucleus and plays multiple roles in transcriptional repression, pre-mRNA splicing, translational regulation and stability8910121351517181921222728More than 40 mutations in TDP-43, mainly in the C-terminal GRR domain, have been identified in familial and sporadic cases of ALS and FTLD723242731in vitro and in cells. Our findings implicate pathological impact of the mutations on the inclusion formation and dysfunction of TDP-43, and bring light to the pathogenesis of the related neurodegenerative diseases.Previously, we identified an amyloidogenic core (residues 311–360) in the C-terminal flexible region of TDP-43E. coli to report the aggregation properties of the fusion proteins. As our previous reportAs preciously described, many mutations in the C-terminal GRR domain of TDP-43, especially in its amyloidogenic core, have been identified in familial and sporadic cases of ALS and FTLD7in vitro by ThT assay. Firstly, we analyzed time courses of the core fragment and its mutants fused with thioredoxin (Trx-TDP(311–360)) upon thrombin cleavage ) forms a helix-turn-helix structure 25. The tWe next investigated structural transformation of the mutant peptides of TDP311–360) during the aggregation processes as detected by circular dichroic (CD) spectroscopy . Previou0 during It has been demonstrated that the C-terminal 35-kDa fragment (TDP-35) of TDP-43 triggers formation of cytoplasmic inclusionsTo confirm the above observation, we transfected FLAG-tagged TDP-35 and its mutants respectively into HEK 293T cells, and then performed supernatant/pellet fractionation analysis . The datAs reported previously, TDP-43 is mainly localized in the nucleus that can be extracted into the supernatant. However, when treated with staurospine (STS), TDP-43 is degraded to fragments that cause formation of the cytoplasmic inclusions and deposit in the pelletAs previously reported, TDP-43 can promote CFTR exon 9 alternative splicing and TDP-35 loses the function of RNA processing811As known, TDP-35 may act as an antagonist against endogenous TDP-43 in RNA processing by a dominant-negative mechanismThere are many pathologically identified mutations in the amyloidogenic core of TDP-4333383932397Structurally, the amyloidogenic core region of TDP-43 forms a helix-turn-helix structure in solution and it is prone to transformation into a hairpin-like β-sheetTDP-43 is a nucleic-acid binding protein with RNA processing function in nucleus. Mutation, fragmentation and mislocation will alter RNA splicing and cause neurodegeneration45Although cytoplasmic inclusions of wild-type TDP-43 are presented in 90% of ALS and 50% of FTLD patients4343313846Nde I/BamH I cloning sites to make the GFP-fusion constructs. The cDNA of TDP(311–360) was subcloned into pET32M to get a thioredoxin (Trx)-fused protein, and it was also cloned into pHGB vector to generate GB1-TDP(311–360) with an N-terminal His tag. The TDP-43 and TDP-35 cDNAs were constructed into pcDNA3.1-Myc/His and pcDNA3.1/ FLAG vectors (Invitrogen) respectively for eukaryotic expression. All mutants  were generated by PCR site-directed mutagenesis. All constructs were verified by DNA sequencing.The cDNAs of human TDP-43 and its fragments were digested and ligated into a pET22b-GFP plasmid by using The method for assaying the aggregation abilities of GFP-fused TDP-43 fragments and their respective mutants were performed as described previously2+-NTA following an FPLC Superdex-75 column (GE Biosciences) respectively, and their concentrations were determined spectrophotometrically by using each extinction coefficients.The Trx and GB1 fused proteins were expressed and purified as described previouslyThe time course of the aggregation process was monitored by ThT fluorescence2/dmol). For the solid-state CD measurements, about 150 μL of the protein solution (ca. 0.50 mg/mL) was cast onto a 2-cm diameter cylindrical quartz glass for evaporating overnight at room temperature, and then the CD spectrum was recorded. The solid-state spectra were presented as ellipticities (mdeg). The secondary structure contents were calculated by using the Model JWSSE-J700 program based on data deconvolution algorithmAll Far-UV CD spectra were recorded on a JASCO J-715 spectropolarimeter (JASCO) at room temperature as described previously15N/13C-labeled GB1-TDP(311–360) mutant (G335D or Q343R) was dissolved in 20 mM phosphate (pH 6.5), 50 mM NaCl and 8% D2O for NMR data acquirements. All NMR spectra were recorded at 25 °C on a Bruker Avance 600-MHz spectrometer equipped with a TCI CryoProbe (Bruker Biospin). The backbone and side-chain chemical-shift assignments were obtained from the spectra of HNCO, HNHA, HNCACB, CBCA(CO)NH, CC(CO)NH, and HCCH-TOCSY. NOE restraints for structure calculations were obtained from 15N- and 13C-edited NOESY spectra. The NMR data were processed by using NMRPipe and analyzed with SPARKY. The backbone dihedral restraints were derived from TALOS program. The structures were calculated using ARIA2.0 and CNS program, assessed by PROCHECK and displayed by MOLMOL. The structural calculation was performed for 9 cycles and a total of 200 structures were finally obtained. Ten lowest-energy structures were selected and displayed.NMR data acquirement and processing, and structure calculation were performed as reported previously2552Cell culture, transfection, Western blotting, immunocytochemistry and confocal microscopy were carried out as described previouslyThe transfected cells were lysed in 100 μL of a RIPA buffer ) on ice for 30 min and centrifuged at 15,000 g for 15 min. The supernatant was added with 100 μL of the loading buffer (2% SDS), while the pellet was sufficiently washed with the RIPA buffer for three times and then added with 50 μL of the loading buffer (4% SDS). Equal volume of supernatant and pellet fractions was subjected to SDS-PAGE with 12% acrylamide gel and transferred onto PVDF membranes (PerkinElmer). The mouse monoclonal antibodies against FLAG or Myc, goat anti-actin (Santa Cruz), goat anti-mouse IgG-HRP antibody and rabbit anti-goat IgG-HRP antibody (Jackson Immuno-Research) were used. The proteins were detected by an ECL detection kit (Thermo scientific). The band intensities were quantified by Scion Image software (Scion Corp).The method for assaying the CFTR exon 9 splicing was same as the literatures1554Accession codes: The coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 2N3X (TDP(311–360)), 2N4G (G335D) and 2N4H (Q343R), respectively. How to cite this article: Jiang, L.-L. et al. Two mutations G335D and Q343R within the amyloidogenic core region of TDP-43 influence its aggregation and inclusion formation. Sci. Rep.6, 23928; doi: 10.1038/srep23928 (2016).


We have quantitatively observed a reduced but still sizable magnetic moments of the epitaxial Fe ML on graphene, which is well resembled by simulations and can be attributed to the strong hybridization between the Fe 3dz2 and the C 2pz orbitals and the sp-orbital-like behavior of the Fe 3d electrons due to the presence of graphene.Successful spin injection into graphene makes it a competitive contender in the race to become a key material for quantum computation, or the spin-operation-based data processing and sensing. Engineering ferromagnetic metal (FM)/graphene heterojunctions is one of the most promising avenues to realise it, however, their interface magnetism remains an open question up to this day. In any proposed FM/graphene spintronic devices, the best opportunity for spin transport could only be achieved where no magnetic dead layer exists at the FM/graphene interface. Here we present a comprehensive study of the epitaxial Fe/graphene interface by means of X-ray magnetic circular dichroism (XMCD) and density functional theory (DFT) calculations. The experiment has been performed using a specially designed FM As a prototypical two-dimensional quantum system, graphene displays a combination of exceptional properties including large charge carrier mobility, high thermal conductivity, strong mechanical strength, excellent optical characteristics, electrically tuneable band gap, as well as the recently discovered long spin coherence length123568910111213111/FM2/SC structure that to a large extent simulates the realistic FM/graphene interface of the proposed graphene-based transistors45In any proposed graphene-based transistors, the best opportunity for spin transport could only be achieved when no magnetic dead layer exists at the FM/graphene interface. Previous studies on various FM/semiconductor (FM/SC) heterojunctions revealed the possibility that the magnetic ordering near a region of the surface or interface of FM/SC may be modified due to interdiffusion, termination and hybridization; and controversial reports make this issue rather complex151617181920Tc)221/FM2/SC structure . The thick FM1 layer provides the FM2 ML with a source of exchange interaction and these two together restore the interfacial behavior of the thick ferromagnetic FM2/SC. Combined with the unique elemental selectivity of XMCD, such structure allows direct observation of the magnetization of FM2 at the FM2/SC interface.Fundamentally all the intriguing spintronic phenomena observed in the FM/graphene heterojunctions strongly depend upon the interfacial hybridization and magnetic exchange interaction8910111213L2,3 absorption edges were performed on beamline I10 at the Diamond Light Source, UK. Circularly polarized X-rays with 100% degree of polarization were used in normal incidence with respect to the sample plane and parallel with the applied magnetic field, as shown in − − σ+, obtained by flipping the X-ray helicity at a fixed magnetic field of 3 T, under which the sample is fully magnetized with little paramagnetic contribution. The X-ray absorption (XAS) and XMCD of the Fe and Ni L2,3 absorption edges were performed and typical spectra obtained at 5–300 K are presented in The XAS and XMCD of the Fe and Ni mspin) and orbital magnetic moment (morb) were calculated by applying the sum rulesL2,3 edges based on3d-band holes, nh, was assumed as 3.7 accounting for the charge transfer from Fe to the atop stabilizing Ni layer (nh = 3.4 in bulk)26mspin, morb, and total magnetic moment  of the interface Fe at temperatures from 5 K to 300 K were plotted in mtotal of Fe displays a decreasing trend with the increasing temperature from 1.23 μB/atom at 5 K to 1.12 μB/atom at 300 K, i.e., ~9% reduction, pointing to a TC close to bulk-like Fe. At the lowest temperature (5 K), we obtained mspin = (1.06 ± 0.1) μB/atom, which is ~50% reduced from the bulk-like Fe (whose mspin = 2.2 μB/atom) and morb = (0.18 ± 0.02) μB/atom, corresponding to an enhancement of ~200% compared to morb = 0.085 μB/atom of that in the bulkThe spin  μB/atom at 5 K to (0.52 ± 0.06) μB/atom at 300 K, pointing to a TC close to the bulk-like Nimorb shows a significant enhancement compared to that of bulk-like Fe, the Ni stabilizing layer shows a nearly quenched morb. This is expected for bulk-like Ni, where the effect of the crystal field plays a dominant role. As an additional note, the graphene is likely to carry induced magnetic moment at the Fe/graphene interface252930The calculated 2), a Fe bcc(100) 5 × 3 supercell (14.33 × 8.60 Å2), and a Fe bcc(110) 5 × 1 supercell (14.33 × 4.06 Å2), respectively, matching the graphene 6 × 2 supercell (14.82 × 8.56 Å2), a 6 × 1 supercell (14.82 × 4.28 Å2), and a 1 × 1 cell (2.47 × 2.47 Å2), are used to model Fe fcc(111), bcc(100) and bcc(110) MLs on graphene. For each superstructure, all atomic positions are relaxed until the atomic forces were smaller than 0.02 eV/Å. Figure S4 presents the initial (upper row) and the relaxed (lower row) geometries of Fe fcc(111), bcc(100) and bcc(110) stacking on graphene, respectively. The calculations suggest that the Fe prefers to follow the fcc(111)-like structure of the graphene substrate. Significant deformations were observed for both Fe bcc(110) and (100) on graphene due to the large lattice mismatch. The averaged Fe-Fe bond length after relaxation changes from 2.73 to 2.35 Å for Fe bcc(110), and from 2.91 to 2.38 Å for Fe bcc(100) ML, respectively, whilst that for Fe fcc(111) remains unchanged from 2.47 Å.In order to determine the electronic and magnetic ground state of the Fe ML on graphene, first it must be addressed where Fe likes to reside with respect to the C atoms. Periodic DFT calculations were performed to obtain the most energetically stable stacking of ML Fe on graphene. Three initial superstructures, namely, a Fe fcc(111) 1 × 1 primitive cell  μB/atom, is more likely a mixture of Fetop (mspin = 1.23 μB/atom) and Febridge (mspin = 0.67 μB/atom), given the small energy difference (∆E = 43 meV) of their calculated total energies.It was found that the Fe atoms deposited on graphene prefer to follow fcc(111) stacking and there exist three inequivalent positions of the fcc Fe allocations on graphene as presented in top/graphene (right column), respectively, together with their corresponding partial density of states (DOSs) are presented in dz2 orbitals (see the white shed areas), which is responsible for the large mspin (2.76 μB/atom). In contract, the Fe 3dz2 orbitals in ML Fetop/graphene become strongly delocalized in the reciprocal space and tend to show an sp-orbital-like partial band character. The exchange splitting sharply reduces from 2.67 eV to 0.94 eV due to the presence of graphene, pointing to a weakened exchange interaction between the 3d electrons of the Fe in Fetop/graphene and consequently a reduced spin polarization near the Fermi level (EF). Further examinations of the symmetry matching and spatial overlap suggest that the strong Fe-C hybridization predominantly originates from that between the C1 pz orbitals and the Fe 3dz2 orbitals, although the interactions between the C2 pz orbitals and the Fe 3dxz and 3dyz orbitals cannot be neglected  and the ML Fetop/graphene. The 3D charge accumulation and depletion was obtained by subtracting the charge density of Fetop/graphene from that of the freestanding Fe ML and graphene as shown in e is transferred from Fe to C atoms per unit cell. This substantial charge transfer mainly occurs at the interfacial region of the Fetop/graphene and predominately via the C1 pz orbitals. The Fe-C hybridization, in turn, slightly polarizes the C atoms in Fetop/graphene in opposite direction to the Fe moments (mspin = −0.02 μB/atom for the C1 and −0.01 μB/atom for the C2 atoms) as can be seen from bridge/graphene (not shown) are qualitatively same but quantitatively different as that of Fetop/graphene (not shown) and mspin of −0.008 μB/atom are induced for both the C1 and C2 atoms.mspin of 1.53 μB/atom for Fe. The inclusion of the topmost 7 MLs Ni has not modified the favorable stacking of the ML Fe in conjunction with the graphene. Furthermore, the calculated average mspin of the 7 ML Ni atop Fe is 0.59 μB/atom, well reproducing the experimental value of mspin = (0.59 ± 0.06) μB/atom obtained from XMCD.As an additional note, the effect of the topmost Ni was also considered. The bulk-like Ni atop the Fe ML serves as a stabilizing layer, in order to make the ML Fe in such configuration to be representative of the interface Fe of a bulk-like Fe on graphene. The two FM layers are expected to ferromagnetically couple with each other, provided an atomically clean interface is prepared, e.g., using UHV growth techniques. The calculation suggests that the stabilizing Ni layer has a limited influence on the underneath Fe by offering only a small charge transfer, i.e. 0.017 e/atom. In the fully relaxed structure, the Ni tends to pull the Fe slightly away from the graphene substrate, resulting in a higher top/graphene interface up to room temperatures. We obtained a reduced but still sizable magnetic moment of the ML Fe on graphene, i.e., (1.23 ± 0.1) μB/atom, which is well resembled by the DFT calculations. The observed suppression of the Fe magnetization is attributed to the strong hybridization between the Fe 3dz2 and the C 2pz orbitals, leading to greatly reduced exchange splitting, and the sp-orbital-like behavior of the Fe 3d electrons due to the presence of graphene. Our study restores a realistic scenario of the proposed graphene-based transistors and addresses the open question of the magnetic and electronic nature of the Fe/graphene interface. Although possibilities such as the discontinuous growth of the ML Fe and the contact of Fe with the SiO2 substrate cannot be fully excluded from the experiment, our study has demonstrated a valuable model of exploring the interfacial magnetism of FM on graphene and the results are encouraging based on the contemporary sample preparation technique. Future work to explore the tuning of the spin polarized band structure of both the FM and graphene via the interface engineering will be of great interest and have strong implications for both fundamental physics and the emerging spintronics technology.To summarize, we have demonstrated an unambiguous ferromagnetic epitaxial Fe/graphene interface by XMCD. This was aided by the inclusion of a Ni stabilizing layer atop the Fe film, which facilitated the study of the temperature dependence of the Fe/graphene interface. We observed strong dichroic XAS spectra of Fe, suggesting a well-maintained ferromagnetic order of the Fe2/Si substrate35The single layer graphene used in this study was prepared by chemical vapor deposition (CVD) on Cu foil and then transferred on top of 300 nm SiOab initio simulation package (VASP)37k-points was used to sample the two-dimensional Brillouin zone for the thin film electronic structure calculations. The total energies were obtained using the tetrahedron method with the Blöchl corrections. In each unit cell, all atomic positions are fully relaxed with the conjugate gradient procedure until the residual forces vanished within 0.02 eV/Å. The vacuum space of 15 Å was set to separate the interactions between neighboring slabs of the unit cells. Test calculations have been performed for the bulk bcc Fe (a0 = 2.87 Å) and the freestanding fcc monolayer Fe (a0 = 2.55 Å) using this method and the results compare well with the pioneering reports25293031414244The first-principle calculations were performed using the projector augmented wave methods implemented in the Vienna How to cite this article: Liu, W. Q. et al. Atomic-Scale Interfacial Magnetism in Fe/Graphene Heterojunction. Sci. Rep.5, 11911; doi: 10.1038/srep11911 (2015).


Transparent electrodes with superior flexibility and stretchability as well as good electrical and optical properties are required for applications in wearable electronics with comfort designs and high performances. Here, we present hybrid nanostructures as stretchable and transparent electrodes based on graphene and networks of metal nanowires, and investigate their optical, electrical, and mechanical properties. High electrical and optical characteristics, superb bendability , excellent stretchability , strong robustness against electrical breakdown and thermal oxidation were obtained through comprehensive study. We believe that these results suggest a substantial promise application in future electronics. Rs) of the randomly distributed mNW networks (lower than approximately 80 Ω sq−1) [Rs of undoped, chemical vapor deposition (CVD)-grown graphene (higher than approximately 1 kΩ sq−1) [−1 with transmittance of approximately 90% at 550 nm) [Transparent conductive films have been widely used in electronics such as touch screen panels, electrochromic smart windows, and displays. In addition, stretchable electronics have attracted tremendous recent attention due to their potential advantages including outstanding suitability on a nonplanar surface and excellent portability. Therefore, encoding stretchability into transparent electrodes has been actively explored as the key to emerge flexible optoelectronic devices. Indium tin oxide (ITO) is the most commonly used material in transparent electrodes. However, it has several critical disadvantages, such as (i) the increasing costs for indium, (ii) need for a complex vacuum process, and (iii) its mechanical brittleness under external stress. These weak points of ITO limit its use for flexible electronics. Thus, there is a clear and urgent need for new transparent conductive materials with superb mechanical properties. Various alternative candidates for ITO have been intensively pursued, including carbon nanotubes -6, condu Ω sq−1) ,20,26 wikΩ sq−1) ,9-11 and 550 nm) . In addi 550 nm) ,27. Seve 550 nm) ,20-22,27Rs of pristine graphene has been estimated to be as low as approximately 30 Ω sq−1 [Rs of undoped, synthesized graphene by using various methods [Rs of ITO. Although chemical doping processes [Rs of the CVD-grown graphene by increasing carrier density, the instability of the interactions between graphene and dopant typically limits the lifetime of this doping effect and leads to a time-variant increase of Rs [Another promising alternative to ITO comes in the form of graphene that can be deformed up to a strain of approximately 4% with negligible cracking  as well 0 Ω sq−1 , but the methods ,10,11,32rocesses ,33 can fse of Rs .In this paper, we report graphene-mNW hybrid nanostructures as high-performance, stretchable, and transparent electrodes. Especially, we focused on the study of the mechanical flexibility of the hybrid film, so various flexible tests such as bending, folding, and uniaxial and multi-axial directional stretching tests were performed. These fabricated hybrid structures present superb mechanical flexibility (folding with bending radius of approximately 3.7 μm) and stretchability .−1) were purchased from Nanopyxis Co., Ltd . The solution was stored at 5 °C, and then spin-coated on substrates. The spin coating condition of AgNWs was fixed at 500 rpm for 30 s, except for the density study of AgNWs, which was done according to the spin rates. After spinning the AgNW suspension on substrates (with or without graphene as a top layer), the samples were annealed at 150 °C for 90 s to evaporate the solvent completely.Silver nanowires (AgNWs) with average length of 30 μm ± 7 μm and average diameter of 20 nm ± 5 nm dispersed in ethanol (3 mg mL2 (500 sccm). The CVD growth was carried out under CH4 (12 sccm) and H2 (500 sccm) flows for 5 min, and then the reactor was rapidly cooled to room temperature under Ar (500 sccm) flow. To transfer the synthesized graphene, we used a 200-nm-thick poly(methyl methacrylate)  film as a supporting layer which was spun on the graphene. The Cu foil was dissolved in a diluted etching solution of FeCl3:HCl:H2O (1:1:20 vol.%) with the PMMA/graphene layer floating on the surface of the solution. Subsequently, the sample was rinsed by floating it on the surface of deionized water (DI water) and then the sample was transferred onto the desired substrates. In addition, the PMMA supporting layer was removed with acetone.The synthesis and transfer of graphene were carried out under the following conditions. A Cu foil  was loaded onto the center of a quartz CVD reactor under low vacuum (100 mTorr). Subsequently, the reactor was heated up to 1,000 °C with introduction of the flow of Ar (200 sccm) and HWe have employed two approaches to assemble the hybrid structures (i) AgNWs on graphene or (ii) graphene on AgNWs: (i) After the CVD synthesis of the graphene layer, the graphene was transferred onto a target substrate. After spin casting of the AgNW dispersions onto the graphene surface, this sample was annealed at 150 °C for 90 s. (ii) AgNW network films were coated on substrate, as described above. Then the synthesized graphene layer was transferred onto the films by using PMMA. Subsequently, the supporting layer was removed with acetone.3PO4:C2H4O2:C6H4NO5SNa:H2O (55:1:4:40 vol.%).The graphene-AgNW hybrid electrodes can be photolithographically patterned by using an etch-back process. First, the hybrid films were dry-etched using oxygen plasma  to remove carbon-based materials. Because the residues of the AgNWs were oxidized and disconnected by oxygen plasma exposure, the exposed areas became electrically nonconductive. Subsequently, these AgNW residues can be completely eliminated by using an etching solution of HThe preparation of a 2-μm-thick polyimide substrate for the bending test was carried out by the following procedures. After casting the 200-nm-thick PMMA sacrificial layer on a bare Si wafer, a polyimide precursor ; Aldrich, St. Louis, MO, USA) was subsequently spin-coated at 3,000 rpm for 30 s. The thermal curing was processed at 250 °C for 6 h. After fabricating graphene-AgNW hybrid electrodes on the prepared polyimide surface, this 2-μm-thick polyimide film together with the hybrid electrodes could be delaminated from the bare Si wafer by eliminating the PMMA layer using acetone.Rs using a probe station with a Keithley 4200-SCS semiconductor parametric analyzer . In addition, the two-point probe method was also used for the R measurements and electrical breakdown test using a Keithley 2425 source meter. Rs can be acquired by calculating it from the measured R by considering the aspect ratio of patterns (Rs = R × (Pattern width)/(Pattern length)). Furthermore, the change in R of hybrid films during uniaxial and multi-axial stretching was measured after fixing the sample on home-built stretching stages.For the electrical, optical, and mechanical characterization of the prepared samples, a four-point probe method was used to measure the The optical film transmittance was assessed by ultraviolet–visible near-infrared (UV–vis-NIR) spectroscopy . The transmittance of the substrates was excluded in all our data. The optical images were taken using an optical microscope . The morphologies of the fabricated films were characterized using field emission scanning electron microcopy (FE-SEM)  and atomic force microscope (AFM) .Rs than higher spin rates  , and this rc can be estimated by measuring the outer diameter of the supports. In this manner, the hybrid samples were wrapped on different cylinder-shaped objects, such as a plastic straw and a glass capillary tube. For extreme bending, the hybrid films on the substrates were folded in half by pressing two pieces of Si wafer together using tape to attach both ends of the polyimide to the wafer  [ε = (ts + tf)/2rc, where ts and tf are the thickness of the substrate and film, respectively. Although this folding leads to approximately 27% bending strain, a negligible change in R occurs for bending to radii of curvatures as small as 3.7 μm.Mechanical flexibility is another key factor of transparent electrodes for next-generation stretchable and wearable electronics. We achieved ultimate flexibility including bending and folding capability of the hybrid structures in Figure rain (ε) ,12 accorR under various tensile strains (σ) was plotted in Figure In addition to the extreme folding, the excellent elasticity of graphene  and the Rs values were measured at specific stretching strains. Figure Rs according to increase of ε, and this graphene-AgNW (spin-coated) hybrid electrode can also be stretched up to 100% tensile strain without significant change. To investigate the durability against consecutive stretching and releasing, the Rs of the sample was measured during cyclic stretching tests  and it was almost constant without notable deformation, as plotted in Figure Rs of our hybrid sample during stretching toward the multi-axial direction at up to 30% by using a homemade cylinder-shaped stretching stage, and the relative difference Rs was recorded at less than 5%  hybrid electrode. Here, a multi-axial direction means the centrifugal force direction parallel with the plane of a fabricated hybrid films. The biaxial direction stretching of film has been reported  but that2 on Si wafer, and then graphene, AgNW networks, and their hybrid structures were formed on this substrate with metal pads, respectively. The patterning of the aforementioned three materials with various widths and lengths was performed by using photolithography and dry and wet etching processes, followed by measurement and comparison of their R . Figure The electrical conductance of the nanostructured films can be changed by pattern size, such as via the widths and lengths of a pattern . For thi2). Figure I-V characteristics of graphene, AgNWs, and the hybrid structures with various channel lengths (L) were measured in an ambient condition. As one of the results, the top graph of Figure y-axis graphs are presented together at the bottom of Figure I-V curves indicate electrical conductance, and the narrow pattern size (channel length = 10 μm) caused the relatively high resistance of AgNW networks, compared to the resistance of the hybrid structures, because only small portions of the conductive pathways along the NWs remained inside the patterns. For all the three cases, currents increase almost linearly with the applied voltage before their breakdown. Based on these results, we compared breakdown bias (Vbreakdown) according to various L of these three different materials. In the case of AgNW networks that can be degraded by electromigration [Vbreakdown < approximately 4 V) for a channel length of approximately ≥30 μm (average length of AgNWs) due to the large aspect ratio of NWs and NW-NW contact resistances. On the other hand, the Vbreakdown of graphene is higher than that of AgNW networks. This polycrystalline graphene can be damaged by Joule heating and oxidation at the defect sites [Vbreakdown of graphene also decreases when the channel length falls below the threshold related to the grain sizes. In the case of graphene-AgNW hybrid nanostructures, the graphene underneath AgNWs can create a path to dissipate heat and electrical stress, which can make AgNWs sustainable against breakdown at relatively high electric bias. Therefore, the hybrid structures exhibit the highest values of Vbreakdown among the three materials . Also, the hybrid electrode can flow the highest current against its breakdown due to its low resistance. The hybridization of the graphene and AgNWs give the hybrid structures strong endurance against electrical load, so this improved robustness is another advantage of the hybrid electrode.In addition, the robustness of conductive materials that can withstand electrical load becomes an important feature for next-generation electronics, as devices are highly integrated and miniaturized. This is because the electric field is inversely proportional to the length of pattern. To assess and compare the robustness of nanostructured films against electrical loads, patterned samples of graphene, AgNWs, and their hybrid structures as channels with a width of 10 and 20 μm were prepared on a metal electrode deposited onto a Si wafer , 10,000 times in stretching cycles with 100% tensile strain toward the uniaxial direction, and 30% stretching in tensile strain toward the multi-axial direction. We believe that the hybridization with two-dimensional and one-dimensional nanomaterials presents a promising strategy toward stretchable, wearable electronics and implantable biosensor devices.The results presented here demonstrate the superior characteristics of graphene-AgNW hybrid nanostructures as flexible, stretchable, and transparent electrodes. From the fundamental study, we confirmed that the hybridization between AgNWs and CVD synthesized graphene could significantly enhance electrical properties such as low


We obtained a profound light modulation over a broad spectrum by using the fabricated large area transparent graphene supercapacitors and we compared the performance of our devices with the one based on graphene from standard copper. We propose that the copper foils used in the lithium-ion batteries could be used to obtain high-quality graphene at much lower-cost, with the improved performance of electrical transport and optical properties in the devices made from them.This work demonstrates an attractive low-cost route to obtain large area and high-quality graphene films by using the ultra-smooth copper foils which are typically used as the negative electrodes in lithium-ion batteries. We first compared the electronic transport properties of our new graphene film with the one synthesized by using commonly used standard copper foils in chemical vapor deposition (CVD). We observed a stark improvement in the electrical performance of the transistors realized on our graphene films. To study the optical properties on large area, we transferred CVD based graphene to transparent flexible substrates using hot lamination method and performed large area optical scanning. We demonstrate the promise of our high quality graphene films for large areas with ~400 cm Carbon based nanomaterials have provided a new perspective in electronics owing to their very high charge carrier mobility and nanoscale dimensions. In particular, the carbon nanotubes7Graphene layers can be isolated chemically from bulk graphite with intercalation of atoms or molecules through the graphite201319et al.et al.26et al.The surface quality of the Cu foils and the transfer process greatly influence the performance and reliability of graphene based devices. Previous works have showed that the morphology of the Cu surface is one of the key parameters to have better quality graphene films2 flexible graphene electrodes and enlarged 160 times the previously reported (2.5 cm2) supercapacitor structureIn this paper, we demonstrate that the commercially available ultra-smooth Cu foils  can help to open new avenues for large area device applications with the improved quality of the graphene films. We studied the synthesis of graphene on commercially available smooth Cu foils and we provided a detailed comparison between the graphene synthesized on ultra-smooth Cu foils and the one synthesized on the standard Cu foils in terms of structural, electrical and optical properties. We observed a stark improvement in the electrical performance of the transistors realized on our graphene films. Then, to demonstrate the advantage of high quality graphene films from ultra-smooth Cu foils, we fabricated 400 cmAlfa Aesar Cu foil which is commonly used for CVD of graphene    in the s B1-SBS)  and scan B1-SBS) . On the  B1-SBS) . We inve2 in the form of a snow-flake  on flexible, 125 μm thick PVC substrate. After the CVD synthesis on ultra-smooth Cu foils, we transferred the graphene to the flexible PVC substrates by using hot lamination method. During the Cu etching, we protected the Cu foils at the edges of the sample to use them as contact electrodes for resistivity measurements and application of bias voltage. The measured total resistance of graphene is superposition of contact resistance and the sheet resistance terms  above the half of the incoming photon energy. This blocks the inter-band transitions and makes the graphene more transparent by blocking optical absorption mechanism of the material. By blocking the inter-band transitions, we achieved the light modulation over a broad range of wavelengths with the large area graphene supercapacitors. We achieve more efficient transmittance modulation in the case of smooth-Cu-graphene based supercapacitors due to the lower sheet resistance and high coverage of the smooth-Cu-graphene film. F. Therefore we were able to extract the Fermi energy values corresponding to each curve. Previously we showed that the supercapacitor device structure can provide an efficient electrostatic doping of graphene which cannot be achieved by graphene based transistors using solid insulating dielectric layersHere the limiting factor for the bias voltage is the electrochemical window of the electrolyte used in the supercapacitor structure. Since our devices use mutual gating between top and bottom graphene electrodes, they are limited with the two times of the electrochemical window of the electrolyte (1-Butyl-3-methylimidazolium hexafluorophosphate) which is around 4.5 V. In mutual gating; the Dirac points of the top and bottom graphene shifts with respect to each other leading to two different dips in the capacitance modulation. As a result, we obtained a more efficient transmittance modulation up to two times (at 700 nm) by using smooth-Cu-graphene in supercapacitors operating as optical modulators. We also observed 27% more efficient Fermi level shift compared to rough-Cu-foils (at 4.4 V). By using electrolytes with broader electrochemical windows, the enhancement in transmittance and Fermi energy shift by using smooth-Cu-graphene can be further demonstrated in optoelectronic devices.2) that we used in this study makes the costs affordable for large scale production of graphene. Cu surface modification techniques also add to the overall cost to the graphene growth with the standard Cu foils which itself costs around 115 $ per m2.The lower price of commercially available ultra-smooth Cu foils  in CVD synthesis holds great promise for high performance in large area graphene based devices. Recent industrial progress of large scale production of smooth Cu foils, for application such as lithium-ion batteries, can also provide opportunities to improve the quality of graphene grown by CVD. After a detailed structural comparison between the rough and smooth-Cu-graphene, we studied the performance improvements in the transistors based on smooth-Cu-graphene by comparing them to transistors based on rough-Cu-graphene. To demonstrate the promises of the smooth-Cu-graphene on large area, we fabricated flexible graphene electrodes and mapped the transmittance by optical scan. These electrodes enabled us to fabricate graphene-based flexible supercapacitors operating as voltage controlled optical modulators. By enlarging the previously demonstrated supercapacitor structure, we examined the effects of usage of rough and smooth-Cu-graphene in the large area electro-optic devices. As against previous works relating the Cu surface morphology to resulting graphene quality, our method does not require chemical treatment or deposition of Cu foils. The effectiveness of our approach is two-fold:We believe that large scale and low cost synthesis of high quality graphene films and the compatibility of our method to the roll-to-roll fabrication would open an avenue through the realization of graphene based flexible optoelectronic systems such as cell phones with roll-up displays, e-paper, radio-frequency identification (RFID) tags, as well as medical patches that can be attached to the skin to deliver drugs or monitor vital signs, and tactile or electronic skin for robotics and prosthetics3637444 and H2 gases are 285 mTorr and 40 mTorr and corresponding rate of flows are 25 sccm and 4 sccm respectively). We annealed the Cu substrates for half an hour at 1035 °C before the growth. Hydrogen flow was kept constant during the heating, annealing, growth and cooling. After 10 minutes of growth, samples were left for fast cooling to room temperature. The growth took place in a 8 inch diameter quartz chamber. We used both the commercially available 20 μm thick smooth Cu foils  and the standard 25 μm Alfa Aesar Cu foil (item #13382) as growth substrates in the same CVD chamber.For single layer graphene, at 1035 °C methane was sent into growth chamber in company with hydrogen  drop casting method. We drop casted thick droplets of S1813 photoresist on graphene holding Cu surfaces and waited overnight at 70 °C to gently harden the PR. Then hardened PR on graphene holding Cu was put into the dense FeCl3 solution yields the graphene on flexible PVC substrates.We also used lamination technique to transfer large area graphene on PVC. At 100 °C PVC becomes active and exhibits conformal sticking to the graphene holding side of ultra-smooth Cu foils when laminated through the hot rollers. Etching the Cu foils in FeClWITec Alpha 300 M Raman microscope system with 50x microscope objectives was used for Raman mapping. The excitation wavelength was 532 nm.In order to fabricate the graphene based FETs, first conventional UV-photolithographic techniques were used to shape the source and drain electrodes on graphene. Then, thermal evaporation technique was used for metallization of the electrodes. The source and drain electrodes are 50 nm gold on 5 nm titanium adhesive layer. After lift-off step, we isolated the graphene layer by using reactive ion etching so that the graphene layers is aligned between the source and drain electrodes to define the channels. Transfer and output characteristics of the graphene based transistors were performed with HP4142B semiconductor parameter analyser. We recorded the drain current as a function of gate voltage at constant drain voltage. We repeat the measurements for devices having various channel lengths.The transmittance measurements were performed by using Bruker Vertex 70 V FTIR spectrometer integrated with silicon photodetector working in visible and near-IR. We worked in the wavelength range between 400 nm to 1100 nm. We biased the graphene based transistors using Keithley 2400 source measure unit during the transmittance measurements. The leakage current is also recorded during the measurement.How to cite this article: Polat, E. O. et al. Synthesis of Large Area Graphene for High Performance in Flexible Optoelectronic Devices. Sci. Rep.5, 16744; doi: 10.1038/srep16744 (2015).


Controlling tunneling properties through graphene vertical heterostructures provides advantages in achieving large conductance modulation which has been known as limitation in lateral graphene device structures. Despite of intensive research on graphene vertical heterosturctures for recent years, the potential of spintronics based on graphene vertical heterostructures remains relatively unexplored. Here, we present an analytical device model for graphene-based spintronics by using ferromagnetic graphene in vertical heterostructures. We consider a normal or ferroelectric insulator as a tunneling layer. The device concept yields a way of controlling spin transport through the vertical heterostructures, resulting in gate-tunable spin-switching phenomena. Also, we revealed that a ‘giant’ resistance emerges through a ferroelectric insulating layer owing to the anti-parallel configuration of ferromagnetic graphene layers by means of electric fields via gate and bias voltages. Our findings discover the prospect of manipulating the spin transport properties in vertical heterostructures without use of magnetic fields. Graphene, a honeycomb-like single layer crystal of carbon atoms, has been attracting a lot of attention in the recent decade both in terms of fundamental interests and technology. Amongst the various aspects of graphene, one of the most promising potentials is that it has extraordinary transport properties such as high carrier mobility and long mean free path124578Recently, an alternative platform has emerged for graphene FETs ,where graphene and other two dimensional layers are stacked vertically1010101015171319222324In this article, we investigate the spin-resolved transport through the vertical heterostructures with ferromagnetic graphene (FMG). We show that the control of the spin transport through the structure can be achieved by electrically manipulating the spin configurations in FMG sheets. The spin-resolved band structure is taken into account to describe the electronic states of FMG, and the spin-resolved tunneling current density is calculated for two different combinations of heterostructures: FMG–normal insulator(NI)–FMG and FMG–ferroelectric insulator (FEI)–FMG. We also show that the giant electroresistance emerges for the anti-parallel configuration of FMGs when the sandwiched insulator is replaced by an FEI.Vb can be applied between two graphene sheets, yelding the tunneling current through the insulating layer. The electronic properties of FMG are characterized by its spin-resolved electronic statesThe system studied in this article is a vertically stacked heterostructure which is formed by FMG and an insulating layer see . The san27282931σ = ±1 for spin-up and down states of Dirac fermions, v↑ = 1.15 × vF and v↓ = 1.4 × vF are Fermi velocities for each spin with vF = 106 m/s, Δ↑ = 134 meV and Δ↓ = 98 meV are the spin-resolved bandgaps, as displayed in where As a starting point, we introduce our vertical transport model used in this study. We consider the elastic tunneling of Dirac fermions in terms of energy, and scattering effects are taken into account by applying the current density formula. Also, we assume that the magnetizations of two FMG layers are parallel in the absence of external magnetic fields. The spin-resolved vertical tunnelig current is formulated with the interlayer transition matrix element based on WKB approximationwhere ties see , and  anm* is the effective mass of the tunnel barrier material, Δ is the barrier height of the tunneling insulator, Vb is bias voltage which is applied via two graphene sheets, and d is the thickness of the tunneling insulator. Note that Γ is an energy-independent prefactor which represents the momentum scattering of Dirac fermions by disorders such as defects or phonons inside the tunnel barrier material. In other words, Γ = 1 means no scattering mechanism while Dirac fermions tunnel through the tunnel barrier, and on the other hand, the smaller Γs indicate more diffusive vertical transport through the tunnel barrier.Here, VTG = VBG ≡ VG. This assumption allows us to simplify the problem with fixed chemical potentials on both graphene layers in equilibrium, i.e., n0 is proportional to the gate voltage VG, i.e., n0 = αVG where α is the proportional constant depending on the substrate (superstrate) materials between a graphene layer and the bottom (top) gate electrode. When bias voltage is applied to both graphene layers, their chemical potentials are shifted and equilibrium is broken, resulting in non-zero tunneling current denslty throughout the vertical heterostructure.Carrier density on graphene layers is controlled by field-effects via gate electrodes. In the absence of bias voltage, the chemical potentials on both graphene layers are in equilibrium, leading to no net tunneling current density. The dual-gated platform is considered to fix and maintain the same carrier densities in the top and the bottom gate electrodesm* = 0.5 mel with the bare mass of an electron mel are used for the calculations, which are approximately compatible with typical 2d materials such as MoS2, WS2, etc.333435Vb| < 0.1 V. Remarkably, within this bias voltage range, it is found that the current density can be fully spin-polarized according to gate voltage. This gate-tunable feature of the spin transport is led by the following mechanisms. When the equilibrium chemical potential μ0 is place in the mid-gap, the small bias voltage cannot lead to a sufficient amount of tunneling current densities for both spins. As bias voltage increases, spin-resolved current densities begin to flow. The spin-down current starts flowing slightly earlier than the spin-up current density because of their different electronic properties, i.e., the amount of band gaps and the position of the band edges. For large bias voltages, the current densities for both spins keep increasing with different increasing ratios associated to the spin-resolved Fermi velocities in SDOS. For VG = 15 V, as plotted in VG = −8 V makes an FMG layer purely spin-up polarized, and the contribution to the tunneling current density is dominated by spin-up Dirac fermions for small bias voltages as shown in VG = 0 V because the majority spin states near both band edges are opposite to each other . For typical ferromagnetic metal (FM)-NI-FM heterojunctions, electrical resistance strongly depends on how the FM configuration is set. While the electrical current flows well with the small resistance in the parallel configuration, a very large resistance is measured in the anti-parallel configuration. To achieve GMR, devices should be asymmetrically fabricated by using different kinds of FM materials, for which magnetization varies with respect to external magnetic fields. This means that controlling the magnetic fields is essential to change FM configuration. In addition, GER has already been introduced in a normal metal (NM)-FEI-NM heterojunction by using the asymmetric electrical response of a sandwiched FEIThe properties of an FEI are described by a simple model of the polarization density as a function of an external electric field,p0 is the saturated polarization density, β is the characteristic coefficient with the physical dimension of inverse electric fields, d, and Ec is the coercive field which is responsible for the hysterisis of the FEI. Here, the factor s = ±1 implies how the electric field varies, i.e. the forward or reverse sweep of electric fields  is the sign function. In equilibrium, the chemical potentials on the FMG layers should be arranged at the same Fermi energy to be consistent with equilibrium in the absence of bias voltage. Therefore, the Dirac cone on each FMG layer is shifted by δμ/2 = (μT − μB)/2, respectively, i.e., δμ/ed. This FEI-induced electric field is reflected in the tunnling probability as below,The shift of the spin-resolved Dirac cones is led by the following mechanism. For dual-gated devices, the carrier densities on FMG layers VG = 0 V where chemical potentials reside near the band edges of FMGs. Further, for the very small bias Vb = 0.01 V, the tunneling current is allowed only by a positive bias, whereas it is strongly suppressed by a negative bias. Such a large GER ratio is led by the following mechanism. For very small bias voltages, the polarization density is almost unchanged from the saturated value, and the resulting Dirac cone shift makes one FMG layer purely spin-up polarized and the other FMG layer purely spin-down polarized, i.e., anti-parallel spin configuration of FMGs is derived. When a small bias is applied in the positive direction, the chemical potential on spin-up polarized FMG becomes lower and touches the lower spin-down band, while the chemical potential on spin-down polarized FMG becomes higher but still reside in the spin-down band only. On the other hand, the FMG configuration remains anti-parallel for Vb = −0.01 V, resulting in the suppression of vertical tunneling by Pauli blocking. Indeed, Vb = 0.01 V is influenced by the spin-down states only. Therefore, the GER ratio ~104 originates from the bias-tunable spin-configuration of FMGs. Also, the GER ratio has dependence on temperature and deteriorates at higher temperatures .