Immunotherapy targeting PD-1/PD-L1 axis showed benefits in cancer. Prognostic significance of tumour infiltrating lymphocytes (TILs) has been determined. We evaluated PD-L1 protein expression in tumour cells and TILs, PD-L1 mRNA level and various histopathologic factors including TILs using 167 formalin-fixed paraffin embedded tissues and 39 fresh tissue of HER2-positive breast cancer. TILs level and PD-L1 expression in tumour cells and TILs were significantly correlated one another. PD-L1 positivity in tumour cells was associated with high histologic grade and high TILs level . High PD-L1 immunoscore in TILs and high total immunoscore (in tumour cells and TILs) of PD-L1 were correlated with high histologic grade , absence of lymphovascular invasion , negative hormone receptor expression  and high TILs level . High PD-L1 mRNA expression was associated with high TILs level . PD-L1 positivity in tumour cells was associated with better disease-free survival in HR−/HER2+ breast cancer (p = 0.039). PD-L1 expression in tumour cells and TILs are significantly associated with TILs level in HER2-positive breast cancer. PD-L1 expression in tumour cells might be positive prognostic factor in HR−/HER2+ breast cancers. Evading immune destruction was added as a component of the hallmarks of cancer by Hanahan and Weinberg in 20112, with emphasis on the tumour microenvironment. There are three mechanisms of immune escape in cancer, including loss of both antigenicity and immunogenicity, and an establishment of an immunosuppressive microenvironment3. To lose immunogenicity, tumour cells use immune checkpoints, which are essential for the maintenance of self-tolerance under normal physiological conditions. Immune checkpoints, including co-stimulatory and inhibitory signals, regulate the level of the T cell response. Inhibitory ligands and receptors, which regulate T cell effector functions, are generally overexpressed in tumour or non-transformed cells in the tumour microenvironment4.Recent progress in the field of immunology supports the theory that the immune system has a role in cancer development through “cancer immunoediting”. This encompasses three processes: elimination, equilibrium and escape13. Remarkably, Schadendorf et al.5 observed a plateau in the survival curves in a meta-analysis of overall survival involving the use of anti-CTLA4 (ipilimumab) in patients with advanced melanoma, which started at approximately three years. This suggests that immunotherapy could be a potentially curative treatment option for advanced malignancies. Despite the evidences surrounding the clinical benefits of immune checkpoint blocking agents, particularly those targeting the PD-1/PD-L1 axis, the value of PD-L1 expression as a predictive biomarker for patient response to anti-PD-1/PD-L1 therapy has not been established. Although the expression of PD-L1 in tumour cells was considered a potential biomarker for PD-1/PD-L1 blocking agents, it has demonstrated effectiveness even in PD-L1 negative patients15. Furthermore, several reports have demonstrated that lymphocyte PD-L1 expression was associated with the response to anti-PD-1/PD-L1 therapy12. Moreover, the prognostic significance of PD-L1 expression has not been conclusively determined.Immune checkpoint blocking agents targeting the cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and programmed death -1 (PD-1)/programmed death-ligand 1 (PD-L1) axis have been developed and possess notable clinical efficacy16. In HER2-positive breast cancers, treatment with trastuzumab appears to boost the immune system response17. A high level of TILs is associated with a better response to trastuzumab, and is a good prognostic factor in HER2-positive breast cancers19. Moreover, there is a consensus regarding the methodology for evaluating histological levels of TILs in breast cancers20.The prognostic impact of tumour infiltrating lymphocytes (TILs) has been reported in various organs, including the breast, and specifically in human epidermal growth factor receptor 2 (HER2)-positive and triple-negative breast cancers (TNBCs)et al.21 classified cancers based on TIL and PD-L1 expression and proposed immunotherapeutic strategies according to their classification. As previously mentioned, immune mechanisms are associated with the efficacy of trastuzumab treatment, and several attempts have been made to use immune checkpoint inhibitors in combination with trastuzumab. Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate consisting of the monoclonal antibody trastuzumab linked to the cytotoxic agent emtansine. The combination of T-DM1 and anti-CTLA4 or anti-PD-1 antibodies, elicited responses in tumour xenografts that had previously been resistant to T-DM1 monotherapy in a mouse model22. An international phase Ib/II trial  involving trastuzumab in combination with the anti-PD-1 antibody is currently being investigated. In a mouse model, the synergistic effect of anti-PD-1 agent on trastuzumab therapy was demonstrated in HER2-positive breast cancer17. However, the immune checkpoints in HER2-positive breast cancers, particularly PD-L1, are not completely elucidated. Whilst reports indicate the significance of PD-L1 expression in TILs for predicting the response to anti-PD-1/PD-L1 inhibitors, many recent studies have focused on the expression of PD-L1 in tumour cells alone. Despite efforts to establish a combination therapy involving trastuzumab and anti-PD-1/PD-L1 inhibitors, PD-L1 expression in HER2-positive breast cancers has not been fully determined.In addition to the progress made in immunotherapy and cancer immunology, Teng In this study, the expression of PD-L1 in tumour cells and TILs and the relationship between the expression levels of PD-L1 and various clinicopathological parameters, including TILs, were evaluated. The prognostic significance of PD-L1 expression was also assessed.in situ (DCIS), the remaining 65 cases (38.9%) were categorized as having a high level of TLSs around the DCIS. Patients were also grouped by abundance of TLSs around invasive margins, 109 patients (65.3%) and the remaining 58 patients (34.7%) were classified as having a high and low level of TLSs respectively (Supplementary Table A cohort of 167 patients with HER2-positive breast cancers was included in our study. Patient age ranged from 23 to 85 years (mean: 52.59 ± 10.126 years) and the size of the breast tumours ranged from 0.2 to 15.5 cm (mean 3.213 ± 1.773 cm). There were 93 (55.7%) and 74 cases (44.3%) of left and right breast cancers, respectively. Eighty-nine cases (53.3%) were of hormone receptor (HR)+/HER2+ breast cancers and 78 cases (46.7%) were of HR−/HER2+ breast cancers. Sixty-one patients (36.5%) had TIL levels of 10% or less, 74 patients (44.3%) had TIL levels between 20% and 60%, and the remaining 32 patients (19.2%) had TIL levels of more than 60%. One hundred and two cases (61.1%) were categorized as having a low level of tertiary lymphoid structures (TLSs) around the ductal carcinoma 23, 28 cases (16.8%) were type I tumours, 82 cases (49.1%) were type II, 53 cases (31.7%) were type III and four cases (2.4%) were type IV (Supplementary Table Eighty-one patients (48.5%) showed positive immunostaining for PD-L1 in tumour cells according to the Allred scoring system , high TIL level (p < 0.001), and an abundance of TLSs around the invasive component (p = 0.002). High PD-L1 immunoscores for TILs was significantly correlated with high histologic grade (p = 0.001), absence of lymphovascular invasion (p = 0.012), absence of HR expression (p = 0.044), high levels of TILs (p < 0.001) and abundance of TLSs around the invasive component (p = 0.034). High levels of total PD-L1 immunoscore (in tumour cells and TILs) showed significant association with high histologic grade (p < 0.001), absence of lymphovascular invasion (p = 0.007), absence of HR expression (p = 0.001), and high levels of TILs (p < 0.001).The association between PD-L1 protein expression and variable clinicopathological factors is shown in Table p = 0.068), though it was not statistically significant. In correlation analyses, the TIL level, each PD-L1 immunoscore for tumour cells and TILs, the total PD-L1 immunoscore in tumour cells and TILs, and mRNA expression levels of PD-L1 showed a significant positive association with one another (Table p = 0.001) and there was no significant association between mRNA levels of PD-L1 and other clinicopathological factors .Twenty-three cases (59.0%) and the remaining 16 cases (41.0%), were categorized as having low and high expression levels of PD-L1 mRNA respectively based on the mean fold difference value. In the t-test, the fold difference value of the PD-L1 mRNA showed difference between the low and high total immunoscore group . During the follow-up period, there were five cases of local recurrence (2.99%) and 14 cases of distant metastasis (8.38%). Five patients died because of the disease (3.0%).23. The expression of PD-L1 was not significant for the prognosis of total HER2-positive breast cancers and HR+/HER2+ breast cancers  is a ligand of PD-1. PD-1 is a transmembrane receptor and an important negative immune controller, regulating the activation, exhaustion, and tolerance of T cells and the resolution of inflammation. The PD-1 pathway is activated upon engagement of its ligands, PD-L1 or PD-L2, and can modulate T cell activity in several ways. The interaction of PD-1 with its ligand induces epigenetic modification in T cells, which alters transcription factor expression and reduces the phosphorylation of T cell receptor (TCR) signalling molecules. Additionally, the PD-1 signal stimulates the expression of proteins that decrease T cell proliferation and cytokine production, thus decreasing T cell survival and killing capacity4. The adaptive immune resistance mechanism of PD-L1 upregulation is an adaptation to an endogenous tumour-specific immune response. The tumour cells use the normal physiology that occurs during pathogen infection to protect the host from immune-mediated damage. Simply, PD-L1 is induced in tumour cells in response to inflammatory signals such as interferons (IFNs). In a previous study of HER2-positive breast cancers, expression of Myxovirus resistance A (MxA), an excellent marker of IFNs activity, in tumour cells was significantly associated with high TIL levels19. Furthermore, the current study found that TIL level, PD-L1 immunoscore in tumour cells and in TILs, and the total PD-L1 immunoscore in tumour cells and TILs, significantly correlated with each other. Innate immune resistance demonstrates that PD-L1 is driven by constitutive oncogenic signalling pathways in the tumour cells. Constitutive anaplastic lymphoma kinase (ALK) signalling has been reported to induce PD-L1 expression25. Phosphatase and tensin homolog (PTEN) loss or downregulation, which reflects the phosphatidulinositol-4,5-biphosphate 3-kinase/protein kinase B (PI3K/Akt) pathway, was related to PD-L1 expression in TNBCs and glioblastoma27. The current study showed that 53 cases (31.7%) involved type III tumours (positive for PD-L1 with low TIL levels) suggesting tumour intrinsic induction. Tokunaga et al.28 demonstrated that Akt activation was significantly associated with HER2 overexpression in breast cancers. However, the intrinsic induction mechanism of PD-L1 in HER2-positive breast cancer should be determined.Tumour cells use two mechanism for expressing PD-L1: adaptive immune resistance (tumour extrinsic mechanism) and innate immune resistance (tumour intrinsic mechanism)35. This study showed that 81 patients (48.5%) were positive for PD-L1 in tumour cells, whereas 51 cases (30.5%) showed high levels of PD-L1 expression in TILs. Overall, 89 patients  were associated with poor disease-free survival, and type III tumours had intermediate disease-free survival rates in HR−/HER2+ breast cancers, although it was not statistically significant. Better disease-free survival of type III tumours than type II tumours in HR−/HER2+ breast cancers suggests probable positive prognostic role of PD-L1 expression regardless of the status of the tumour microenvironment. Further in vitro studies are required to clarify the prognostic value of PD-L1 in cancer. Type II tumours can be described as tumours with immunological ignorance21. For such tumours, adoptive T cell therapy with the delivery of costimulatory signals, the blocking of inhibitory signals and vaccination could be considered. Adoptive T cell therapy is a form of immunotherapy whereby ex vivo isolation and expansion of antigen-specific T cells is performed and they are transferred back into patients that already showed clinical efficacy in recurrent viral infections37. Furthermore, Bald et al. showed that reversion of immune ignorance with peritumoural injections of the immunostimulant, polyinosinic:polycytidylic acid (poly:IC), induced upregulation of PD-L1 expression in a mouse model of human immune cell-poor melanoma. Additionally, they demonstrated the synergistic effect of anti-PD-1 therapy with immunostimulants38. In the present study, type III tumours showed intermediate outcomes in disease-free survival compared with the remaining tumour types in patients with HR−/HER2+ breast cancers. As mentioned above, intrinsic tumour expression of PD-L1 is associated with constitutive oncogenic signalling. However, these results suggest that blocking anti-PD-1/PD-L1 is unlikely to induce T cell response to the tumour because the tumour microenvironment does not have enough TILs to elicit effective anti-tumour immunity. An approach similar to that used for type II tumours involving the recruitment of TILs into the tumour microenvironment can also be used for this group of patients.Results regarding the prognostic significance of PD-L1 in breast cancers are conflicting. Several reports suggest a good prognostic role of PD-L1 in breast cancer40. Moreover, Lee et al. recently demonstrated that HLA expression was negatively associated with estrogen receptor expression and positively correlated with high TILs level and IFN-associated molecules in breast cancer41. However, the biologic determinants of PD-L1 expression in tumour cells as a positive prognostic factor in HR negative tumours, but not in HR positive tumours, should be further explored.IFNs, inducers of PD-L1, stimulate the expression of human leukocyte antigen (HLA)Studies with large cohorts with long term follow up periods are required to classify all consecutive breast cancers, as each type should have a different treatment strategy. Furthermore, functional studies are required to understand the intrinsic tumour expression of PD-L1 in breast cancer. The importance of defining the molecular pathways that regulate the expression of immune checkpoints cannot be overemphasized, as immune checkpoint intervention will likely lead to clinical benefits.In conclusion, TIL levels and PD-L1 protein expression in tumour cells and in TILs are significantly correlated with one another. High expression of PD-L1 mRNA was significantly associated with high TIL levels. Protein expression of PD-L1 in tumour cells was significantly associated with a better disease-free survival rate in HR−/HER2+ breast cancers.A total of 167 patients with HER2-positive breast cancer were included in our study. Patients were recruited following surgery for primary breast cancer between 2011 and 2013 at Pusan National University Hospital. All included patients were pre-operatively chemo- and radiotherapy naïve. Also, all of them received adjuvant treatment. All patients were treated with 4 cycles of anthracycline and cyclophosphamide (AC) and 1 year of trastuzumab as a standard treatment. Paclitaxel or docetaxel was added between 4 cycles of AC and 1 year of trastuzumab based on the risk stratification encompassing the size of the tumour, lymph node metastasis, Ki-67 proliferation index, histologic grade, age, family history of breast cancer, and general condition. Among them, 14 patients discontinued trastuzumab during treatment, because 4 patients died, 11 patients had recurrence or metastasis and 3 developed cardiac toxicity. Formalin-fixed, paraffin-embedded tissue specimens were available for all patients. Clinicopathological information was collected by reviewing each patient’s electronic medical records and pathology reports. Among these, 39 fresh tissue samples were provided by the Pusan National University Hospital, a member of the National Biobank of Korea, supported by the Ministry of Health, Welfare, and Family Affairs. All samples from the National Biobank of Korea were obtained with informed consent under institutional review board-approved protocols.20. TILs level was evaluated by visual estimation. In brief, TILs level was calculated as the percentage of stroma within the invasive area covered by mononuclear cells over total intratumoural stromal area. A full evaluation of average TILs in the tumour was performed without focusing solely on hotspots. The TILs level was evaluated as a percentage in 10% increments. If the TIL level was less than 10%, a 1% or 5% criteria was used18. The ducts affected by DCIS that had TLSs surrounding themselves of the total ducts affected by DCIS were assessed, and the amount of TLSs as a percentage of the total circumference of the invasive front was evaluated.Histological evaluation of full face haematoxylin and eosin (H&E) slides was performed. Various histopathological factors, including histological grade, nuclear grade, necrosis, lymphovascular invasion, lymph node metastasis, TIL level, TLSs around the DCIS and invasive component were evaluated. Histological and nuclear grades were determined by the modified Bloom-Richardson classification. TILs level was assessed based on the recommendation by the International TILs Working GroupFor statistical analysis, patients were subdivided into three categories  or two categories (≤60% and >60%) according to the TIL level as appropriate. TLSs level was divided into two subgroups (low and high) based on the mean value.All available H&E stained slides were reviewed to define a representative tumour area. A tissue arraying instrument was used to array each sample with a tissue core of 2 mm in diameter. To reduce tissue loss and the effects of tumour heterogeneity, all samples were arrayed in duplicate.in situ hybridization (SISH) for HER2 was performed in cases of equivocal immunostaining with a score of 2. An automated immunohistochemical staining device  was used for immunohistochemical analysis. The EZ prep was used for deparaffinization with an auto-staining device on 3 µm paraffin sections. For antigen retrieval, cell conditioner 1  or cell conditioner 2  was used, and optimally formulated antibodies targeting ER , PR , and HER-2/neu  were used. An anti-PD-L1 antibody  was applied to the tissue microarray sections. A HER2/CEP17 chromosome dual-probe  was used for SISH.At the time of diagnosis, immunohistochemical staining of the oestrogen receptor (ER), progesterone receptor (PR), and HER2 was performed, and silver 42. Tumours were classified into two subtypes according to HR (ER and PR) expression: HR+/HER2+ and HR−/HER2+.ER and PR positivity was defined as at least 1% of tumour nuclei showing positive staining. Tumours that were positive for ER or PR were classified as the HR-positive group. HER2 positivity was defined as an immunostaining score of 3, or by gene amplification identified by SISH. SISH was performed in cases of equivocal results, such as in the event of an immunohistochemical staining score of 2. The HER2 testing was interpreted according to American Society of Clinical Oncology/Cllege of American Pathologists Guideline 2007, which was used at the time of diagnosis43. TILs expression levels were divided into two categories (low and high) based on the mean value of the PD-L1 immunoscore of the TILs. Additionally, tumours were classified into two categories (low and high) based on the mean value of the total PD-L1 immunoscore of tumour cells and TILs. For survival analysis, tumours were categorized into four groups according to the PD-L1 expression of the tumour cells and the TILs level, which is similar to the criteria of Teng’s classification, which are as follows: type I, positive for PD-L1 and high TIL level; type II, negative for PD-L1 and low TIL level; type III, positive for PD-L1 and low TIL level and type IV, negative for PD-L1 and high TIL level23.PD-L1 expression was evaluated separately for tumour cells and TILs. Membranous stains were assessed for PD-L1 positivity, using human placental tissue as a positive control. The intensity and the percentage of membranous positivity were considered collectively . After blocking non-specific binding sites, the membrane was immunoblotted with the anti-PD-L1 antibody , followed by incubation with the secondary antibody conjugate. The bands were detected using an LAS-3000 Imaging System .® universal PCR Master mix  and TaqMan® Gene Expression Assays . An ABI 7500 Real-Time PCR System  was used for qRT-PCR. Each assay was performed in duplicate. The comparative CT method (ΔΔCT method) was used to determine the relative expression levels of PD-L1 mRNA. Relative expression levels of PD-L1 mRNA were normalized to those of beta-actin, and fresh human placental tissue was used for calibration. Expression levels of PD-L1 mRNA were subdivided into two categories (low and high) according the mean fold difference value.Total mRNA was isolated from 39 cases of HER2 positive breast cancers provided by the National Biobank of Korea, using an RNeasy mini kit  according to the manufacturer’s instructions. Extracted mRNA was reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kits  and amplified using TaqManp-value of less than 0.10 in the univariate analyses. The likelyhood ratio test was done to ensure the proportional hazard assumptions in the Cox regression model. Differences with p-values of less than 0.05 were considered statistically significant.All statistical analyses were carried out using SPSS statistical software program . The t-test, Chi-square test, Fisher’s exact test, linear-by-linear association tests, Spearman rank correlation test, Kaplan-Meier survival analyses, log-rank test and Cox proportional hazards regression model were used as appropriate. For multivariate analysis, a forward stepwise selection method was used with covariates showing Exemption from informed consent after de-identification of information was approved by the Institutional Review Board of Pusan National University Hospital.Supplementary Information


Distributed measurement of humidity is a sought-after capability for various fields of application, especially in the civil engineering and structural health monitoring sectors. This article presents a method for distributed humidity sensing along polymethyl methacrylate (PMMA) polymer optical fibers (POFs) by analyzing wavelength-dependent Rayleigh backscattering and attenuation characteristics at 500 nm and 650 nm wavelengths. Spatially resolved humidity sensing is obtained from backscatter traces of a dual-wavelength optical time domain reflectometer (OTDR). Backscatter dependence, attenuation dependence as well as the fiber length change are characterized as functions of relative humidity. Cross-sensitivity effects are discussed and quantified. The evaluation of the humidity-dependent backscatter effects at the two wavelength measurements allows for distributed and unambiguous measurement of relative humidity. The technique can be readily employed with low-cost standard polymer optical fibers and commercial OTDR devices.  The measurement of humidity or moisture is an important task in various fields of applications, for example in industrial process control, agriculture, domestic applications, air condition control, or the civil engineering and structural health monitoring sectors. Especially for the monitoring of extended structures and water ingress detection, a distributed  measurement is important. This cannot be adequately achieved with conventional humidity point sensors. Common principles for the measurement of humidity are summarized in ,2. ApartThe approach presented in this article has the advantage of true distributed  humidity measurement using intrinsic scattering and absorption effects in polymer optical fibers (POFs). Distributed measurement is achieved by analyzing backscattering and attenuation parameters along the fiber. We previously proposed distributed relative humidity (RH) measurement in standard poly(methyl methacrylate) (PMMA) POFs at 650 nm wavelength ,19: backIn this article, we present the detailed results of Rayleigh backscattered power dependencies and transmission dependencies on RH changes at 500 nm and 650 nm wavelengths. The combination of these two effects and their differing characteristics at different wavelength measurements  are analyzed for unambiguous identification and localization of RH changes and to distinguish other backscatter-dependent effects.υ-OTDR by Luciol Instruments) with a sampling resolution of 2.6 cm and a spatial resolution of about 10 cm. The pulse optical wavelength of the OTDR can be switched between 500 nm and 650 nm, the favorable optical transmission windows for PMMA POF. The photon counting technique provides very high sensitivity and spatial resolution [All measurements have been conducted with standard step-index PMMA POFs (Mitsubishi’s Eska GK-40) with a 980 µm diameter PMMA core, a 10 µm thin fluorinated polymer cladding and a numerical aperture of NA = 0.5. The OTDR technique provides spatially resolved backscatter measurement analogous to the radar technique: short optical pulses are sent into the fiber and backscattered power is sampled as a function of time or distance. The OTDR measurements have been performed with a photon counting OTDR (solution . Howeverl ≈ 21–37 m and l ≈ 58–69 m). The POF sections in the water basins have a saturated water content and act as a reference during the RH cycles presented in Our aim was to investigate all backscatter effects at different optical wavelengths (in this case at 500 nm and 650 nm) and analyze their differing humidity-dependent characteristics for distributed RH sensing. Since the observed response times of the humidity-dependent backscatter changes are relatively slow, the integration time for each backscatter measurement has been chosen to be 1 h. OTDR traces at both wavelengths have been recorded alternatingly at constant temperature and varying RH settings. All OTDR measurements have been conducted with the setup depicted in T = 30 °C.PMMA can take up to about 2% water by mass. Investigations on different PMMA materials show that the rate of water sorption increases with increasing temperature but the equilibrium concentration of water does not depend on the temperature . We alsoIn this section, the measured RH-dependent effects of spectral transmission change, transmission change at the OTDR wavelengths, backscattered power change and detected fiber length change are presented. Each effect is briefly discussed at the end of the respective sub-section. A more general discussion of the POF RH sensor characteristics and the impact of cross-sensitivities is given in fl = 40.5 m long POF. Three transmission measurements have been conducted during saturated RH values of 30%, 60% and 90%. In order to verify the measured OTDR transmission dependence and identify favorable wavelength measurements for future sensor designs, spectral transmission changes have been conducted. υOH and 4υOH + δOH [a/b,OTDRP at the two wavelengths are calculated from the measured transmission spectra (counts) of RH,a(λ)P at RH a relative to RH,b(λ)P at RH b and the actual OTDR source spectra OTDR(λ)P at 500 nm and 650 nm (Pnm500(λ) and Pnm650(λ), respectively) as generalized in this equation:The RH-dependent transmission change results in PMMA POF show increasing transmission loss with increasing RH for wavelengths exceeding about 513 nm with distinct absorption peaks at 612 nm and 665 nm. The cause for the transmission changes are impacts of the OH vibrational absorptions, presumably 5OH + δOH ,31, and P, calculated from measured transmission and emission spectra, are summarized in The transmission changes ΔAs expected from l ≈ 21–37 m and l ≈ 58–69 m and are used as a reference power level for relative backscatter change evaluation.Distributed backscatter change and transmission change is obtained from relative backscattered power evaluation of OTDR measurements. l = 35 m and l = 65 m). Only the backscattered power change can be observed at l = 37–58 m and l > 69 m. Two effects are evident at 650 nm: increased backscattered power level and superimposed transmission loss related to increased water content in the fiber at higher RH settings. As visible in the spectral transmission results in y-axis for direct comparison with the spectral transmission results in All following calculations of backscatter change and transmission change are conducted by comparing OTDR traces relative to a reference OTDR trace at a certain RH setting. The averaged backscattered power level of a fiber section subjected to varying relative humidity is compared to the averaged power level of a saturated fiber section in the water basin. The calculation of backscatter changes at 650 nm has additionally been adjusted by proportionally subtracting the measured optical loss changes over the compared fiber sections. It has to be noted that all relative change values are defined as change of backscattered power as it is detected by the OTDR. That means that transmission change over a given fiber length constitutes optical power loss that is accumulated by passing the fiber section twice: as forward-propagating pulse and as backscattered power. Transmission measurement readings at the end of the fiber would show half of that value for changes over the same fiber length. This definition corresponds to twice the “transmitted intensity” changes and “scatter intensity” changes presented in ,19. FiguThe results show reproducible and reversible characteristics. As indicated in The same backscatter and transmission change evaluation as above has been conducted during gradual RH change cycles between 30%and 90% while maintaining constant RH for 1 day at the reversal values. The resulting backscatter change and transmission change characteristics are shown for slopes of 2 days (a) and 4 days (b) at 500 nm in The results show good reproducibility and reversibility and agree well in magnitude with the results obtained during step-wise humidity changes between 30% and 90% RH in The apparent “hysteresis” is due to the response time of the sensor and is less distinct for lower RH gradients during the 4-day slope. Saturated water content in the fiber shows reproducible results without hysteresis, as presented in −9 cm2s−1 and 9 × 10−9 cm2s−1 for sorption and desorption [Significant response time differences between sorption and desorption are not visible in sorption , respectsorption  reportedsorption . It is asorption . We assume that the Rayleigh backscattered power dependence may be partly caused by the reduction of refractive index differences at the interfaces between the polymer network and the microvoids as well as within the polymer structure itself due to water uptake and the filling of gaseous voids by water molecules. This may result in a reduced inhomogeneity of the refractive index along the fiber and, as a consequence, decreased scattering. It can also be speculated that the humidity-induced swelling of the polymer causes a mechanically forced closing of the microvoids and micro-crazes which in turn leads to an increase of material homogeneity and, therefore, reduced scattering. The exact reasons, however, are unknown. The response time is further discussed in l, normalized to the fiber length that is exposed to RH changes, is obtained from the interpolated shift of the rising slope of the fiber end reflection peak . The measured length changes during step-wise RH changes (a) and gradual changes (b) are shown in The absorption of water into the fiber has also an effect on the optical runtime of the light. Lower RH values result in decreased runtime, meaning a reduced measured fiber length. This is visible in the shift of the fiber end reflection peak see a. The le−6 (%RH)−1) more significant than the opposite impact of the RH-dependent refractive index change of about Δn ≈ −11 × 10−6 (%RH)−1 [n/ΔRH = f(T) and a change of sign (from positive to negative) at temperatures exceeding about 60 °C [n/ΔRH for PMMA may also exhibit a significant temperature dependence.The measurements indicate reduced “fiber length” at lower RH values and a response time comparable to that of the backscattered power change and transmission change effects in  (%RH)−1 ,35. Measut 60 °C . This inn/ΔRH dependence of PMMA, the temperature-dependent refractive index change of PMMA is negative with about −137 × 10−6 K−1 at 20.1 °C [−6 K−1 [−6 K−1 to 92.8 × 10−6 K−1 at 50 °C [−6 K−1 [If the measured fiber length change is intended to be used for humidity measurement, temperature variations of the sensor also have to be considered: besides the unknown Δ [−6 K−1  and alsoat 50 °C . We dete [−6 K−1 . DependiThe aim of this article is to identify the suitability of PMMA POF for distributed humidity measurement in general, and at different wavelengths in particular. The results are very promising. The resolution of the RH changes depends on the signal-to-noise ratio of the OTDR trace, i.e., the fiber distance, the OTDR integration time and the length of the evaluated fiber section. For a saturated condition, a resolution in the order of 1–2% RH could be achieved. The following cross-sensitivities and limitations have to be considered in an application scenario. temperature. We already investigated this effect for PMMA POF [A relevant cross-sensitivity is the fiber’s backscatter dependence on PMMA POF ,38 and lPMMA POF ,24. Tempstrained fiber sections cause increased backscatter levels [It has to be noted that also r levels ,25. Therresponse time of the sensor is slower than that of most other humidity sensors. The field of application is thus limited to slowly varying RH changes or applications where the immediate response to humidity change is not crucial. However, the response time can be improved to a few hours or less by using commercially available standard PMMA fibers with smaller core diameters, for example 500 µm or 250 µm diameters. Response times below one hour may be possible with a 250 µm PMMA fiber. For comparison: a response time of 30 min has been reported for a FBG in a 200 µm PMMA fiber [The MA fiber . FurtherMA fiber  and evenMA fiber . Also, fMA fiber .referencemeasurement or relative to a reference fiber section with a known RH or water content. We used water-saturated fiber sections as a reference. If such a reference condition cannot be provided, only relative changes along the sensor fiber can be measured. This could be viable for applications where water ingress or moisture change has to be detected in an otherwise homogeneous structure. For direct RH measurement, however, one or more reference sections are necessary. Such a reference backscatter power level may also be provided by inscribing constant scattering damage structures into the POF core using focused femtosecond laser pulses [The RH measurement is based on the evaluation of backscattered power changes relative to a r pulses . In thisThe advantage of the approach presented in this article is the distributed measurement capability over an extended sensor length of up to 200 m. The technique has the potential for humidity or moisture measurement not only in gases but also in solid state materials and structures. Due to the distributed measurement along a flexible sensor fiber, continuous and spatially resolved humidity measurement can be conducted. Also humidity changes of fiber sections shorter than the spatial resolution could be detected since power changes are accumulated on the backscatter trace . The combined advantages of distributed and gapless measurement is beneficial in various fields of application.Application examples can be found in the structural health monitoring sector for humidity/moisture monitoring in building foundations, the measurement of moisture content in concrete and screed or moisture ingress detection in sealed structures such as bridges. Other applications that can benefit from distributed RH measurement are seepage line monitoring in dykes and dams or the detection of leaks in landfills, industrial facilities and reservoirs. In most of these applications, the humidity ingress mechanisms are either slow or the required response time to take action is not crucial. Moisture penetration, for example in buildings and foundations, is a very slow process with critical consequences on the long-term structural integrity or public health, for instance due to mold formation. The limited response time is not an issue for these applications. As mentioned above, decreasing the response time by using smaller diameter fibers would further extend the range of applications.The distributed sensing capability is essential if local water ingress or leakage in extended structures is to be detected. These applications can often not be sufficiently serviced with a point sensor approach which may require the installation of hundreds of point sensors and the associated wiring effort. Distributed sensing, on the other hand, allows for precise localization of humidity changes as well as the spatial spreading of the humidity influx over time along a single fiber. Another application example is the distributed measurement of concrete curing and setting processes inside large concrete infrastructures. The information of the spatial humidity distribution inside the structure can be used to optimize the moisture content during curing as well as the construction progress.The increased cost of an OTDR unit can be economical for applications where water ingress or leakage has to be monitored over extended distances and a great number of point sensors can be substituted. OTDR-based sensing could also be cost-efficient if the unit does not have to be installed permanently, for example during a temporary curing monitoring application, or when regular service interval measurements are sufficient. The fiber sensor itself is low-cost and can be installed in any orientation (linear or meandering) to monitor an area, volume or along extended structures. Sensor fibers can also be installed without the initial intent of monitoring the structure at close to zero cost, providing the potential for later activation for distributed humidity measurement. A reduction in cost can be further achieved in large-scale applications since multiple sensors can be multiplexed in a sensor network and silica fibers could be used as lead fibers to extend the attenuation-limited measurement distance of a POF RH sensor network.


Intron retention (IR) occurs when an intron is transcribed into pre-mRNA and remains in the final mRNA. We have developed a program and database called IRFinder to accurately detect IR from mRNA sequencing data. Analysis of 2573 samples showed that IR occurs in all tissues analyzed, affects over 80% of all coding genes and is associated with cell differentiation and the cell cycle. Frequently retained introns are enriched for specific RNA binding protein sites and are often retained in clusters in the same gene. IR is associated with lower protein levels and intron-retaining transcripts that escape nonsense-mediated decay are not actively translated.The online version of this article (doi:10.1186/s13059-017-1184-4) contains supplementary material, which is available to authorized users. Alternative splicing (AS) affects up to 95% of multi-exonic genes in humans . The thrWe recently discovered that IR combined with the NMD pathway is not a by-product of faulty splicing but rather a major driver of the cellular differentiation of granulocytes . In thishttps://github.com/williamritchie/IRFinder) and a database of IR in over 2000 human samples is freely available at IRBase (http://mimirna.centenary.org.au/irfinder/database/).Here we have developed a significantly enhanced program in terms of sensitivity and speed for detecting retained introns and filtering samples that are inappropriate for IR analysis in terms of library preparation and quality. This novel program was validated using quantitative RT-PCR (RT-qPCR) against retained introns and a NMD knockdown experiment. IRFinder correctly identified IR events and measured the ratio of retained introns to correctly spliced introns with great accuracy. Using IRFinder, we analyzed 3435 human samples, of which 2573 were suitable for analysis. We found that IR occurs in hundreds of genes in all tissues analyzed, affects over 80% of all coding genes, and is associated with cell differentiation and cell cycle. Retained introns in the same genes are frequently adjacent to each other and intron-retaining genes cluster closely on the genome, suggesting a global mechanism that regulates multiple introns simultaneously. By analyzing mass spectrometry and ribosome sequencing data we discovered that intron-retaining genes have lower protein output and that IR transcripts that escape NMD are not actively translated. Finally, by comparing introns that are frequently retained amongst the 2573 samples with introns that are rarely retained, we discovered a distinct primary sequence signature amongst frequently retained introns that is enriched in RNA binding protein sites. These proteins modulate the level of IR and thus the level of repression of frequently retained introns. Our program to analyze IR from sequencing data is available at GitHub  were suitable for analysis in this study. This was mainly due to the incorrect use of the term “mRNA-Seq”, which was frequently used in whole RNA experiments and CHIP-Seq experiments. Our approach also uses a series of programmatically fast steps written in C++ that automatically detect and trim adapters from sequencing reads. Of the 3435 initial samples we analyzed, 3096 (91%) still had adapters in the sequencing reads despite having been already processed by an adaptor-trimming algorithm.IRFinder was capable of estimating IR events with low coverage or low mappability as confirmed by RT-qPCR .The IRFinder algorithm and instruction manual are available at GitHub (http://www.ebi.ac.uk/ena). These were annotated as mRNA-Seq experiments in the archive with over 50 million reads each. The IRFinder quality control filter (see “Methods”) labeled 1201 of these as unfit for further analysis because they were either not RNA-seq experiments or not enriched for poly(A) tailed mRNAs. We searched for retained introns in 2573 remaining samples. We focused on introns that were retained in more than 10% of transcripts (IR ratio >0.1) with at least a coverage of three reads across the entire intron after excluding non-measurable intronic regions (see “Methods”). We found that 87.9%  of all multi-exonic protein coding genes with sufficient coverage had retained introns in at least three samples in our dataset. None of these 16,307 was retained in all of the samples and the majority (95%) of introns were retained in less than 7% of samples, suggesting that the IR events we detected were specific to tissue or cell types. All samples had over 100 retained introns with a median of 926 retained introns  and found that the average IR ratio was homogenous within each tissue , we calculated the distance between genes with retained introns Fig. c and howhttp://mimirna.centenary.org.au/irfinder/database).We created an online database of IR calculated in these samples, which is available at IRBase . These data were coupled with mRNA sequencing and quantitative translation initiation sequencing (QTI-seq), which uses lactimidomycin to preserve initiating ribosomes and puromycin to deplete elongating ribosomes . These dWe previously discovered that IR was essential for granulocyte , erythrop = 2.5 × 10−5). We also found numerous genes involved in neurogenesis with high levels of IR amongst which were the neural transcription factors SOX15 and POU4F1. SOX15 is known to be highly expressed in undifferentiated cells and repressed upon neuron differentiation [hiPSCs can be differentiated into highly homogenous neurons within 4 days by overexpressing the transcription factors Neurogenin-1 and Neurogenin-2 . This alntiation . In agrentiation . In agreWe then analyzed another set of data where the authors used genetically matched hESCs and hiPSCs derived from fibroblasts using the Sendai virus reprogramming method to prove that these cells are equivalent at a gene transcription level . To asse+ T cells. IR has recently been shown to be an integral regulator of T-cell activation [+ T-helper cells that were induced to differentiate toward a TH2 subtype [p = 2E-17 and p = 8E-6 after Benjamini–Hochberg correction, respectively  consisting of a shRNA-mediated knockdown (KD) of RNA binding proteins in HepG2 cells. We used IRFinder to calculate IR before and after KD of proteins for which the motifs were enriched in frequently retained introns , treated with DNaseI (Life Technologies), and converted to cDNA using Superscript III (Life Technologies). Quantification of intronic expression shown in Additional file To compare the efficiency of IRFinder with other existing tools that can detect IR events, we measured the average change in expression of retained introns predicted by IRFinder, MISO , and DEXUpf2fl/fl) and Upf2 knockout  mouse (GSE26561) was downloaded from the Gene Expression Omnibus (GEO). The single-end reads from both samples were mapped to the transcriptome annotation version 73 of Ensembl mouse genome GRCm38 using STAR (version and options described in IRFinder methods).Total RNA-seq of liver tissue from wild-type  less than 0.05 in the DEXSeq report was recognized as differentially retained.DEXSeq 1.14.0 with default parameters was applied to determine differential intron usage between the wild-type and UPF2 knockout samples. Each intron with q value  the Bayesian factor was above 19 (the likelihood that this intron is retained is 19 times higher than that of not being retained); 2) at least one read covered this intronic region; 3) at least ten reads covered the two flanking exons of this intron.


Moreover, anti-hPD-L1 treatment also led to profound inhibition of Treg expansion and shifting of myeloid cell profiles, showing bona fide induction of multilateral anti-tumor responses by anti-hPD-L1 blockade. Thus, this hPD-L1 mouse model system would facilitate the pre-clinical investigation of therapeutic efficacy and immune modulatory function of various forms of anti-hPD-L1 antibodies.Huge efforts have been devoted to develop therapeutic monoclonal antibodies targeting human Programmed death-ligand 1 (hPD-L1) for treating various types of human cancers. However, thus far there is no suitable animal model for evaluating the anti-tumor efficacy of such antibodies against hPD-L1. Here we report the generation of a robust and effective system utilizing hPD-L1-expressing mouse tumor cells to study the therapeutic activity and mode of action of anti-human PD-L1 in mice. The model has been validated by using a clinically proven hPD-L1 blocking antibody. The anti-hPD-L1 antibody treatment resulted in potent dose-dependent rejection of the human PD-L1-expressing tumors in mice. Consistent with what have observed in autochthonous mouse tumor models and cancer patients, the hPD-L1 tumor bearing mice treated by anti-hPD-L1 antibody showed rapid activation, proliferation and reinvigoration of the cytolytic effector function of CD8 Recently monoclonal antibodies targeting immune checkpoint molecules have achieved unprecedented success in clinic for the treatment of a broad range of the most prevalent human cancers12313412313PD-L1 was originally identified and cloned as a B7 family of co-stimulatory/co-inhibitory molecule, called B7-H114812Human peripheral lymphoid cells101115+CTLs and repressed Treg cell enrichment, as well as facilitated the expansion of tumor infiltrating myeloid cells in tumor tissues. Taken together, we construct an hPD-L1 tumor animal model and we hope this model could contribute to quick pre-clinic antibody screening. Additionally, this model could also be used for pre-clinic screening of other immune checkpoint inhibitors.In this study, we constructed an hPD-L1 expressing MC-38 tumor animal model and observed an evident anti-tumor effect by treating with MPDL-3280A, the hPD-L1 monoclonal antibody. Flow cytometry analysis revealed antibody treatment increased the frequency and the cytotoxicity of infiltrated CD8in vivo is in favor with the general public. This study reported a human PD-L1 tumor model which was constructed by replacing mouse PD-L1 with human PD-L1 in a common transplantable mouse tumor cells , MC-38-hPD-L1 cells in vitro  to mouse PD-1 (mPD-1) by ELISA. The OD value indicated that the hPD-L1 bound to mPD-1 very well . Further+T cell infiltration. To address whether this model also hit this point, the phenotype of immune cells in tumor microenvironment were analyzed by flow cytometry. The frequency of CD8+T cells and the ratio of CD8 to CD4 T cells were both remarkably up-regulated  assay  assay . ConsistT) assay . To summ+, Ly6G+or Ly6C+myeloid cells in tumor tissues by flow cytometry. FACS data revealed CD11b+myeloid cells were not affected by anti-hPD-L1 antibody treatment. Amazingly, the frequency of CD11b+Ly6G+cells was slightly increased at day15 but significantly increased at day21. Similarly, though the frequency of CD11b+Ly6C+was not affected at day15, it was significantly increased at day21 . All experiments were carried out in accordance with the approved guidelines.http://crispr.mit.edu), cloned into sgRNA expression vector (FG-BB-U6-sgRNA) at BbsI restriction site, and confirmed by sequencing. CRISPR/Cas9 knockout was performed by co-transfection of the anti-mPD-L1 sgRNA construct with Cas9 expression plasmid (FG-hEF/HTLV-Cas9-PGK-Puro-WPRE) carrying a puromycin selection marker and into the parental MC-38 cells using Lipofectamine 3000 (Invitrogen), and the sgRNA sequence is 5′-GCTTGCGTTAGTGGTGTACT-3′. The transfected cells were transient selected with puromycin for 2 days, then expanded and cloned to screen for target gene knockout by flow cytometry and TA cloning-sequencing. For TA cloning-sequencing analysis, genomic DNA was extracted from the pooled cells by DNeasy Blood&Tissue kit (Qiagen), forward primer is 5′-TGGTTCCTTTTAAACAAGACTGGG-3′, and reverse primer is 5′-CGCACCACCGTAGCTGATTA-3′. The sgRNA targeted DNA fragment was amplified by Taq DNA polymerase (NEB) and cloned into pTZ57R vector using Ins TAclone PCR Cloning Kit (Fermentas Thermo). Numbers (5 clones) of individual resulting TA plasmid clones were sequenced, and the sequencing results were analyzed by ClusterW software. Frameshift Indels in mouse PD-L1 gene were identified by Blast (NCBI), and functional knockout was confirmed by flow cytometry for the absence of PD-L1.C57BL/6 derived mouse colon tumor cell line MC-38 was originally obtained from ATCC (ATCC#CRL-2638). Short-guide RNAs (sgRNA) specific to mouse PD-L1 (CD274) was designed according to online design tool  carrying human PD-L1 ORF was constructed and verified by sequencing. High titer recombinant lentivirus particles were produced from HEK293T cells by calcium phosphate transfection with the third generation of lentiviral packaging plasmids according to Qin The plate was coated with mPD-L1-Fc or hPD-L1-Fc protein in high pH coating buffer overnight at 4 °C. Added diluted biotin labeled mPD-1 protein into each well with dilution buffer for 1 h at room temperature after incubated by blocking buffer. Added diluted Neutavidin-HRP (1:5000 dilution) to wells and incubated for 1 hr in room temperature. Read OD value of the plate at 450 nm by automatics plate reader .We performed flow cytomtry analysis to determine whether mouse PD-L1 could bind to human PD-L1. Briefly, 1.0*10^6 MC-38-hPD-L1 cells were incubated with 5.0 μg/ml or 2.5 μg/ml mouse PD-1 protein for 25 min after the cells were blocked by FcR antibody. Next, these cells were washed by staining buffer for 3 times and then stained by anti-Fc-APC for 20 min. These cells were subjected to flow cytometry analysis. All of these proteins and antibodies are obtained from Mabspace Biosciences (Suzhou) Co., Ltd.Cell proliferation activity was determined by Cell Counting Kit 8 (CCK8) purchased from Beyotime  according to the manufacturer’s instruction. Briefly, the 96 well plate was seeded with 20,000 cells per well and cultured in 100 μl 10% FBS RPMI-1640 medium for 24 h. We next added 10 μl CCK8 reagent and incubated for 4 h. The plate was measured by automatics plate reader .7 cells/mL (2 × 106 cells/mouse). Tumor growth was monitored and measured every two days using digital caliper , and the tumor volume was calculated according to the following formula: M12*M2 /2 . When the tumor volume reached 100 mm3 (day 7), mice were treated with 1.0 mpk, 3.0 mpk or 10.0 mpk of anti-hPD-L1 antibody (the antibody was synthesized by MabSpace Biosciences (YW243.55.S70 or MPDL3280A as disclosed in the US Patent No. 8217149) or PBS vehicle control by intraperitoneal (i.p.) injection, and the second dose of treatment was given 4 days later. The mice were sacrificed for immune phenotype and function analyses either 4 or 10 days after the second dose of treatment.Female C57BL/6 mice were purchased from Vital River Laboratories . All mice were maintained in a barrier facility at Soochow University. To establish subcutaneous tumor, C57BL/6 mice were inoculated with various tumor cell lines in the right side axillae with 0.2 mL/mouse of the cells adjusted to 1.0 × 10Flow cytometry was performed with Life Attune Nxt FACS machine . Fluoro-chrome conjugated anti-mouse CD45 , CD3 , CD4 , CD8 , Foxp3 , Ki-67 , Granzyme B , Ly-6C , Ly-6G , mPD-L1 , hPD-L1  and IFN-gamma (XMG1.2). Granzyme B and IFN-gamma staining was performed with intracellular staining kit from BD Bioscience. While Ki-67 and Foxp3 staining was done with nuclear protein staining kit from eBioscience . FACS data were analyzed using FlowJo software .5 tumor tissue cells were added to the plate per well in 100 μl and then incubated the plate at 37 °C for 12 h in a 5% CO2 incubator. Discard the cell suspension and wash every micro-well by Elispot wash buffer. Added the diluted detection antibody at 2.0 μg/ml in 100 μl and incubated the plate at room temperature for 2 h. After washing with PBS, the plate was incubated with 100 μl streptavidin-HRP conjugate for 1 h at room temperature. Then washed the plate for 3 times by Elispot by wash buffer and added 100 μl final substrate solution for antibody detection. Stop the substrate reaction by washing wells with DI water. Enumerate spots of every micro-well by automatical ELISPOT plate reader (Celluar Technology Ltd. USA).The IFN-γ ELISPOT assay was performed according to manufacturer’s protocol from BD life science. Briefly, the ELISPOT plate was coated with anti-mouse IFN-γ capture antibody diluted in PBS at 5.0 μg/ml overnight at 4 °C. 2.0*10Statistical analysis was performed for all experiments using Student’s t-test. Values of p inferior to 0.05 were considered significant. All tests were done using Prism 6 software (Graph Pad Prism6).How to cite this article: Huang, A. et al. A human programmed death-ligand 1-expressing mouse tumor model for evaluating the therapeutic efficacy of anti-human PD-L1 antibodies. Sci. Rep.7, 42687; doi: 10.1038/srep42687 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Supercapacitors, as one of the energy storage devices, exhibit ultrahigh capacitance, high power density, and long cycle. High specific surface area, mechanical and chemical stability, and low cost are often required for supercapacitor materials. Graphene, as a new emerging carbon material, has attracted a lot of attention in energy storage field due to its intrinsic properties. Polymers are often incorporated into graphene for a number of enhanced or new properties as supercapacitors. In this paper, different polymers which are used to form composite materials for supercapacitor applications are reviewed. The functions, strategies, and the enhanced properties of graphene and polymer composites are discussed. Finally, the recent development of graphene and polymers for flexible supercapacitors are also discussed. Origina2(SO4)3] . With th2(SO4)3] . The rol2(SO4)3] .Fig. 8SE−1 [−1 in HCl and H2SO4 electrolytes, respectively [−1, and RGO/PEDOT composite electrode has a specific capacitance of 209 Fg−1 with 87% capacitance retention over 2000 cycles [Pristine PEDOT supercapacitors have a specific capacitance in the range from 70 to 130 Fg−1 , 60, 61 ectively . Wen et 0 cycles . The rolGraphene and its derivatives can have great influence on the morphologies, electrical properties, and the structural stabilities of the CPs when they are made into composites, and thus lead to drastic enhancement of the electrochemical properties of CPs. However, for a graphene/CPs supercapacitor, there are many factors which can determine the ultimate supercapacitor performance  such as polymerization method, intrinsic properties of individual CPs, electrolyte, dispersion/aggregation of the graphene in the composites, different properties of graphene used, etc. Generally speaking, for the aforementioned three types of CPs , there are distinct advantages and disadvantages of using individual CPs to form the composite. PEDOT has the largest molecular molar mass, hence, the specific capacitance of the graphene/PEDOT composites supercapacitor is normally smaller than the ones formed with PANI or PPy. On the other hand, the larger particles formed in the PPy film make it less porous than PANI or PEDOT, thus the cycling performance of PPy is normally the worst among these three CPs. In addition, the conductivity comparison of these CPs is PEDOT > PPy > PANI , which c−1 at the current density of 0.3 Ag−1, respectively. Figure Zhang et al. compared the supercapacitor performance of the composites formed of RGO nanosheets with PANI, PPy, and PEDOT polymers using similar polymerization and mixing methods . The speTable -1 for the 100-nm-thick graphene film. There are other types of flexible supercapacitor electrodes based on graphene materials, such as graphene paper [The recently developed flexible electronic devices such as flexible displays, curved smartphones, flexible implantable medical devices, and wearable electronic devices imply that flexible devices are beginning to emerge as the leading revolution in next generation of electronics. Several advantages of flexible electronic devices compared to conventional electronic devices include lighter weight, wearability, bendability, environmental friendliness, reduced cost, etc. In order to match the fast growth of flexible electronic devices, energy storage systems that are light, thin, and flexible should also be developed. Recently, considerable attempts have been made to develop flexible supercapacitor electrode based on carbon materials including activated carbon , 66, carne paper , 78, grane paper , 80, andne paper –83. Howe3PO4 gel was used as the solid electrolyte in this device, and gold was sputter coated on one side of the electrode as the metal current collector [Graphene/conducting polymers (CPs) composite film, due to its flexible nature, becomes a viable option for flexible supercapacitor applications as it can also adapt to the roll-to-roll manufacture. Graphene/CPs film can be assembled with solid electrolyte to form a flexible supercapacitor device which can be subject to high degrees of bending or twisting without losing the device integrity. Meanwhile, as it is discussed previously, CP itself can provide pseudocapacitance due to the redox reaction, increasing the specific capacitance, as well as the capacity of the device. Figure ollector . Polymerollector .Fig. 12SBending test is usually necessary to examine the structural and electrical integrity of the flexible supercapacitor device subject to different bending angles and cycles. Bending or twisting can cause stress on the films which may lead to material loss, material fatigue  or even material failure . CV and/or charge-discharge curves are monitored at several of bending angles during the bending cycles , 86–90 tThe emergence of graphene has undoubtedly changed the scope of supercapacitor field due to its outstanding electrochemical properties along with other unique properties such as large surface area, high electrical conductivity, light weight, and mechanical strength. The recent development of graphene and polymer composites showed very promising features of these composite materials for supercapacitor applications. Compounded with binder polymers, graphene would compensate the undesired features of insulating polymers . On the other hand, when graphene makes composites with conducting polymers, it would provide mechanical support for the framework of the polymers and thus greatly improve the cycling performance as well as the specific capacitance. In addition, the flexible nature of the graphene/polymer film makes it possible for flexible, wearable, conformable energy storage devices. Despite the innovative ideas and techniques that have been demonstrated for the graphene/polymer supercapacitor device with unique features not possessed by current state-of-art technology, there remain a lot of challenges for graphene/polymer composite-based supercapacitors to reach their full potentials. One of the main challenges is to find a feasible way for the low-cost mass production of graphene/polymer supercapacitor electrode without compromising the micro/nanostructures of graphene due to the restacking or aggregation. Many studies have demonstrated the compatibility of large-scale coating or roll-to-roll manufacture capability of graphene/polymer supercapacitor electrodes, however, there is still a need to have a rational design of the porous structures inside the supercapacitor electrodes to form hierarchical interconnected porous microstructures and avoid the formation of dead volume or the collapse of the porous microstructures . Further


The magnetic circular dichroism and the Faraday rotation are the fundamental phenomena of great practical importance arising from the breaking of the time reversal symmetry by a magnetic field. In most materials, the strength and the sign of these effects can be only controlled by the field value and its orientation. Furthermore, the terahertz range is lacking materials having the ability to affect the polarization state of the light in a non-reciprocal manner. Here we demonstrate, using broadband terahertz magneto-electro-optical spectroscopy, that in graphene both the magnetic circular dichroism and the Faraday rotation can be modulated in intensity, tuned in frequency and, importantly, inverted using only electrostatic doping at a fixed magnetic field. In addition, we observe strong magneto-plasmonic resonances in a patterned array of graphene antidots, which potentially allows exploiting these magneto-optical phenomena in a broad THz range. Time reversal symmetry breaking gives rise to magnetic circular dichroism and Faraday rotation in graphene. The authors use terahertz magneto-electro-optical spectroscopy to demonstrate that electrostatic doping at a fixed magnetic field allows inversion of magnetic circular dichroism and Faraday rotation. Terahertz waves are actively explored for fundamental research and applications1345678111214151617181920214237Here we combine MO THz spectroscopy with electrostatic gating and graphene patterning. This allows us to observe several new phenomena, including an electrostatic control of the THz cyclotron frequency, a purely electrostatic inversion of the magnetic circular dichroism (MCD) and the Faraday rotation (FR) and strong gate-tunable magneto-plasmonic resonances in periodic antidot arrays. We argue that these effects potentially allow a broad spectral tunability and enable novel functionalities in graphene-based passive terahertz devices.n (positive for electrons and negative for holes) with magneto-transport and MO measurements in a perpendicular magnetic field B (positive towards the light source). The THz transmission T(ω) and the FR spectra θF(ω) were obtained while graphene was illuminated by a linearly polarized light with the angular frequency ω. The Fabry–Perot interference in the substrate was suppressed intentionally to simplify the spectra analysis. From these measurements, we extracted the transmission spectra for the right-hand (RH)/left-hand (LH) circular polarizations, T±(ω)We first discuss the intrinsic MO terahertz effects in continuous (unpatterned) graphene. A basic device used in our experiments is a large-area CVD -graphene field-effect transistor (g-FET) sketched in Vg in all our devices has a characteristic peaked shape /TCNP(ω), which are representative of the optical absorption in graphene , τ is the scattering time and ± refer to RH/LH circular polarizations. A relation between σ± and T± that takes the substrate into account is given in where Before we analyse the dependence of the Drude parameters on the doping and the magnetic field, we stress that the Dirac-fermion theory  predictsandvF is the Fermi velocity, ħ the reduced Planck's constant and e the (positive) elementary charge. This behaviour is different from the case of conventional 2DEGsn, while the cyclotron frequency is n-independent. Notably, where τ extracted by fitting the zero-field extinction curves (where ωc=0) are strongly doping dependent  for the LH circular polarization, where the CR is observed in this case, are shown in B .In a magnetic field, the terahertz spectra are dominated by the cyclotron resonance (CR) . The extinset of  and a ren changes sign. The inversion of the MCD is thus due to the fact that the polarization where the CR is observed is defined by the doping type. This new effect that enables novel applications (as discussed below) is thus directly related to the unique possibility of ambipolar doping control in grapheneA) as in continuous graphene and two additional peaks (B and C) due to Bragg scattering of graphene plasmons on the periodic structure. Our previous study revealed that peak B is mostly due to the -order Bragg reflection, while peak C has a mixed – character. Importantly, they both demonstrate a As demonstrated above, the non-reciprocal MO effects in unpatterned graphene are especially strong at low frequencies (below 5–7 THz). However, in the higher THz region the Drude response is less efficient. In order to broaden further the tuning range of the magneto-teraherz response, one can use the new physical properties arising from the excitation of magneto-plasmons  in graphene nanostructures23343536fermions 41415 thaA comparison of A shifts upwards in the LH polarization while keeping the intensity constant. Although this resembles the behaviour of the CR peak in continuous graphene . Most notable is a strong increase/decrease of their intensity for the LH/RH polarization, accompanied by field-induced shifts as indicated by the dashed lines in B, where opposite shifts are observed in the two polarizations. Associated with these spectacular magneto-plasmonic effects are the resonant MCD and FR structures  between the experimental and the simulated frequencies. Nevertheless, overall our simulations based on the Dirac-fermion theory prove to be quantitatively accurate also in patterned graphene.To give a further physics insight into the origin of the observed MO resonances, we show in μ≈3,500 cm2 V−1 s−1 in our present g-FETs is largely limited by the substrate (SiO2/Si), and high magnetic fields (up to 7 T) are needed to attain reasonably good device specifications452 V−1 s−1 at room temperature was recently obtained in h-BN-encapsulated CVD graphene using a dry-transfer techniqueOur results indicate that combining terahertz spectroscopy, electrostatic gating, magnetic field and pattering reveals interesting properties of the Dirac fermions, such as a broadband electrostatic control of the cyclotron frequency and relativistic magneto-plasmons, and give rise to qualitatively new phenomena, such as a purely electrostatic control of the magnetic effects (MCD and FR). Importantly, the experiments were done close to room temperature, in a striking contrast to the existing 2DEG studies3839As we demonstrated, the unusual properties of Dirac fermions allow a wide tuning of the working (cyclotron or magneto-plasmon) frequency, potentially in the entire THz range, by the doping, magnetic field and patterning parameters. Moreover, the possibility to invert the MCD and the FR electrically opens the door to completely new functionalities, such as electrically switchable isolators, and to combining several basic functions in a single device.Importantly, in the present case the MO effects occur in an atomically thin layer, therefore the device thickness is limited only by the substrate and external optical elements . This potentially allows a miniaturization of the graphene-based elements, in a stark contrast with conventional ones, where light has to travel a centimetre-scale distance. The scope of the MO terahertz applications of graphene is certainly not limited to free-space wave propagation, as in the present experiments, and we anticipate that its remarkable properties will be employed in miniaturized chip- and THz waveguide-integrated2 that served as a back-gate dielectric. Au/Ti electrodes were deposited outside the illuminated region (5 × 5 mm2) allowing transport measurements and back-gate control simultaneously with MO studies. The carrier concentration was calculated using the formula n=α, where α=7.2 × 1010 cm−2 V−1 is the gate capacitance and Vg, CNP is the gate voltage corresponding to the maximum resistivity. A perpendicular magnetic field up to 7 T was applied using a superconducting split-coil system. The sample was illuminated by linearly polarized light and the terahertz transmission and FR spectra using the technique presented in ref. Large-area monolayer CVD graphene was grown on copper and transferred to a weakly doped Si substrate with a 300 nm layer of SiOFinite-element electromagnetic simulations of antidot arrays were performed using the COMSOL software. The graphene layer was modelled as a surface current in the boundary conditions. In order to achieve convergence, the mesh element size in the vicinity of graphene was much smaller than the magneto-plasmon wavelength.The data that support the findings of this study are available from the corresponding author upon request.How to cite this article: Poumirol, J.-M. et al. Electrically controlled terahertz magneto-optical phenomena in continuous and patterned graphene. Nat. Commun.8, 14626 doi: 10.1038/ncomms14626 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Notes and Supplementary References


The spectral singularity existing in PT-synthetic plasmonic system has been widely investigated. Only lasing-mode can be excited resulting from the passive characteristic of metallic materials. Here, we investigated the spectral singularity in the hybrid structure composed of the photoexcited graphene and one-dimensional PT-diffractive grating. In this system, both lasing- and absorption-modes can be excited with the surface conductivity of photoexcited graphene being loss and gain, respectively. Remarkably, the spectral singularity will disappear with the optically pumped graphene to be lossless. In particular, we find that spectral singularities can exhibit symmetry-modes, when the loss and gain of the grating is unbalanced. Meanwhile, by tuning the loss (gain) of graphene and non-PT diffraction grating, lasing- and absorption-modes can also be excited. We hope that tunable optical modes at spectral singularities can have some applications in designing novel surface-enhanced spectroscopies and plasmon lasers. Beyond some non-Hermiticity threshold, typically called the exceptional point, PT-symmetric systems can display an abrupt phase transition and the corresponding eigenspectra become complex. Recently, the concept of PT-symmetry has been fruitfully extended to wave optics. Photonic platforms are ideally suited to implement the structures with PT-symmetry, which is directly translated into a requirement for the arrangement of elements with balanced gain and loss25. Various intriguing optical phenomena have been revealed in PT-symmetric systems, such as asymmetric light propagation9 and invisibility11, Bloch oscillation of energy12, coherent perfect laser absorber17, PT-symmetric metasurfaces21, single-mode laser23, reversing the pump dependence of a laser24 and loss-induced suppression and revival of lasing25.Since the pioneering work of Bender 30. Besides the exceptional point, the spectral singularity32 existing in the PT-plasmonic system, which is related to scattering resonance of the non-Hermitian Hamiltonian and manifests itself as giant transmission and reflection with vanishing bandwidth, also has unique features. For example, hugely anisotropic optical scattering33, three-dimensional light confinement34, and loss-induced super scattering36.More recently, PT-symmetric systems built using plasmonic elements have attracted increased attention. The strong interaction between surface plasmons and electromagnetic field can enhance the extraordinary properties associated with exceptional points38. For example, nanopatterned graphene can be used as an active medium for infrared electro-optic devices40. Meanwhile, loss induced amplification of graphene plasmons41, regulation of energy distribution in graphene42, and singularity-enhanced sensing based on the PT-graphene metasurface43, as characteristics of exceptional point behaviors, have been demonstrated theoretically. However, characters of spectral singularities in graphene-based PT systems has not been discussed.On the other hand, there is a rapid progress in the field of graphene plasmonics motivated by the unique electrical and optical properties of graphene47. Such diverse surface dynamic conductivity responses can be used to manipulate optical modes of spectral singularities in graphene-based PT-systems. When the grating possesses perfect PT-symmetry, the spectral singularity will present the feature of lasing-mode (electric fields concentrate mainly on the gain element) or absorption-mode (electric fields concentrate mainly on the loss element) with the loss of optically pumped graphene being negative or positive, respectively. It is noted that spectral singularities are vanished if the loss of optically pumped graphene becomes zero. In particular, the spectral singularity may exhibit symmetry-modes , when the loss and gain of the grating is unbalanced. In this case, the spectral singularity on the lasing or absorption-mode also appears with the loss and gain for the grating exceeding the value of the corresponding symmetry-modes.In this work, we systematically investigate characteristics of the eigenmode at spectral singularity when the electromagnetic field, in terahertz (THz) regime, is incident on an optically pumped monolayer graphene underneath the one-dimensional gain-loss diffractive grating. The intrinsic loss of graphene can exhibit negative, zero, and positive values resulting from the population inversion produced by cascaded optical-photon emissionuFe and holes uFh of the same absolute value uFe = −uFh = uF. Since the relaxation time for intraband transition is much faster than the recombination time for electron-hole pairs, the population inversion can be achieved with optical pumping. In this condition, the complex intraband and interband conductivities, 48:e is the electric charge, T is the temperature, and T = 3 K and quasi-Fermi-level is uF = 100 meV. As can be seen, the loss of graphene 49. The amplifying view of a unit cell is clearly presented in the inset of Fig. p = 8 um. The sizes of the grating along x- and y-axis are 2.0 um and 1.8 um, respectively. The relative permittivities of the gratings are given by Fgain and Floss being the non-Hermiticity coefficients of the system. It is noted that the whole structure possesses the PT-symmetry, only when distributions of gain/loss elements are balanced (Fgain = Floss) and the dynamic conductivity of graphene becomes zero . Only two modes are considered here with eigenfrequencies  locating within 6–8 THz. Although the system has no loss or gain element, the imaginary part of eigenfrequency is still non-zero resulting from the existence of radiation loss, which is largest at the Brillouin center and vanished at Brillouin boundaries. As we turn on the gain and loss (Fgain = Floss = 0.05), the gap of the real part of eigenfrequency at the Brillouin boundary will be closed and the imaginary part separated, giving rise to the exception point (red arrow), as shown in Fig. Firstly, we proceed to investigate the interaction between the PT-diffraction grating and lossless monolayer graphene , as shown in the inset of Fig. F, as shown in Fig. In order to observe the evolution of eigenmode with the variation of non-Hermiticity coefficient, in Fig. kx = 0 in a complex frequency plane are shown in Fig. F being increased. The black dot line represents the condition that the real part of the surface conductivity of graphene is positive . The evolutions of eigenfrequencies for the states at F = 0.06613 and F = 0.06455, respectively. It is presented that the giant transmittance and reflectance appear, and corresponding near-field distributions are shown in Fig. The spectral singularity manifests itself as giant transmission and reflection with vanishing bandwidth. In Fig. Fgain = Floss = F, and the optically pumped graphene. The evolutions of the eigenfrequencies for the states at kx = 0 in a complex frequency plane are shown in Fig. F being increased. When the surface dynamic conductivity of graphene is active 1, 2, 3) and each possesses different eigenmodes, shown in right insets of Fig. 1, 2) possess symmetric eigenmodes before the eigenfrequency passes through the inflection points (marked by the blue arrows). Beyond the inflection points, the lasing-mode, which previously left away from the lasing threshold, will approach to it again and the corresponding spectral singularity reappears. This phenomenon follows the main characteristics of the quasi-PT-systems with exceptional points, like loss-induced suppression and revival of lasing15, and reversing the pump dependence of a laser14. It is noted that spectral singularities only exhibit symmetry-modes, when the distributions of the loss and gain for the diffractive grating are unbalanced. Moreover, in Fig. Finally, we will investigate the interplay between a non-PT diffraction grating, 2Fgain = 2Floss = F. Characteristics of the corresponding spectral singularities in a manner analogous to the above case are shown in Fig. 1, 2) possess symmetric eigenmodes and another one presents features of the absorption-mode. Similarly, the giant transmission and reflection are also excited at frequencies of the corresponding spectral singularities.We also consider the condition, when non-Hermiticity coefficients satisfied the relationship of In conclusion, we have demonstrated numerically that eigenmodes at spectral singularities can be conveniently tuned by a suitable variation of the loss and gain in the graphene-based quasi-PT systems. When the diffractive grating, which assisted graphene plasmonics excitation, has the perfect PT-symmetry, the spectral singularity can present the feature of lasing- or absorption-modes, which is decided by the intrinsic property of loss or gain characteristic for the surface conductivity of the pumped graphene. These spectral singularities vanished if the surface resistance of graphene becomes zero. In particular, the spectral singularity may exhibit symmetry-mode only with the asymmetric distribution of the loss and gain for the diffractive grating. Furthermore, with the increasing of non-Hermiticity coefficients, the spectral singularities with asymmetric eigenmodes reappeared. In contrast to the previous method to control the near-field by modifying the geometrical parameters of the plasmonic structures, we can tune the near-field distributions around the graphene-based PT-system by just varying the surface dynamic conductivity of graphene. We hope that our finding may have some applications in designing novel surface-enhanced spectroscopies and plasmon lasers.All full wave numerical simulations and dispersion relations were done using finite element method (Comsol Multiphysics). In the simulations, the graphene is modeled as a two-dimensional surface with complex conductivity. The mesh size inside the graphene layer is 0.5 nm, which is fine enough for convergence.


We also prove that multiple carriers exist in vertically aligned graphene.We investigated how to control the growth of vertically aligned graphene on C-face SiC by varying the processing conditions. It is found that, the growth rate scales with the annealing temperature and the graphene height is proportional to the annealing time. Temperature gradient and crystalline quality of the SiC substrates influence their vaporization. The partial vapor pressure is crucial as it can interfere with further vaporization. A growth mechanism is proposed in terms of physical vapor transport. The monolayer character of vertically aligned graphene is verified by Raman and X-ray absorption spectroscopy. With the processed samples, d Graphene is a 2D carbon allotrope with honeycomb monolayers. This kind of unique structure exhibits amazing electronic, photo-electric, magnetic, mechanical, and chemical properties3675678Vertically aligned graphene1216171819201416171920211511122729Although the properties of vertical graphene have been studied well, its growth on SiC is hard to control and its growth mechanism is not clear, which will hinder its further development and application. To promote the potential application, it is necessary to determine how process parameters (such as temperature and pressure) affect the graphene growth. Here, we investigated the growth of vertically aligned monolayer-like graphene on C-face SiC. The influence of temperature, annealing time, temperature gradient, pressure, and crystalline quality of SiC substrates was studied. Based on the growth, the magnetism and transport properties in as-grown and Cu-implanted vertical graphene were explored.To confirm the temperature threshold required to grow the vertically aligned graphene, the substrates were annealed at different temperatures for 2 hours between 1400 °C and 1900 °C. No graphene was obtained using temperatures of 1400 °C to 1600 °C. Vertically aligned graphene begins to grow when the temperature reaches 1700 °C. With further increasing temperature, graphene growth speeds up. We find that the growth rate increases with temperature. The height of the obtained graphene in different samples is listed in Usually, epitaxial planar graphene is compact and it will prevent the vapor release, so planar graphene cannot grow too thick. However, the situation for vertically aligned graphene is unclear. Can tunnels inside the vertical graphene increase or decrease the sublimation rate? The vertically aligned graphene was prepared with annealing times varying up to 40 hours, as shown in When the C-side is faced up with the initial setup (6 mm of susceptor thickness and 175 mm of mount height), the C-side faces towards the temperature maximum and the vapor cannot escape as it usually moves from high to low temperature. So no graphene can grow during annealing.When the C-side is faced down with the initial setup, the substrates cross the position of temperature maximum, the vapor can escape easily and then vertically aligned graphene can be obtained.When the susceptor thickness or the mount height increases, the substrates move away from the temperature maximum, the temperature gradient increases and graphene can grow faster.When the susceptor thickness or the mount height continuously increases, the substrates move further away, the temperature drops and then the growing rate decreases, considering that the difference is dramatically increased between the sample temperature and the measured temperature.The crystal growth is sensitive to the temperature distribution in the crucibleTherefore, altering the position of the substrates is an important means to control the growth of vertically aligned graphene.The substrates were loaded both in the middle and at the edge of the susceptor to grow graphene simultaneously for 8 hours. The height of the graphene grown in the middle is 22 μm compared with 58 μm at the edge. As the heat is generated only on the outer lateral surface of the susceptor and the lid, the edge will have both higher temperature and higher temperature gradient than the inner part, which can accelerate graphene growth. However, the direction of temperature gradient is not perpendicular to the substrate surface, which may result in a height inhomogeneity in lateral direction. It is also noticed that, the substrates are located in the middle of the susceptors when graphene grew with different susceptor thicknesses, while they are at the edge of the susceptors when different mount heights are applied. Therefore, the former graphene heights are smaller than the latter ones because of the relatively low temperature and relatively gradual temperature gradient See .The residual pressure in the chamber also affects the growth of vertically aligned graphene. For this investigation, we used a mechanic pump instead of the turbo molecular pump. The pressure maintained at about 9 mbar. With the susceptor thickness of 6 mm and the mount height of 178 mm, the graphene height reduces from 15.7 μm to 9.4 μm. It can be understood that when the pressure increases, the vapor partial pressure around the substrates rises and the convection decreases the temperature gradient, both of which will suppress the sublimation. If several pieces of substrates are annealed at one time, the height of the obtained graphene will also decrease. For instance, the height was 28 μm after annealing 8 pieces for 8 hours, compared with 40 μm for annealing only 1 piece, as the total Si-evaporation from 8 pieces raises the Si-partial pressure higher than that of 1 piece and it will suppress the sublimation on each single sample. This result reveals the evacuation of the vapor created from the surface of substrates is critical to the growth of vertically aligned graphene.It is known that the arrangements of vertical graphene arrays are different when grown on substrates with different crystallographic orientation2, Si2C, Si and C) can be transported rapidly with the proper temperature gradient discussed above (Some may deposit on the inner surface of the lid.), the sublimation will continue. As the saturation vapor pressure of Si is higher than that of C at the same temperature, carbon will be left and will reconstruct. The reconstruction of C atoms resulting in vertically aligned graphene is more complicated. Considering the difference between the temperature, the temperature gradient, and the pressure utilized to prepare planar graphene and those of vertically aligned graphene, the vaporization rate could be the key factor in determining the structure after reconstruction. When the vaporization is high enough, the initial C atom sheets will vertically stand due to the strong gas flow. So graphite or planar graphene cannot form but only the low density of vertical graphene makes the crystallization possible. The guide of the high speed gas flow, the annealing under a high temperature, and the etching of Si atoms will further improve the standing thin film into vertically aligned graphene. The schematic is shown in According to the results above, the growth of vertically aligned graphene can be understood in terms of SiC single crystal growth using the physical vapor transport method with a single wall crucible343537G and W2D) from 24 to 29 cm−1 and from 47 to 53 cm−1 shows the similarly high quality of all samples. The 2D peaks are symmetric and well match a Lorentzian line, so the interaction between the adjacent layers is weak and graphene layers exhibit a monolayer behavior. It is heeded that the difference between measured samples is not only limited to their graphene height. They were prepared under different conditions: the 241 μm sample has grown for 40 hours; the 40 μm sample used 1700–1800 °C for 10 hours; and the Si-face of the 1.3 μm sample was up during the process  and the carrier concentration of about 3 × 1018 cm−3, and the hole of 1 × 104 cm2/(Vs) and 1 × 1019 cm−3 according to a two-band model454 cm2/(Vs) while the concentration reduces slightly to 2 × 1018 cm−3. The hole Hall mobility and concentration almost remain unchanged after implantation. It is speculated that the impurity band of Cu is located a little bit below the major negative carrier so Cu doping can shift the Fermi level towards the Dirac cone and thus increase the mobility.In as-grown samples, only diamagnetism is observed. After implantation, the magnetism after removing the diamagnetic background is shown in 0 magnetism of 0.97 μB per Cu at 300 K is revealed in Cu-implanted vertical graphene. The negative magnetoresistance due to weak localization after implantation is observed. Vertical graphene possesses both electron and hole as major carriers, whose Hall mobility is more than 1 × 104 cm2/(Vs). In implanted samples, its electron Hall mobility is further improved to 9 × 104 cm2/(Vs). The results will be helpful to extend the research area and the potential application of vertically aligned graphene.In conclusion, the growth control of vertically aligned graphene on C-face SiC has been investigated. The influence of processing conditions is demonstrated. A temperature higher than 1700 °C is indispensable and the growth will accelerate with temperature increase. The graphene height is basically proportional to the annealing time. Proper temperature gradient and good crystalline quality of SiC substrates can further associate the vaporization of substrates and promote the growth. Pressure does not directly affect the growth while the partial pressure of vapor is crucial as it can interfere with further vaporization. The growth mechanism is proposed in terms of physical vapor transport. The monolayer character is verified by Raman and XAS spectra regardless of the graphene height, which indicates the robustness of the growth method. With the obtained samples, the d3.We used commercially available SiC substrates (Beijing TankeBlue) to grow vertically aligned graphene. Without special notation, the substrates are conductive C-face 4H-SiC of dummy level with dimensions of 10 × 5 × 0.33 mm−6 mbar. SiC substrates with C-side facing up are loaded into a graphite susceptor with a lid. The susceptor is placed in the middle of the chamber supported by a graphite tubular mount  with an induction coil and a turbo molecular pump, which can reach temperatures as high as 2000 °C and a vacuum of ~10ar mount . The tem14 to 1 × 1016 cm−2 are labeled according to their fluence. Cu distribution is demonstrated in inset of 20 cm−3 for the sample with the Cu fluence of 1 × 1016 cm−2.Some vertical graphene samples were implanted with Cu ions after annealing. The incident energy and angle were 190 keV and 7°, respectively. The samples with the Cu fluence varying from 1 × 10−1. X-ray absorption spectroscopy (XAS) was performed at beamline 6.3.1 of the Advanced Light Source using the total electron yield mode at room temperature. Rutherford backscattering (RBS) spectrometry with ~1.7 MeV He ions was employed to determine the composition distribution. The magnetic properties were measured by a vibrating sample magnetometer equipped with a commercial superconducting quantum interference device  with a sensitivity limit of 10−7 emu in the field range of 5 kOe at 300 K. The transport measurements were executed using a current of 1 mA under the magnetic field of 50 kOe at 5 K with a Lakeshore system.Scanning electron microscopy (SEM) micrographs were captured using an S-4800 field-emission SEM (Hitachi) operated at an acceleration voltage of 10 kV. Room temperature Raman measurements were carried out at a LabRAM (HORIBA Jobin Yvon) using a 532 nm Nd:YAG laser as excitation source with a resolution of ~0.5 cmHow to cite this article: Liu, Y. et al. Controllable growth of vertically aligned graphene on C-face SiC. Sci. Rep.6, 34814; doi: 10.1038/srep34814 (2016).