For neutral graphene films soaked with different amounts of electrolytes (1 M TEABF4/ACN), weakly and strongly adsorbed anions are identified based on the resonances at different 11B chemical shifts. Unlike other porous carbonaceous materials, the strongly adsorbed anions are found as the major electrolyte anions components in graphene films. Further measurements on the ion population upon charging are carried out with applying different charging voltages on the graphene films. Results indicate that the charging process of graphene-film based EDLCs can be divided into two distinct charge storage stages  for different voltages. The as-obtained results will be useful for the design and fabrication of high performance graphene-film based EDLCs.Graphene film has been demonstrated as promising active materials for electric double layer capacitors (EDLCs), mainly due to its excellent mechanical flexibility and freestanding morphology. In this work, the distribution and variation pattern of electrolyte ions in graphene-film based EDLC electrodes are investigated with a  With ex situ magic-angle spinning (MAS) NMR, comprehensive information on the anions, cations, and remaining solvent molecules inside or outside the porosity of active carbons were provided by Deschamps et al.4 in acetonitrile (ACN) electrolyte, Wang et al.20in situ measurements were also conducted on carbons soaked in different organic33358173738NMR studies have been applied to porous materials for the understanding of the adsorption of molecules in micro- and nano-sized pores. Harris Graphene is a two-dimensional nanomaterial with huge specific surface area and a series of unique physical, chemical and mechanical properties40421044454411B MAS-NMR spectroscopy was applied to graphene-film based EDLCs using TEABF4/ACN electrolytes. The adsorption behaviors of BF4− anions in neutral graphene films with different electrolyte feedings were investigated. Graphene-film based EDLCs were then assembled and the changes in the population of anions with different adsorption states in electrode materials were measured at different charging voltages. The as-obtained results provide useful insights into the charge storage mechanisms in graphene nano-channels, which could be helpful in designing high-performance EDLCs.In this work, solid-state 11B MAS-NMR spectra of graphene films soaked with different amounts (50~200 μL) of 1 M TEABF4/ACN electrolytes. Each spectrum is fitted with mixed Gaussian/Lorentzian lineshapes, where the as-obtained NMR spectra are shown in black, the fitted components are shown in dark blue and purple, and the total fitted lineshapes are shown in red. At a relatively low electrolyte loading of 50 μL, a single resonance was detected at the 11B chemical shift of −10.02 ppm. With the increase of the electrolyte loading, this resonance increased in intensity and shifted in peak position to higher frequencies , reaching to −5.17 ppm with the electrolyte loading volume of 200 μL. Meanwhile, a second resonance at higher frequency emerged with the electrolyte loading volumes lager than 75 μL. The chemical shift of the second resonance (−2.75~−2.28 ppm) is close to that of crystalline TEABF4  arise from the 11B atoms of strongly bound BF4− anions on carbon surface, which is herein referred to as “strongly adsorbed state”. Density functional theory (DFT) calculation indicates that the “ring current” effects on adsorbed ions increased rapidly with the decreasing ion-surface distance, which is strongly related to the size of graphene domains, pore width, and curvature of carbon surface18e.g., −2.75 ppm for electrolyte loading volume of 75 μL) are assigned to the weakly bound BF4− anions. These anions situated further from the graphene surfaces, corresponding to the “weakly adsorbed state”. The anions at this position were weakly affected by the graphene surfaces and therefore presented a chemical shift close to that of crystalline TEABF4. Due to the rapid dynamic exchange of electrolyte ions between the weakly and strongly adsorbed sites on the NMR timescale30The different chemical shifts of the resonances provide an indication of their origin in graphene materials4− anion population in graphene film was obtained, providing a relationship to quantify the absolute number of ions for a given resonance intensity in the NMR spectrum. 4/ACN. Both the strongly and weakly adsorbed resonances intensities increased with the increasing electrolyte loadings. For each certain electrolyte loading, the strongly adsorbed resonances presented a higher intensity than that of the weakly adsorbed resonances. This observation is different from previous NMR results on some other porous carbon materials, where the weakly adsorbed  resonances presented a higher intensity than the strongly adsorbed  counterparts2931e.g., prepared by mixing 95 wt% carbon powder with 5 wt% PTFE2032334− anions were the NICS averaged over the pore space and had a less negative value than that of anions adsorbed on graphene surfaces234− anions are preferentially adsorbed on the graphene surface . This phenomenon could be attributed to the high adsorption ability of graphenei.e., the ex-pore) adsorption does not occur until the strongly  adsorption has attained saturationi.e., the strongly adsorption was not saturated), when the weakly adsorbed anions were observed. Both the weakly and strongly resonances intensities increased with the increasing electrolyte loadings, indicating that the strongly and weakly adsorption were carried out simultaneously. For the relatively high electrolyte loadings of more than 75 μL, the intensities of both the strongly and weakly adsorbed resonances increased as a function of electrolyte loadings, indicating that the number of BF4− anions in this two adsorption sites increased gradually. This observation could be attributed to two possible adsorption processes of electrolyte ions. With the increase of electrolyte loadings, the electrolyte ions entered into the inner space of the graphene channels, and the volume occupied by the electrolyte ions increased gradually. Meanwhile, the packing density of the ions within graphene nano-channels increased with the increasing electrolyte loadings, and therefore more ions were stockpiled in graphene films .η was introduced:To further describe the ion packing behavior during the adsorption process, a coefficients Vion and Vpore represent the volume of electrolyte ions and the pore volume of graphene film, respectively. According to N2 adsorption-desorption isotherms, the graphene film used in the current work has a pore volume (Vpore) of 2.11 cm3 g−1, which is in agreement with the results of other researches49where Vion, can be calculated as:Assuming that there are equal numbers of anions and cations in graphene film, the total volume occupied by full-solvated electrolyte ions, dcation represents the diameter of the full-solvated cations (TEA+), danion represents the diameter of the full-solvated anions (BF4−), and λ refers to the packing factor of full-solvated electrolyte ions. For TEABF4/ACN electrolyte, dcation and danion are 1.3 nm (TEA+) and 1.16 nm (BF4−), respectivelydcation nearly equals to that of danion, the minimum value of Vion can be estimated by considering a maximum packing density of solvated anions and cations where c is the electrolyte concentration, V represents the loading volume, Na refers to the Avogadro’s number and m is the sample weight.where η were calculated as 0.63 and 0.95, respectively. These values are less than 1, which means that the volumes occupied by full-solvated electrolyte ions are less than the pore volume of graphene film. The coefficients η were calculated as 1.3, 1.6, 1.9 and 2.5 at the electrolyte loading volumes of 100, 125, 150 and 200 μL, respectively. It suggests that the total volume occupied by electrolyte ions is higher than the pore volume of graphene films, indicating the partial desolvation of ion solvation shells during the adsorption process.For the electrolyte loading volumes of 50 and 75 μL, the coefficients 11B MAS-NMR spectra of positive and negative electrodes at different charging voltages. For the positive electrodes, the peak intensity of NMR lines first decreased and then increased with an increasing applied voltage from 0 to 2.5 V. Two resonances corresponding to weakly and strongly adsorbed BF4− anions were unveiled by deconvolution of these as-obtained NMR spectra. The strongly adsorbed resonances played a predominant role on the charging process. For the negative electrodes, similar observations were also obtained upon charging.4− anions were departed from the graphene channel during the charging process. It suggests that the charge storage in graphene electrode materials was not driven by the adsorption of anions as the traditional theory supposed+ cations was the primary mechanism for the charging process. At the charging voltage higher than 1.0 V, an abrupt increase in the intensity of strongly adsorbed resonances was observed. It indicates that a large number of BF4− anions were adsorbed into the graphene nano-channels upon charging. Therefore, the adsorption of anions played a more significant role in charge storage between 1.0 and 2.5 V. Similar trends were observed for the negative electrodes  within graphene nano-channels decreased firstly and then increased sharply with the charging voltages higher than 1.0 V.+ cations in electrodes, the TEA+ ionic charge stored on positive electrodes was calculated based on the principle of charge conservation. + ionic charge  within the graphene channel decreased greatly at the charging voltage lower than 1.0 V. As discussed in the previous section, the expulsion of cations was the primary mechanisms of charge storage in graphene electrodes. Excess cations have been expelled from the graphene nano-channels upon charging to balance the electronic charge that accumulated in the graphene surface. Meanwhile, the population of anions decreased abnormally, possibly because amounts of BF4− anions were taken out of the channel by the cations during the charging process. Similar observation was also reported by Luo et al.+ and BF4− on negative charging above 0.6 V was observed. This abnormal phenomenon was caused by the competing effect between the ion-ion correlations and the ion-surface electrostatic interactions. In this case, the decreasing TEA+ cations concentration favors the dragging of BF4− anions out of the graphene channels because of the strongly ion-ion correlations between electrolyte ions. As the charging voltage increased from 1.0 to 2.5 V, a gradually increase in the TEA+ ionic charge was observed, which means that a number of TEA+ cations were brought into graphene films by anions upon charging.In order to understand the migration of TEA+ cations. Meanwhile, a number of anions were taken out of graphene films by cations because of the ion-ion correlations between BF4− anions and TEA+ cations . An opposite phenomenon showed up at the charging voltages of 1.0 V~2.5 V. In such a voltage range, the charge storage process is dominated by the adsorption of BF4− anions. At the same time, a number of TEA+ cations were taken into the graphene nano-channels by BF4− anions upon charging because of the strongly ion-ion correlation electrolyte ions . The charge storage in negative electrodes has the similar process with the increasing charging voltage  played an important role in the charge storage process. In this stage, the co-ions were expelled from graphene nano-channels with the increasing of charging voltages. At a higher charging voltage (1.0~2.5 V), the charge storage process was dominated by the adsorption of counter-ions . Large numbers of counter-ions were adsorbed in to the graphene channels to balance the electronic charge that accumulated in the graphene surface. During the charging process, the abnormal decrease or increase in the number of counter-ions or co-ions within graphene channels arised from the strongly ion-ion correlation between cations and anions.Two distinct charging stages were observed on the graphene-film based EDLCs charged at voltage values varying from 0 to 2.5 V. At low charging voltages (0~1.0 V), the expulsion of co-ions . The ion population of weakly and strongly adsorbed anions increased as a function of electrolyte loadings, and partial desolvation of ion solvation shells was found at higher loading volumes (>100 μL). Unlike previous NMR studies on other porous carbons and CNTs, the number of strongly adsorbed anions was much more than that of the weakly adsorbed anions at each certain electrolyte loading, mainly due to the unique two-dimensional layered structure of graphene films. The NMR experiments carried out at different voltages showed that the charging process can be divided into two distinct charge storage stages for different voltage ranges . The abnormal decrease or increase in the number of counter- or co-ions  could be attributed to the ion-ion correlation between cations and anions. The results obtained in the current NMR measurements provide useful insights for advancing the optimization of graphene-film based EDLCs.In summary, Graphite oxide (GO) powder (250 mg) synthesized by modified Hummer’s method was dispersed in deionized water (1 L) and ultrasonicated  for 1.5 h. The resulting GO dispersion was then refluxed in a 95 °C oil bath for 2 h with 4 mL ammonia solution  and 206 μL hydrazine hydrate . Graphene film was fabricated by vacuum filtration of the resulting dispersion through a membrane filter of 0.22 μm in pore size, and dried in desiccator for a week before transferred to glovebox.4, 99.0%, Sigma-aldrich Co.) in acetonitrile  electrolyte were added into as-prepared graphene films (60 mg) and soaked for 12 h in an airtight container. Then these samples were taken out of the container and dried in argon environment for 10 min.In an argon glovebox, a series amounts  of 1 M tetraethylammonium tetrafluoroborate  for 30 min and then disassembled quickly within 1 min. Then the graphene films electrode materials were separated from the current collector and dried under argon environment for 10 mins. The electrochemical measurements were performed on an electrochemical workstation  at room temperature.Graphene-film based EDLCs were assembled in a two electrode system with 1 M TEABF11B, and equipped with a double-resonance magic-angle spinning (MAS) probe, supporting MAS rotors of 3.2 mm outer diameter. 11B Slid-state NMR spectra were recorded at MAS frequency of 10 kHz, employing the direct excitation method with rf-nutaion frequency of 140 kHz for 11B. 11B chemical shift was referenced to NaH4B at −42.06 ppm. A recycle delay of 5 s was used to collect a total of 64 scans for each sample. NMR spectra with a spectral width of 608.37 ppm (78125.00 Hz) were recorded in all cases. To ensure the experimental parameters were proper in the current work, the spin-lattice (T1) relaxation time for 11B were measured with an inversion-recovery pulse sequence in separate experiments (see Slid-state NMR experiments were performed on a Bruker AVANCE III 400 MHz spectrometer operating at 128.42 MHz for n (=4) refers to the number of data points for each case, xi is the value of these data point, and xi.where How to cite this article: Li, K. et al. Solid-state NMR Study of Ion Adsorption and Charge Storage in Graphene Film Supercapacitor Electrodes. Sci. Rep.6, 39689; doi: 10.1038/srep39689 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Atmospheric doping of supported graphene was investigated by Raman scattering under different pressures. Various Raman spectra parameters were found to depend on the pressure and the substrate material. The results are interpreted in terms of atmospheric adsorption leading to a change in graphene charge carrier density and the effect of the substrate on the electronic and phonon properties of graphene. It was found that adsorption of molecules from the atmosphere onto graphene doped with nitrogen (electron doping) compensates for the electron charge. Furthermore, the atmosphere-induced doping drastically decreases the spatial heterogeneity of charge carriers in graphene doped with nitrogen, while the opposite effect was observed for undoped samples. The results of this study should be taken into account for the development of sensors and nanoelectronic devices based on graphene. Graphene is a promising material for a variety of applications due to its unique physical properties . Among iA natural way for probing the adsorption properties of graphene is to conduct experiments under a low-pressure environment. Various high-pressure measurements for graphene are presented in the literature –6, whileRaman spectroscopy is a versatile tool for studying graphene and graphene-based nanomaterials –12. SeveThe purpose of this study is to investigate the pressure-dependent behavior of the density of charge carriers in graphene and the effect of the substrate on the adsorption properties of graphene at pressures below atmospheric pressure.I2D/IG intensity ratio shows values of up to 1.5 for the monolayer and about 0.9 for bilayer graphene  can be attributed to several effects: substrate-induced strain [−1 and the considerably low I2D/IG ratio of 1.3–1.4 for graphene transferred onto copper and Al2O3 could be explained by similar effects, which in this case, tend to be less pronounced after the transfer. For doped graphene (1.11% of nitrogen from XPS data), the D band intensity increases compared to pristine graphene, the second disorder-induced peak (the D’ band) also becomes evident, the G peak position is upshifted by ≈3 cm−1, while the 2D peak is about normal, naturally indicating electron doping together with I2D/IG values of 1.4–1.6 [Comparing the spectra for as-grown and transferred graphene on copper, one can distinguish the upshifted positions of the G and 2D peaks. The stronger upshift for as-grown graphene  graphene lattice compressibility  [ [ [The G and 2D peak experimental pressure shift ibility  , (2) strility  [ ,14, and ity  [ [ ,13. ThusThe first term is defined by the following expression : and  are normal G and 2D peak positions, respectively; γG,2D is a Grüneisen parameter for corresponding E2g and A1g phonon modes . As it was shown in [a(p)/a0 estimation:where ectively ), a is tshown in , a relatshown in  is appliwhere0 and β' values of 1250 GPa and 1 were used, respectively [is the linear compressibility and β' is its pressure derivative. In the present study, βectively .The strain-induced shift, by analogy with thermal expansion strain –818, can8where−1/% for G and 2D peak, respectively [r is a substrate atomic bond length.is a biaxial strain rate /r0 relation can be approximately defined through the substrate bulk modulus B0 in the following way:In  it was s can on one hand be calculated from I2D/IG intensity ratio [A value for the doping contribution ty ratio . does not lead to a substantial change in the G and 2D peak positions – less than 10−2 cm−1 as the pressure changes from 0 to 1 atm (≈1·105 Pa). The same is valid for the strain-induced shift  . Since these results dramatically exclude the first two terms from During the calculation, it turned out that the graphene lattice compression contribution n obtained from Raman spectra [n values were obtained for nitrogen-doped graphene, while for bilayers on SiO2/Si and graphene transferred to copper, weaker desorption occurred. It is important to note that doping has various effects on the Raman spectral parameters [I2D/IG ratio values, while the G and 2D peak positions depend on carrier density non-linearly. spectra  of variorameters : stronge−1 distance between the plots. This observation can be explained by substrate-induced strain in as-grown graphene . This explains the similar shift but different intercept for pristine and transferred graphene on Cu in In order to analyze the obtained results, we first compare the peak position shift for pristine and transferred graphene on Cu in ositions ): The vaositions  which arositions  and a co2O3 as typical conducting and dielectric materials, a stronger carrier density change was obtained for the latter. As the pressure decreases, removal of adsorbates from the surface of graphene on Al2O3 results in the Raman spectra parameters becoming close to those typical for completely undoped graphene [I2D/IG increase seen in 2 against ≈0.47 J/m2 for graphene on Al2O3 substrate [Comparing graphene transferred to Cu and Algraphene  – in parubstrate –21) and ubstrate . Besidesubstrate –23 can cubstrate , leadingubstrate .13 cm−2 hole, which is close to the case of graphene transferred to Cu. Being a van der Waals structure on the scale of adsorption effects (in terms of interlayer bonding), bilayer graphene is known to have a slightly greater energy barrier for the adsorption process [I2D/IG did not indicate a strong doping presence. Thus, the overall greater G and 2D peak position values most likely are strain-related rather than an indication of greater atmosphere-induced doping.In the case of bilayer graphene, the desorption process leads to a density decrease of about 0.4 × 10 process . However process . BesidesI2D/IG corresponds to an increase of electron density, according to dependencies presented in [A very interesting situation is observed for nitrogen-doped graphene. The results of pressure-dependent studies presented in ented in . This faAs seen from −5 mbar), as seen in It is important to note that the opposite situation was observed for undoped graphene: as the pressure decreased (with desorption leading to doping decrease), the G peak (in addition to the downshift) evolved from a distorted lineshape at normal pressure to an almost symmetric one in vacuum  or assuming exposure of air to the device functional elements.−5 mbar of supported graphene have shown that the G and 2D peak positions and the ratio of the 2D and G peak intensities change with the pressure, whereby the substrate material affects the dependencies. Performed calculations showed negligible lattice compressibility and strain contributions to the observed effect, and thus the shift was fully attributed to atmosphere-induced adsorption doping. During the analysis, the effect of the substrate on graphene electronic and phonon properties was taken into account. For nitrogen-doped graphene (electronic doping), adsorption was found to counterbalance the excess charge carriers. In addition, it turned out that atmosphere-induced doping decreases charge carrier inhomogeneity in nitrogen-doped graphene. For the undoped graphene samples, the opposite effect occurs. The results of the present study are useful for graphene-based sensor design, graphene functionalization techniques, and taking into account adsorption effects during nanoelectronic device engineering.Pressure-dependent Raman spectroscopy studies in the range from 1000 to 5 × 104 of 40 sccm and H2 of 10 sccm. Cu foil  was pre-annealed at 1060 °C with a hydrogen flow of 300 sccm and an argon flow of 2,000 sccm at a pressure of <10−4 Torr for 1–2 hours inside the chamber. After the growth of graphene, the Cu foil was cooled to room temperature. The cooling rate was described in detail elsewhere [Graphene was synthesized on Cu foil at 1020 °C by a chemical vapor deposition (CVD) method using a mixture of CHlsewhere .4)2S2O8 aqueous solution was used to etch Cu and a water/isopropyl alcohol mixture was used to wash graphene from etching products [The grown graphene was transferred to various substrates using a “PMMA-mediated” method . PMMA . A laser power of 2 mW was used, and the laser spot diameter was about 1.5 µm. Pressure-dependent measurements were carried out in a typical graphene monolayer and a bilayer low-defect region.The Raman spectra were obtained with a confocal Raman spectrometer Nanofinder HE (LOTIS TII) with a spectral resolution better than 3 cm


Programmed cell death ligand 1 (PD-L1) is a transmembrane protein that is expressed on tumor cells that suppresses the T cell-mediated immune response. Therapies targeting the PD-L1 pathway promote anti-tumor immunity and have shown promising results in some types of cancers. However, the functional and therapeutic roles of PD-L1 in osteosarcoma remain largely unknown. In this study, we found that PD-L1 protein was expressed in osteosarcoma cell lines and tissue microarray of patient tumors. Tissue microarray immunohistochemistry analysis showed that the overall and five-year survival rates of patients with high levels of PD-L1 expression were significantly shorter than patients with low levels. High levels of PD-L1 expression were also associated with metastasis in osteosarcoma patients. Furthermore, we applied the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system to target PD-L1 gene at the DNA level in osteosarcoma cell lines. We found that the expression of PD-L1 could be efficiently disrupted by CRISPR/Cas9 system and PD-L1 knockdown increased drug sensitivities for doxorubicin and paclitaxel. These results suggest that PD-L1 is an independent prognostic factor in osteosarcoma and that PD-L1 knockout by CRISPR/Cas9 may be a therapeutic approach for the treatment of osteosarcoma. Osteosarcoma is the most common type of primary malignant bone tumor affecting both children and adults. Current treatment for osteosarcoma is effective but limited. While surgery alone can only result in long-term survival rates around 10–20%, surgery combined with chemotherapy has increased the disease-free survival rate to more than 60% [Programmed cell death ligand 1 (PD-L1) is the ligand for programmed cell death 1 (PD-1). As an immune inhibitory moiety, PD-L1 is expressed by most cell types including cancer cells. PD-L1 is able to deliver an inhibitory signal to PD-1 expressing T cells, resulting in immune system impairment . Recent PD-1 and PD-L1 based cancer immunotherapies normally require continuous treatment with anti-PD-1 or anti-PD-L1 antibody, which may be costly. Alternative therapeutic strategies of targeting PD-1 or PD-L1 are needed. The clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9-based RNA-guided DNA endonuclease technology has been widely adopted for its affordability, versatility, ease of use, and low cost , 12. CRIIn this study, we detected the expressions of PD-L1 protein in osteosarcoma cell lines and patient specimens. We also evaluated the association between PD-L1 expression and clinical characteristics in osteosarcoma patients. The efficiency of a CRISPR/Cas9 system on disruption of PD-L1 expression was assessed in osteosarcoma cells. Furthermore, the effects of PD-L1 knockout on osteosarcoma cell growth, migration, invasion, and drug resistance to doxorubicin and paclitaxel were determined.PD-L1 expression has been demonstrated to play a crucial role in some tumor cell metastasis , 19. HowP < 0.001). In addition, patients with high expression of PD-L1 had a trend of poor response to preoperative chemotherapy (P = 0.1642). However, there were no significant relationship between PD-L1 expression and the other clinic pathological features of the human tumor samples, such as age, gender, or the recurrence (Table P = 0.0048) . Univariate Cox regression analysis indicated that PD-L1 expression was the independent prognostic factor of overall and five-year survival rates (P = 0.045 and 0.009)  . Taking in vitro was performed. A sgRNA consists of a crRNA sequence that binds to a specific DNA target, and a tracrRNA sequence that binds to Cas9 protein. When a sgRNA binds to a recombinant form of the Cas9 protein that has double-stranded DNA endonuclease activity, the resulting complex will produce target-specific double-stranded cleavage. Cellular repair, which is error-prone, will take place at the cleavage site, and may result in a mutation that can knock out a gene. In Figure Before the transfection of plasmid with different sgRNA into osteosarcoma cells, verification of PD-L1 CRISPR/Cas9 in vitro verification, we chose two different sgRNAs (#2 and #3) individually targeting at exon 2 and 3 of PD-L1 gene for the generation of osteosarcoma cell lines with constitutive knockout of PD-L1 expression. Transfection of osteosarcoma cells (KHOS and MNNG/HOS) with PD-L1 CRISPR/Cas9 plasmid and GFP resulted in transfection of approximately 50–75% of the cells as observed by green fluorescence . Similar results were seen in MNNG/HOS cells. The diameter of MNNG/HOS spheroids formed by PD-L1 CRISPR/Cas9 intervention was 64% of control cells (P = 0.012)  Figure . TherebyMigration and invasion capabilities are crucial prerequisites for metastatic osteosarcoma. In this study, we evaluated whether knockout of PD-L1 expression could affect the migratory and invasive activities of osteosarcoma cells by the wound healing assay and Matrigel invasion assay. In the wound healing assay, wounds were almost recovered after 24 hours of migration both in blank control cells and KHOS PD-L1-Cas9 and MNNG/HOS PD-L1-Cas9 cells Figure . In MatrIn this study, we successfully established a PD-L1 CRISPR/Cas9 system and generated osteosarcoma cell lines with constitutive knockout of PD-L1 expression. By CRISPR/Cas9 system, PD-L1 has been verified to mediate osteosarcoma cell, growth and drug resistance to doxorubicin and paclitaxel. In addition, PD-L1 expression was also confirmed to be associated with metastasis in osteosarcoma and can be considered as a predictor for five-year and overall survival in osteosarcoma patients. This is the first report demonstrating the PD-L1 CRISPR/Cas9 system in osteosarcoma, which offers a new treatment approach for PD-L1 in osteosarcoma.+ T cells at the lymph nodes and suppresses the immune response [It is well-known that the immune regulatory receptor PD-1 and its ligand PD-L1 are important mechanisms of tumor immune tolerance and escape. PD-1 is a cell-surface receptor expressed on subsets of T and B lymphocytes, as well as on other immune cells. PD-L1 is a transmembrane protein expressed on antigen present cells (APC), such as on various cancer cells. The PD-1/PD-L1 complex transmits an inhibitory signal, which reduces the proliferation of CD8response . Previouresponse . In chorresponse . Howeverresponse . Anotherresponse . Thus, tin vitro. The efficient enrichment of cells enabled the isolation of permanent cell lines expressing the mutations via fluorescent cell sorting. These results demonstrate that PD-L1 sgRNA-guided CRISPR/Cas9 is able to specifically knockout PD-L1 expression. Meanwhile, the establishment of a constitutive PD-L1 knockout cell line offers a good model for the study of osteosarcoma pathology and provides a good basis for the application of CRISPR/Cas9 technology in osteosarcoma treatment. Our data also confirm that the CRISPR/Cas9 system for genome editing is a robust technology that makes it possible to generate cellular models that recapitulate those cooperative alterations rapidly and at low cost.The immune therapeutics targeting the PD-1/PD-L1 axis in cancer patients with a wide variety of malignancies is rapidly evolving with successful clinical studies . Recentlin vivo environment of osteosarcoma cell growth, 3D cell culture was applied in this study. The diameter of spheroids formed by PD-L1-Cas9 cells was significantly decreased compared with the control cells, which implied that PD-L1 might also be involved in the growth and tumorigenicity of osteosarcoma cells. As migration and invasion capabilities are crucial prerequisites for metastatic osteosarcoma, we compared those functions between PD-L1 knockout cells and control cells. However, there were no significant differences between PD-L1 knockout cells and control cells. Our result showed that migration and invasion of osteosarcoma might not be mediated by PD-L1 alone, but by cell growth or by the PD-1/PD-L1 complex. The function of PD-L1 may be related to the PD-1/PD-L1 complex, which transmits an inhibitory signal to reduce the proliferation of CD8+ T cells [With the application of CRISPR/Cas9 by PD-L1 sgRNA, we found that PD-L1 was an important factor involved in osteosarcoma cell growth and drug resistance. In order to mimic the  T cells . With si T cells . Beside  T cells . In lymp T cells . ConsistThis study confirms that PD-L1 is highly expressed in osteosarcoma cell lines and tissues. Moreover, this is the first application of CRISPR/Cas9 targeting the PD-L1 gene in osteosarcoma. Furthermore, we have successfully established the PD-L1 CRISPR/Cas9 system and generated osteosarcoma cell lines with constitutive knockout of PD-L1 expression. With the simplicity and flexibility of CRISPR/Cas9, it is expected that the PD-L1 CRISPR/Cas9 system may be a promising therapeutic approach for the treatment of osteosarcoma in the future.2, 95% air atmosphere at 37°C.The human osteoblast cells NHOst and HOB-c were purchased from Lonza Walkersville  and PromoCell GmbH , respectively. The human osteosarcoma cell lines, U-2OS, SaOS, MG63, MNNG/HOS, and 143B were obtained from the American Type Culture Collection . The human osteosarcoma KHOS cell line was kindly provided by Dr. Efstathios Gonos . The osteosarcoma cell lines were cultured in RPMI-1640  supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin . The osteoblast cell lines were cultured in osteoblast growth medium (PromoCell) with 10% fetal bovine serum. All cells were maintained in a humidified atmosphere containing 5% COOsteosarcoma TMAs were generated as described previously , 33. PD-® 800CW and IRDye® 680LT were obtained from LI-COR . Western blot analysis was performed as previously reported [Osteosarcoma and osteoblast cells were lysed with RIPA Lysis Buffer  plus complete protease inhibitor cocktail tablets . Protein concentrations were calculated by the Bio-Rad Protein Assay . Antibodies against PD-L1 (1:1000 dilution), Cas9 (1:1000 dilution) were purchased from Abcam, and β-actin (1:2000 dilution) were purchased from Sigma-Aldrich, respectively. Secondary antibodies IRDyereported . WesternThe CRISPR/Cas9 and green fluorescent protein (GFP) fusion protein expression vector guided by PD-L1 single guide RNA (sgRNA) (abbreviated as PD-L1-Cas9-GFP) was obtained from DNA 2.0 . GFP is co-expressed from the same mRNA as the Cas9 protein via a NLS peptide linkage, which enables tracking of transfection efficiency. The exon of the PD-L1 gene selected for guiding RNA design is located at the second and third coding exons. The oligo nucleotides used for construction of the PD-L1 gene sgRNA are: 5′AGCAAATATCCTCATCTTTC3′NGG (#1); 5′TCTTTATATTCATGACCTAC3′NGG (#2); 5′ ATTTACTGTCACGGTTCCCA 3′NGG (#3); 5′ TACCATACTCTACCACATAT3′NGG (#4); 5′ AATAGACAATTAGTGCAGCC3′NGG (#5).In Vitro Transcription and Screening Systems” kit was purchased from Clontech Laboratories Inc. (CA). Firstly, a DNA template that contained the sgRNA encoding sequence under the control of a T7 promoter was generated by performing a PCR with the included Guide-it Scaffold Template and the designed primer. The template was then transcribed with the included Guide-it T7 Polymerase Mix to create a sgRNA containing the target PD-L1 sequence. Subsequently, we purified the sgRNA using digestion with DNAseI, phenol: chloroform extraction, and ethanol precipitation, and measured the concentration. After that, the cleavage template that contained the target sequence in an asymmetric position was expanded by PCR. A cleavage reaction on this template was then performed by using the purified sgRNA in combination with the included Guide-it Recombinant Cas9 Nuclease. Finally, the efficiency of the cleavage reactions was analyzed on an agarose gel to determine if the target sequence had been successfully incorporated.In order to verify the effectiveness of the PD-L1 sgRNA, we performed the genome editing with “Guide-it technology”. The “Guide-it sgRNA The stable osteosarcoma cell lines (KHOS and MNNG/HOS) were transfected with the specific PD-L1-Cas9-GFP vector using Lipofectamine3000  according to the manufacturer's instructions, and the cells were allowed to recover for 48 hours before fluorescence activated cell sorting (FACS). Forty-eight hours after transfection, delivery of sgRNA plasmids was confirmed by visualizing green fluorescent marker expression by fluorescence microscopy. Cells dispersed from the culture plate were pelleted and resuspended in 1ml PBS. Using FACS, green fluorescent cells were collected. Individual GFP-positive cells were diluted to 5 cells/ml and the clones in 96-well plates were allowed to expand for seven days. Westernblot was used to identify PD-L1 protein expression and to confirm the cell clones with constitutively knockout PD-L1 expression after FACS. The clones confirmed with constitutive knockout of PD-L1 expression were used for the following functional assays.3 cells/mL  were seeded into each well. Complete growth medium without antibiotics was added into each well to a volume of 100 μL in triplicate. Osteosarcoma cells were dosed with doxorubicin in increasing concentration up to10 μM or paclitaxel in increasing concentrations up to 1 μM. After 96 hours of culture, 20 μL of MTT (5 mg/mL) was added to each well and the cells were incubated for 4 hours at 37°C. Absorbance at a wave length of 490 nm was measured on a SPECTR Amax Microplate Spectrophotometer from Molecular Devices . All results were analyzed by GraphPad Prism 5 software .Doxorubicin and paclitaxel, which are the most common chemotherapy choices for osteosarcoma treatment, were provided by the pharmacy at the Massachusetts General Hospital Cancer Center. The stock solutions of these drugs were prepared according to the manufacturers’ specifications and stored at −20°C. Effects of PD-L1 on osteosarcoma drug resistance to doxorubicin and paclitaxel were assessed by the MTT assay. In 96-well plates, 5 × 10in vivo and is a more accurate model to determine the behavior of cancer cell growth. Following the manufacturer's protocol, osteosarcoma cell spheroid formation was established in HDP1096 Perfecta3D® 96-Well Hanging Drop Plates (3D Biomatrix). Hanging drops were formed by pipetting 40 μL of cell suspension (1 × 105/mL) into each well. Medium was changed every other day to provide enough nutrients for cells and to prevent osmolality shift of the medium. After seven days culture, the spheroids were harvested from the bottom side of the plate by gently pipetting 100 μL PBS into each well. After 15 minutes of incubation with 1 μM Calcein AM (Life Technologies), the spheroids were imaged on a Nikon Eclipse Ti-U inverted fluorescence microscope . The size of spheroids was calculated using ImageJ.A 3D cell culture is an improved method that can simulate the cell growth environment ™ Matrigel™ Invasion Chambers  were used according to the manufacturer's instructions. Osteosarcoma cells (5 × 104 cells/plate) were planted into the upper chamber of each well in serum-free medium, and complete medium was put into the bottom chamber. After incubation for 24 hours, non-invaded cells were removed by scrubbing from the upper surface of the membrane with cotton-tipped swabs. After fixation in 100% methanol and staining with hematoxylin, the invaded cells were counted in three images of each membrane under a microscope using a 200× objective.Matrigel invasion assay was applied to evaluate the role of PD-L1 in the process of osteosarcoma metastasis. BioCoat5 cells/well) were seeded into 12-well plates. Three parallel lines were made in confluent cell cultures with a 200 μL tip. The wounds were observed at 0, 8, and 24 hours after scratching separately, and photographed at each time point using a 10× objective. The cell migration distance was defined as the wound width at the 0 h time point minus the wound width at each time point and then divided by two. The distance between the two edges of the wound width was randomly quantified at 10 sites in each image.Migration activity is another method to identify the potential role of PD-L1 in the metastasis of osteosarcoma. In this study, migration activity was detected by the multiple scratch wound assay. Osteosarcoma cells  in the univariate survival analysis were included in the multivariate analyses.GraphPad Prism5 software  was used for statistical analyses. Results are expressed as mean ± SD and


Dynamic wavelength tunability has long been the holy grail of photodetector technology. Because of its atomic thickness and unique properties, graphene opens up new paradigms to realize this concept, but so far this has been elusive experimentally. Here we employ detailed quantum transport modeling of photocurrent in graphene field-effect transistors  to show that wavelength tunability is possible by dynamically changing the gate voltage. We reveal the phenomena that govern the behavior of this type of device and show significant departure from the simple expectations based on vertical transitions. We find strong focusing of the electromagnetic fields at the contact edges over the same length scale as the band-bending. Both of these spatially-varying potentials lead to an enhancement of non-vertical optical transitions, which dominate even in the absence of phonon or impurity scattering. We also show that the vanishing density of states near the Dirac point leads to contact blocking and a gate-dependent modulation of the photocurrent. Several of the effects discussed here should be applicable to a broad range of one- and two-dimensional materials and devices. Photodetectors based on graphene have been extensively studied both experimentally123457optical properties of graphene can be modulated using a back gate11photocurrent, it could be harnessed for tunable photodetector applications. Unfortunately the experimental realization of this concept remains elusive, and to our knowledge the only experimental report of spectral photocurrent tunability (by a few tens of nanometers) was realized by inducing conformal changes in graphenemultiple graphene layers, such as bilayer graphene photogatingGraphene opens interesting possibilities to realize this concept, as recent work has shown that the In this paper, we address the challenges of identifying promising tunable detector designs and the necessity for more detailed quantum modeling by developing and implementing a non-equilibrium quantum transport approach for photocurrent calculations in graphene devices, including realistic electromagnetic fields. Our main result is that dynamic hyperspectral imaging should be possible in such devices, but a wealth of phenomena renders the system rather complex. For example, we find that translational symmetry breaking due to spatially-varying band-bending and electromagnetic fields leads to a dominance of non-vertical transitions that significantly change the behavior expected from pure vertical transitions and Fermi blockinglmfp/L where lmfp is the electron scattering mean free path and L is the channel length.We consider a subwavelength graphene field-effect transistor (FET) as illustrated in To calculate the photocurrent, we employ the non-equilibrium Green’s function (NEGF) approach, where we explicitly include the electron-photon interaction at the quantum level. We previously developed and applied this approach to carbon nanotube p-n junctionsdark graphene FET using our previously developed approach for carbon nanotubes2022Our calculations consist of two preliminary steps and a final step to obtain the photocurrent, as illustrated in more detail in the Iph is the photocurrent at a source-drain bias Vsd.The full expression for σph can be found in the where In our simulations, we model the graphene using a tight-binding Hamiltonian with overlap integral of 2.5 eV and nearest-neighbor bond length of 0.142 nm. The zigzag direction of the graphene is perpendicular to the electron transport direction. The source and drain electrodes are perfect metals vertically separated from the graphene sheet by a distance of 0.3 nm. Charge transfer between the metal and the graphene is naturally captured in our simulations by using a metal workfunction 0.5 eV larger than graphene. The electron-photon interaction is included by modifying the tight-binding Hamiltonian with the usual e2/h. This result is in good agreement with experimental data for short channel devices25Ax at the contacts to that in the middle of the device, which is about 36 at the longer wavelengths and increases to more than 60 at a wavelength of 1 micron. These results suggest that the properties of photodetectors based on two-dimensional materials should be particularly sensitive to near-contact effects even under flood illumination.We obtained the vector potential The near-contact focusing of the electromagnetic field is spatially co-located with the device band-bending. As discussed in the context of Ay component and consider only the spatially-varying Ax component. Having obtained the gate-dependent dark band-bending and the vector potential resulting from illumination, we can calculate the photoconductance. Since the optical absorption in graphene for the out-of-plane contribution is very smallE. If only vertical transitions were present, the transmission would show a peak at energy EF and KBT above and below.)The above effects have their origin in two main factors: non-vertical transitions and gate-dependent contact blocking. Non-vertical transitions can arise when the translational symmetry is broken; in the case of the graphene FET translational symmetry is broken by the spatially-varying electrostatic potential and spatially-varying magnetic vector potential. The missing momentum that allows non-vertical transitions arises from the infinite number of Fourier modes that make up these spatial variations. To illustrate the importance of non-vertical transitions, we plot in For the case of This effect is in part responsible for the shape of the photoresponse versus gate voltage observed in Vg = 1V. To further determine the factors that lead to the unusual importance of non-vertical transitions, we calculated the transmission for a gate voltage of 1 V and illumination at In conclusion, we find that monolayer graphene FETs open a new avenue to realize photodetectors with dynamic spectral tunability, and display a number of novel underlying phenomena that should be general. For example, electromagnetic field focusing at the contacts plays a key role in governing photoresponse behavior, a phenomenon that should apply to a broad range of one- and two-dimensional materials. We also expect that non-vertical transitions induced by symmetry breaking due to the spatial variations of the band-bending and the vector potential should also become prominent in other low-dimensionality materials. Finally, additional studies are needed to assess the robustness of the wavelength tunability to non-ideal factors such as substrate charged impurities. Nonetheless, our work establishes the first framework to guide and interpret experimental and theoretical efforts.How to cite this article: Léonard, F. et al. Dynamic Wavelength-Tunable Photodetector Using Subwavelength Graphene Field-Effect Transistors. Sci. Rep.7, 45873; doi: 10.1038/srep45873 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The majority of the proposed graphene-based THz devices consist of a metamaterial that can optically interact with graphene. This coupled graphene-metamaterial system gives rise to a family of resonant modes such as the surface plasmon polariton (SPP) modes of graphene, the geometrically induced SPPs, also known as the spoof SPP modes, and the Fabry-Perot (FP) modes. In the literature, these modes are usually considered separately as if each could only exist in one structure. By contrast, in this paper, we show that even in a simple metamaterial structure such as a one-dimensional (1D) metallic slit grating, these modes all exist and can potentially interact with each other. A graphene SPP-based THz device is also fabricated and measured. Despite the high scattering rate, the effective SPP resonances can still be observed and show a consistent trend between the effective frequency and the grating period, as predicted by the theory. We also find that the excitation of the graphene SPP mode is most efficient in the terahertz spectral region due to the Drude conductivity of graphene in this spectral region. For normally incident radiation of a wavelength λ that is much larger than the structural dimensions of the metamaterial patterns, the resonance frequencies of 1D grooves with h < d1, 1D slits with h = d1, 2D holes, and 2D rings, as shown in the insets of v ≈ c/4neffd1v ≈ c/2neffd1v ≈ c/2neffwv ≈ c/2πrneffneff is the effective index of the mode and r is the radius of the ring. If the ring is cut so that the symmetry is broken, the resulting SRR structure can be regarded as an LC circuit with a new resonance frequency of μ, we effectively change the boundary condition on one side of the gap, and thus the excitation can be either suppressed or enhanced. If a graphene SPP mode is also excited, there can be a coupling of the graphene SPP mode and the spoof SPP mode. As a result, the spoof SPP mode is split into two coupled modes, as can be seen in the inset of 5The graphene-metamaterial hybrid devices studied in the literature have a structure similar to the one shown in 1367Another possible excitation for the device shown in 10μ2 > μ1. Therefore, by adjusting the chemical potential of graphene, the resonance frequency can be tuned and thus the transmitted signal can be switched on and off. Compared to the devices based on other types of surface excitation, the device shown in −1, the modulation depth is around 90% at 0.6 THz, as shown in the inset of 111013For the periodic structure shown in d1 (right panel) and d2 (left panel). The parameters are the same as those used for the insets in w ≪ Λ, the resonance frequency ω of the mth-order SPP mode is given by16The dispersion curves of graphene SPP, spoof SPP, and FP modes can be found by locating the resonance frequency in the transmittance spectrum. For normally incident light, the dispersion map calculated using the modal methodK = 2π/Λ is the wave number of the periodic metallic grating that has a period of Λ. In where d1; then, the dispersion curve is altered as shown in As the optical frequency increases, the excitation of the metal SPP mode becomes possible. In the case of d1 of the grating increases, the excitation of spoof SPP modes in THz spectral region becomes possible. We consider the first-order grating because the effect of the high-order gratings is hardly observable in the transmission spectrum3λ ≫ Λ and λ ≫ w is given by4As the thickness kd = ndω/c and K ≫ kd, the left-hand side of kdd1/2 on the right-hand side of π/2, which gives v = mc/2ndd1 for the resonance frequency of the mth-order mode. The first two modes are plotted as dotted curves labeled (f) as a group, which agree well with the simulation results in the right panel of 17where d2, a FP cavity is formed between the graphene and the metallic grating with the boundary conditions determined by the chemical potential of graphene. The mth-order resonance frequency of a FP cavity characterized by a dielectric constant n2 and a thickness d2 is given by v = mc/2n2d2, where m is a positive integer. The first two modes are plotted as dotted curves labeled (g) as a group in the left panel of n1 and n3 that make the FP cavity imperfect. The transmittance is tuned by adjusting the chemical potential of graphene to achieve the functionality of the device shown in When the wavelength of light is comparable to the distance w = 0.5 μm and d1 region where graphene SPPs dominate. As can be seen in v = mc/2ndd1, which are plotted as the dashed curves. To clearly see the dispersion curves of the cavity modes and the metal SPP mode in this coupling region, we tune the structural parameters so that the excitation of either the cavity modes or the metal SPP mode is suppressed. In v = mc/2ndd1. In d1 region. As can be seen, the dispersion curve of the metal SPP mode is characterized by a low transmittance and a weak dependence on d1. For a metal of a finite conductivity, the resonance frequency redshifts away from the Rayleigh condition of For the completeness and the later discussion of graphene SPP coupling, we first discuss the coupling between the metal SPP mode and the cavity modes. As discussed in the preceding section, a spoof SPP mode that is far away from the metal SPP dispersion curve is essentially a cavity mode with its energy confined within the gaps. As the dispersion curve of the cavity modes approaches the resonance frequency of a metal SPP mode, the resultant spoof SPP mode becomes a mixed state of metal SPP and spoof SPP, with the field distribution in the gaps and also on the metal grating. The dispersion curves of two spoof SPPs, characterized by a high transmittancev = mc/2ndd1 in the absence of the coupling effect. Similar to the case of metal plasmons, the dispersion curves of graphene SPPs are characterized by the low transmittance. This low transmittance effectively suppresses the transmittance at the intersections of the dispersion curves and thus creates multiple bands in the dispersion curves. This gap opening is also shown in μ to zero, there is no graphene SPPs, and the aforementioned gap openings are closed, as shown in A similar study can be performed on the coupling between the graphene SPP modes and the cavity modes. The coupling of the graphene SPP and the cavity modes are shown in γ = 1 ps−1 for the data shown in the above figures. The resonance frequency ω of graphene SPPs, found by using d1 = d2 = 10 nm in Kd2 ≪ 1, the resonance frequency varies as ω ∝ Λ−1 according to The graphene has been assumed in good quality with a scattering of γ = 1 ps−1, 5 ps−1, and 16 ps−1, respectively, with other simulation parameters being the same as those used for γ = 16 ps−1, the resonances become so broad that they merge and become a broad trough. Nevertheless, the absorption of graphene SPPs is still apparent, as can be seen by comparing the transmittance curve calculated using μ = 0 in the upper panel of μ = 100 meV in the lower panel of ω, we can still observe the trend that shows the increasing ω with decreasing Λ, approximately following the relation ω ∝ Λ−1. This trend is shown in μm, 5 μm, and 10 μm, respectively. The increasing ω with decreasing Λ is due to the fact that the broad transmittance trough is the collective result of the many resonances given by ω ∝ Λ−1.In the case of a high scattering rate, the resonances of graphene SPPs become broad; eventually the individual resonances cannot be distinguished for a sufficiently high scattering rate. This phenomenon is shown in Eair, at room temperature, where ϕ is the azimuth angle of the incident light (see method). The values of the ratio ReEg/ReEair are plotted as dotted curves in μm and Λ = 4.9 μm, respectively. For the 2D grating, the values of the transmitted electric field ratio ReEg/ReEair for the incident THz fields polarized at the angle of ϕ = 0° along the long period of Λ = 4.9 μm and at the angle of ϕ = 90° along the short period of Λ = 1.5 μm, respectively, are plotted as solid curves in To demonstrate the concept device based on Egg is divided by ReEg so that the resonance locations of the graphene SPP mode can be observed. The experimental and simulation results are plotted in μ = 60 meV and γ = 16 ps−1 except for the period Λ = 4.9 μm of the 2D grating, which is fitted using a scattering rate of γ = 29 ps−1. The high scattering rate might be due to the ripples caused by the uneven surface of the 2D grating structure, or due to angle-sensitive scattering centers such as grain boundaries that prefer to grow in a certain direction resulting in a high scattering rate at specific polarization angles. By rotating the angle from 0° to 90° for the 2D sample, as plotted in Egg/ReEg at different angles. This further indicates the angle-dependent scattering rate and the polarization-sensitive nature of our 2D structure. It can also be seen that the resonance frequency of the SPP mode shifts to a higher frequency for a shorter grating period, which is consistent with the simulation results shown in −1 is physically possible as has been already measured for a graphene sheet that has a mobility of 230,000 cm−1V−1s−1 and a carrier density of 2 × 1011 cm−2 Monolayer graphene grown by chemical vapor deposition (CVD) is then transferred onto the grating structures so that we have a system shown in The effect of Drude conductivity is observed using Fourier-transform infrared spectroscopy (FTIR). The transmittance of 1D gratings with periods of Λ = 1.5 μm and Λ = 3 μm are measured with and without the graphene layer. As can be seen in We have shown the characteristic transmittance curves of various graphene-based THz devices utilizing the excitation and coupling of different surface modes. The dispersion curves of these modes are found through both simulation and analytical solutions. The coupling of graphene SPPs with the cavity modes is also discussed. The coupling results in the gap openings in the dispersion curves of the cavity modes. The locations of the gaps can be tuned by tuning the chemical potential of graphene, and therefore can be utilized in a graphene-based modulator.ω. The effective ω approximately follows the relation ω ∝ Λ−1 due to the fact that the broad transmittance trough is the collective result of the resonances given by the relation ω ∝ Λ−1. The effective ω of structures with different periods are experimentally measured in a THz-TDS experiment. As predicted, the effective ω increases with decreasing grating period. The angle-dependent scattering rate and the polarization-sensitive nature of the 2D structure are shown by rotating the polarization direction of the incident THz wave. It is also shown that the excitation phenomenon gradually disappears at high frequencies due to the decreasing Drude conductivity of graphene, consistent with the simulation results. The decrease of the Drude conductivity with increasing frequency imposes another limitation in the operational frequency range of a graphene-based THz device besides the losses of the SPP mode due to Landau damping at high frequencies.In the case of a very high scattering rate, the individual resonances of graphene SPPs become so broad that they merge and become one broad trough with a new effective resonance frequency y direction were developed on a Si wafer. For the samples of 1D slit grating, the spacing between neighboring trenches was 0.4 μm, and the width was Λ–0.4 μm, where Λ was the period of the structure. Various periods for different 1D samples were developed on the Si wafer. For the samples of 2D slit grating, trenches along the x direction were also developed, as shown in the right panel of Following the standard photolithography process, 50-nm deep trenches along the ϕ of the polarization is varied by carefully rotating the sample. The temporal electric-field-amplitude profile of the transmitted THz pulse was sampled by scanning the delay between the pump and probe optical pulses. For this sampling measurement, the transmitted THz pulse being measured was focused by another pair of off-axis parabolic mirrors on an antenna of the same type as that for the THz generation. In order to avoid any undesirable effects caused by the humidity in the laboratory air, the THz-TDS system was placed in a vacuumed Plexiglas box. The collected time-domain data was then Fourier transformed to the frequency-domain data.The generation and detection of THz pulsed radiation was set up on a mode-locked Ti:sapphire laser operating at 800-nm wavelength with 42-fs pulses at a 75-MHz repetition rate. The femtosecond-pulse laser beam was split into a pump beam and a probe beam. The THz pulses were generated by using the pump laser pulses to trigger a low-temperature-grown GaAs photoconductive dipole antennas characterized by a wire distance of 20 μm and a gap size of 5 μm. The generated THz-pulse beam was focused by a pair of off-axis parabolic mirrors onto a sample at normal incidence so that the polarization is parallel to the sample surface. The angle ϕ = 0° was considered for the 1D structures. The transmitted electric field for the polarization at ϕ = 90° was too small, and the signal-to-noise ratio was too low for any meaningful data, indicating negligible depolarization through the grating. The value of ϕ, Eg and Eair are measured separately. The same procedure was also taken for the measurements of Eg and Egg.Because the metallic grating also works as a polarizer, only the incident field along the grating direction at How to cite this article: Lin, I.-T. et al. Family of graphene-assisted resonant surface optical excitations for terahertz devices. Sci. Rep.6, 35467; doi: 10.1038/srep35467 (2016).


This study was intended to determine the efficacy of nivolumab, we evaluated treatment response with respect to PD-1/PD-L1 SNPs among patients with NSCLC. A total of 50 patients with NSCLC were treated with nivolumab and were also evaluated for PD-1/PD-L1 single nucleotide polymorphisms (SNPs) from plasma DNA. We investigated the association among PD-1/PD-L1 SNPs, objective response rate (ORR) and progression-free survival (PFS). Two of seven SNPs studied showed association with ORR and PFS, with maximum evidence at the marker rs2282055. The ORR was 25%, 15%, and 0% for the G/G, G/T and T/T genotypes of PD-L1 rs2282055, respectively. The G allele of PD-L1 rs2282055 was significantly associated with better clinical response compared with the T allele (P = 0.0339 [Cochran-Armitage trend test]). The median PFS time was 2.6 months  for the G/G and G/T genotypes and 1.8 months  for the T/T genotype (P = 0.0163). Moreover, the C/C and C/G genotypes of PD-L1 rs4143815 were significantly associated with better ORR and PFS in NSCLC patients treated with nivolumab. These results suggest that rs2282055 and rs4143815 may be a biomarker for the efficacy of nivolumab. Although lung cancer remains a major cause of cancer-related mortality worldwide, targeted therapies and immunotherapy contribute to better clinical outcome in non–small-cell lung cancer (NSCLC) patients223Some patients with lung cancer show marked responses to nivolumab, whereas others do not respond (ORR ~20%). It is currently difficult to predict treatment response to nivolumab since there are no precise biomarkers for it. Expression of PD-L1 in the tumor microenvironment is thought to be crucial for therapeutic activity7Recently, the application of next-generation sequencing (NGS) has emphasized the potential role of somatic DNA sequence markers, which may be used as independent markers, novel therapeutic targets, or as tools to refine expression markers1821Patient characteristics are listed in Among the 50 patients, 8 (16%) had PR, 16 32%) had stable disease (SD), 26 (52%) had PD , and non% had staFive PD-L1 SNPs and two PD-1 SNPs were genotyped in 50 NSCLC patients who were administered with nivolumab. The SNP genotype, gene information, genotype frequency, and the associated clinical response are shown in Of the 50 patients treated with nivolumab, 12 were G/G (24%), 15 were C/C (30%), and 23 were C/G (46%) of PD-L1 rs4143815. Genotype was not associated with clinicopathologic variables . The C/CWe also assessed PFS among the PD-L1 rs2282055 genotypes. Objective responses were observed in 5 of 20 (25%), 3 of 20 (15%), and none of 10 patients of PD-L1 rs2282055 genotypes G/G, G/T and T/T, respectively . Kaplan-2 = 0.292) in this study. In the PD-1 gene, the D’ value and r2 between rs2227981 and rs2227982 were |D’| = 1.0 and r2 = 0.355, respectively.Three polymorphism of the PD-L1 gene, rs822339, rs2282055, and rs1411262, were generally under strong linkage disequilibrium (LD) from the pairwise disequilibrium map (LD map) . The twoWe assessed the efficacy of nivolumab according to PD-1/PD-L1 SNPs. Interestingly, our study showed that patients with the C/C or C/G genotype of PD-L1 rs4143815 and the G/G or G/T genotypes of rs2282055 had a significantly longer PFS with nivolumab treatment than patients with the G/G genotype and T/T genotype respectively. In addition, none of the patients obtained treatment effect with the G/G genotype of PD-L1 rs4143815 and the T/T genotype of rs2282055.Currently, since the known candidate biomarkers of nivolumab mainly originate from the tumor itself and the surrounding tumor environment, it was suggested that the PD-L1 expression in tumor cells measured by immunohistochemistry may predict responses to immune checkpoint inhibitors62 < 0.8). This might explain the clinical significance of PD-L1 rs2282055. Conversely, PD-L1 rs822339, PD-L1 rs1411262, and PD-L1 rs2282055 were in strong LD  and binds to miR-57025trong LD , therefoIn this study, we focused on the PD-1/PD-L1 SNPs as a predictive biomarker of nivolumab since the PD-1/PD-L1 pathway is involved in the immune system. The PD-1 receptor, which is broadly expressed in multiple cell types, including T- and B-cells, dendritic cells, and macrophages37This study had several limitations. First, this was a retrospective study with a small sample size and the patients who had received nivolumab were already relatively heavily pre-treated. Nevertheless, we showed a clinically meaningful difference in response rates and PFS among the PD-L1 rs4143815 and rs2282055 genotypes. Second, since this study lacks in immunohistochemical information from biopsies, the association between the PD-L1 rs4143815 genotypes and the PD-L1 expression in tumor cells is unknown in this cohort. Although PD-L1 expression is an important biomarker, we did not assess its expression in this study. The reason for this is due to the use of different antibodies in immunohistochemistry assays and the varying cut-off values to evaluate the expression of PD-L1, which make it difficult to compare with other studies. Third, we selected seven PD-1/PD-L1 polymorphisms that are thought to be functional based on previous reports; however, there may be other, more efficient predictive SNP biomarkers.The association between PD-L1 SNPs and the efficacy of nivolumab in a large-sized study needs to be investigated in the future. We are planning future studies to assess the association of PD-L1 expression, PD-L1 SNPs and efficacy of anti-cancer treatment in order to confirm the observations presented in this study.In conclusion, we found that in advanced stage NSCLC patients who received nivolumab, the C allele of PD-L1 rs4143815 and the G allele of rs2282055 were significantly associated with better ORR and PFS. This is the first report that PD-L1 SNP, which was thought to increase PD-L1 expression, is associated with a response to nivolumab. Therefore, these results suggests that PD-L1 rs4143815 and rs2282055 may be a biomarker for identifying patients for whom nivolumab may be particularly beneficial, which, as a result, may contribute to the clinical setting in the treatment of lung cancer.Between December 2015 and August 2016, a total of 67 consecutive patients with histologically or cytologically confirmed NSCLC were treated with nivolumab. Among these 67 patients, we enrolled 50 patients in this study, all of whom were registered at Kyoto University Hospital. The remaining 17 patients were excluded from this study as a result of lack of DNA samples, discontinued observation due to change in hospital, the inability to obtain informed consent, or the patient had a history of double cancer. Participants in this study have provided a written informed consent and all experimental methods were in accordance with the 1975 Declaration of Helsinki. The study was approved by the Review Board of Kyoto University Hospital. A 10-mL peripheral blood sample was collected from each study participant. This procedure was performed in accordance with the ethical principles for medical research.® genotyping assay  and analyzed with an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems). The polymerase chain reaction (PCR) solution included 12.5 μL of 2X TaqMan Universal PCR Master Mix, 0.3125 μL of primer probe mix, 11.2 μL of nuclease-free water, and 1 μL of DNA sample. After baseline fluorescence measurements at 25 °C, the PCR protocol included a 10-min incubation at 95 °C, 40 cycles of denaturing at 92 °C for 15 s and annealing and extending at 60 °C for 1 min, and a final measurement of fluorescence at 60 °C. SNPs with previously described associations or putative functional effects were selected242526272839404142Genomic DNA was extracted from peripheral blood leukocytes using the GENE PREP STAR NA-480  according to the manufacturer’s instructions. Genotyping was performed using the TaqManMedical records were reviewed and data extracted based on clinical features and treatment histories. These data have been updated as of October 2016. The initial dose of nivolumab was 3 mg/kg every two weeks and was intravenously administered until the assessment of progressive disease (PD) or an unacceptable toxicity level was reached. Radiographic imaging was done every 4 to 6 weeks. Response was assessed as per the Response Evaluation Criteria in Solid Tumors (RECIST) (version 1.1) by an investigator. The PFS was measured from the start of nivolumab administration until the date of RECIST PD. Patients without documented clinical or radiographic disease progression were censored on the date of the last follow-up. The ORR, as determined by the RECIST criteria, was calculated as the total percentage of patients with a complete or partial response (PR).The JMP Pro statistical software version 12.1.0  was used for all statistical analyses. Differences in characteristics among the genotypes were compared using Fisher’s exact test for categorical data. Genotypes were analyzed separately and as combined allelic groups. Clinical responses among the genotypes were analyzed using the Cochrane-Armitage test. The patient’s baseline characteristics and the genotype of rs4143815 or rs2282055 were analyzed using one-way analysis of variance (ANOVA) for continuous variables.2.The survival curves were estimated using the Kaplan-Meier method. Statistical significance of the relationship between PFS and genetic polymorphism was assessed using the trend log-rank-test. Multivariate Cox proportional hazards models were used to estimate adjusted hazard ratios (HR) with 95% confidence intervals. Multivariable regressions analysis was adjusted for age, gender, and histology. Haploview v4.2 was used to calculate the linkage disequilibrium (LD), expressed as D’ and rHow to cite this article: Nomizo, T. et al. Clinical Impact of Single Nucleotide Polymorphism in PD-L1 on Response to Nivolumab for Advanced Non-Small-Cell Lung Cancer Patients. Sci. Rep.7, 45124; doi: 10.1038/srep45124 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In the era of semantic web, life science ontologies play an important role in tasks such as annotating biological objects, linking relevant data pieces, and verifying data consistency. Understanding ontology structures and overlapping ontologies is essential for tasks such as ontology reuse and development. We present an exploratory study where we examine structure and look for patterns in BioPortal, a comprehensive publicly available repository of live science ontologies.We report an analysis of biomedical ontology mapping data over time. We apply graph theory methods such as Modularity Analysis and Betweenness Centrality to analyse data gathered at five different time points. We identify communities, i.e., sets of overlapping ontologies, and define similar and closest communities. We demonstrate evolution of identified communities over time and identify core ontologies of the closest communities. We use BioPortal project and category data to measure community coherence. We also validate identified communities with their mutual mentions in scientific literature.With comparing mapping data gathered at five different time points, we identified similar and closest communities of overlapping ontologies, and demonstrated evolution of communities over time. Results showed that anatomy and health ontologies tend to form more isolated communities compared to other categories. We also showed that communities contain all or the majority of ontologies being used in narrower projects. In addition, we identified major changes in mapping data after migration to BioPortal Version 4. Ontologies are used for tasks such as the standardization of terminology, the verification of data consistency, and the integration of heterogeneous databases. Ontologies have been actively applied to areas including, but not limited to Biology and Medicine , Crisis ontology mappings identify existing concepts with similar meaning. These ontology mappings are useful in tasks like finding new annotations, supporting other data integration methods, combining related ontologies, or ontology reuse. When ontologists build new ontologies they often search for existing ontologies to avoid redundancy of concepts as recommended, for example, by the OBO Foundry principles (Life science inciples . Identifinciples .evolution of ontologies (communities) that tend to overlap more often than others. Please note that in this paper we use the word community for a set of ontologies that tend to overlap. In contrast, the OBO  project defines a community as a set of ontologies that work together and reduce mutual overlap. We also recognize hub ontologies, i.e., ontologies that connect many other ontologies/communities. Since BioPortal data often changes , we analyse the mapping data at different time points. We propose an alignment of similar communities, define stable communities and perform a time transition analysis. Our work aims to answer questions like “In my area of interest, what ontologies already exist and how are they related to each other?” or “In my area of interest, which sub-areas are stable and which are not in terms of ontology development?”. Answering these questions can assist in tasks like ontology reuse and development. The results of the data gathered and analysed at five different time points are presented.In this paper we analyse and evaluate NCBO BioPortal  ontologytologies . To helpRelated work can be categorized in the following (often overlapping) two groups: (1) analysis of ontology mappings and (2) evolution of ontologies. Below we introduce the most relevant papers from these groups.Similar to our work, Changes in ontologies have been previously studied and tools such as GOMMA  have beestable and similar communities), which is the focus of this paper. We also perform analysis of project and category data and discuss alignment with identified communities. In addition, we try to validate generated clusters with information found in MEDLINE abstracts : •NCBO mappings are periodically calculated with a computer algorithm. The algorithm finds mappings for terms with close lexical match or mappings for terms with the same URI from different ontologies. The majority of the mappings is from this group. •Unified Medical Language System (UMLS) mappings link terms with the same UMLS concept unique identifier (CUI) or terms from the UMLS MRMAP.RRF data. •Mappings between ontology terms related by an OBO  xref property.test in their full name, and (3) restricted or private ontologies. From data gathered in December 2013 and July 2014 we removed summary ontologies . The filtered data was then processed with Gephi  ontologies with the retired or alpha status, (2) ontologies that contain the keyword th Gephi , an openfromOntology: the name of the source ontology,(1) toOntology: the name of the target ontology(2) numberOfMappings: number of directed mappings between source and target ontology.(3) Modularity Analysis  to the data. Modularity Analysis (MA) is a measure of structure in graphs. Gephi implements Louvain method  metric  metric  to identWe used information from MEDLINE abstracts , to analWe downloaded the 2016 version of MEDLINE in XML format and developed an algorithm to find pairs of ontology names in all abstracts published before August 2014 (our latest version of the graph is July 2014). Ontology names and abstracts were transformed to lower case characters before the comparison. Simple exact string matching was used to look for ontology names mentions. For example, in the following text “… we introduce GoPubMed, a web server which allows users to explore PubMed search results with the Gene Ontology…”, Gene Ontology would be identified.t contain at least some ontologies from the previous time point t − 1. The challenge is to align similar communities to compare graphs at multiple time points. With aligned communities we can identify communities that changed their size, new communities, or disappearing communities.Running the community detection algorithm at five time points provides us with different number of communities for each time point. Our previous research  showed tThere are several ways to find similar communities in evolving graphs  and no mcommunity core ontologies. In addition, we should consider ontologies that are not shared between two communities. Based on these issues, we first define several terms that are explained in the following paragraphs.Therefore, we wish to use a more stable method for identifying similar communities. We expect that ontologies with the highest BC scores play an important role in BioPortal as they will likely stay in the repository in the future. We call these ontologies C1 contains communities identified at time point t1 and C2 contains communities identified at time point t2 (t2 > t1). First, we define the importance score of ontology o as: BCo,t1 and BCo,t2 represent BC scores for ontology o at time points t1 and t2 respectively.Let us imagine that we identified two groups of communities where group similarity score SCcx,cy between two communities cx ∈ C2 and cy ∈ C1. The similarity score is based on a weighted version of the Dice coefficient  communities and their main hub ontologies, present results of validation with MEDLINE abstracts, analyse transition (evolution) between different time points, analyse the coherence of communities, and present results of measuring effects of ontology sizes on community detection. •MAV represents Modularity Analysis values, •#All is the number of all ontologies in the graph, •#Map is the number of ontologies with at least one mapping (source or target), •%Map is percentage of ontologies with at least one mapping (source or target), •#NoMap is the number of ontologies with no mappings, •%NoMap is the percentage of ontologies with no mappings, •x < 8) values represent the number of ontologies in each identified community. The number of ontologies in each community orders communities.#Com is the number of identified communities. The #Cx , we can notice that pairs where each ontology belongs to a different community tend to be found in lower number of abstracts . On the other hand, ontology pairs that can be found together in large numbers of abstracts  tend to belong to the same community.We found 3,020 ontology pairs  that were mentioned together in at least one abstract. These results imply that identified communities contain ontologies that appear more often together in the literature. However, since most ontologies pairs were not found in the abstracts, different methods should be explored . This is an area for future investigations.NoMap row represents ontologies that had no mappings in the previous version of the graph but are members of one of the communities in the newer version. The New row represents ontologies that did not exist in the previous version of the graph.When observing heat maps on For example, let us consider the heat map A . Row names represent hub ontologies for the old version (Oct12), while column names represent ontologies for the new version (Feb 13) of the graph. The third row shows that all ontologies from the UBERON community stayed in the same community, i.e., the community did not split. On the other hand, observing row 2 shows that although the majority of Oct12 NCIT ontologies stayed in the closest community in Feb13 , some ontologies also migrated into the NIF (column 1), UBERON (column 3), RADLEX (column 4) and SNOMEDCT (column 5) communities. The third column illustrates merging of parts of three different communities  into the new UBERON community. The heat map A also shows that the new identified community  mainly consists of ontologies from the old EP (row 1) community and some ontologies that had no mappings in Oct12 (row 6).With the heat maps we can find pairs of closest communities, which are identified with the most intensive shades of green in each column. For example, on the heat map A, the NIF column contains three coloured squares. However, the square in the EP row is the most intensive shade of green, which identifies the closest community to NIF. In An interesting community is the NCBITaxon community, which appears the Feb13 and Dec13 versions. We already learned that some taxonomy ontologies formed their own community in February 2013 . HoweverConsidering categories such as Anatomy, Health, Ethology, and Gene Product. In addition, information about projects that use BioPortal ontologies is available. We use these two types of information to discuss the coherence of identified communities in our methods. BioPortal groups ontologies into 41 Charts on Another distinct community is G2, where a large proportion of ontologies belongs to the Health category. Almost 70% of Oct12 G2 ontologies are categorised as health ontologies and present the majority in G2 future graphs as well. Health ontologies are distributed through other identified communities in the future and present large portions of G4 and G6. Communities G1 and G3 are more heterogeneous with a mixture of ontologies from all categories in all graph versions.We also investigated the BioPortal project data to analyse its alignment with identified communities. Each project has a list of ontologies that it uses and we investigated how these lists correspond to the identified communities for the Jul14 version. Project with the highest number of ontologies used 35 ontologies, while the majority of projects used a single ontology. We ignored the latter projects in our analysis since it was obvious that they will be aligned with a single community. An important factor that influences the number of mappings between two ontologies is the size  of both ontologies. It is more likely that larger ontologies have higher number of mappings when compared to smaller ontologies. Unfortunately, we did not collect ontology sizes for each time spot in our analysis and historical data is not available through BioPortal’s API. We downloaded old versions of ontologies at the time of writing this paper and tried to manually parse the ontologies with the OWL API to calculate their sizes. The OWL API is a Java API and reference implementation for creating, manipulating and serialising ontologies . HoweverIn this paper we focused on investigating a comprehensive repository of biomedical ontologies  using graph theory concepts. We performed the exploratory study of BioPortal’s mapping data over different time points. As far as we know, this is the first attempt of this kind. With investigating mapping data gathered at five different time points using graph theory methods, we identified similar and closest communities of overlapping ontologies, and demonstrated evolution of communities over time. We also tried to validate communities through mentions of their ontology members in MEDLINE abstracts.The five communities identified in the first version of the graph changed their size. We showed how communities appear, disappear, split or merge over time. Based on similarity scores we determined closest communities between pairs of different graph versions. We then analysed the stability of these closest communities. We discussed how identified communities align with BioPortal’s category and project information. We also identified core ontologies of the closest communities.When studying our conclusions, we should take into consideration some limitations of the work. First, the BioPortal repository can be publicly modified and no evaluation of the uploaded ontologies or mapping data is done. In addition, although we tried to identify them, there are probably some “test” ontologies left in our data. Therefore, we should expect some data noise. Our analysis also showed large differences in data between the Aug13 and Dec13 when BioPortal 4 was announced. Second, our method for identifying communities might favour larger ontologies since we do not consider ontology sizes when calculating edge weights. Although our analysis of data gathered in November 2015 implies that normalising edge numbers results in small changes in final graph, this remains an area for future investigation. Next, due to limitations of BioPortal’s web service API, we were not able to distinguish between different types of ontology mappings in older versions. For example, the MESH and RH-MESH ontologies have same concepts and only differ in syntactic translation, which has not been picked up by our methods. Finally, our observations highly depend on the Louvain method for community detection. We accept this method as a “ground truth” quality metrics of our clusters. The Louvain method was the only available method in Gephi and it is considered as the fastest and most accurate method in terms of modularity score .In the future, we plan to address the above issues, especially distinguishing between different types of mappings and considering ontology sizes. We also plan to consider other graph centrality measures and methods for community detections. Finally, we plan to perform a deeper analysis of changes in the underlying ontologies to investigate how these affect the broader graph clustering patterns.10.7717/peerj.2990/supp-1Supplemental Information 1Mappings between pairs of ontologies for different versions of graphs.Click here for additional data file.


High-resolution transmission electron microscopy (HRTEM) and selected-area electron diffraction (SAED) confirm the formation of the GO layer structure and the hexagonal framework. This is a promising method for fast and effective synthesis of GO from sugarcane bagasse intended for a variety of energy and environmental applications.A new method of graphene oxide (GO) synthesis via single-step reforming of sugarcane bagasse agricultural waste by oxidation under muffled atmosphere conditions is reported. The strong and sharp X-ray diffraction peak at 2θ = 11.6° corresponds to an interlayer distance of 0.788 nm ( Graphenree form , thermalree form , and higree form ,6,7,8.4 and NaNO3 in concentrated H2SO4 acid.Graphene oxide (GO) has also attracted major interdisciplinary attention because of the broad range of envisaged applications across several scientific and engineering fields including physics, chemistry, biology, and medicine ,11,12,132, N2O4, and ClO2, toxic gases which are also explosive. In the past few years, various techniques such as mechanical exfoliation techniques, chemical vapor deposition (CVD) techniques and other chemical techniques have been developed for the synthesis of graphene and graphene oxide materials. However, many of these techniques are highly sophisticated and expensive [All these procedures involve the generation of NOxpensive . Most ofxpensive ,19.Here we report on the synthesis of graphene oxide by directly oxidizing sugarcane bagasse under muffled atmosphere. The GO produced by this method is called SOMA-GO (sugarcane oxidized under muffled atmosphere for graphene oxide). Furthermore, the structural characteristics of the obtained product are confirmed by X-ray diffraction, Fourier transmission infrared spectroscopy, field emission scanning electron microscopy, high-resolution transmission electron microscopy, and Raman spectroscopy.Sugarcane bagasse is a type of agricultural waste (agro waste). After juice was extracted, the remaining fiber was taken. The fiber was crushed and ground well in order to produce powder. This crushing and separating process was repeated several times to obtain fine powder. About 0.5 g of ground sugarcane bagasse powder mixed with 0.1 g of ferrocene was placed in a crucible and put directly into a muffle furnace at 300 °C for 10 min under atmospheric conditions. Next, the as-produced black solid was collected at room temperature. The black solid product was subjected to further analysis .−1 scan rate. Fourier transform infrared spectroscopy (FT-IR), Avatar 370, Thermo Nicolet  was carried out to study the bonding configuration in the synthesized graphene oxide. High resolution transmission electron microscopy (HRTEM) was performed using a JEOL JEM-3000F microscope  operating at 300 kV to study the microstructure of the GO structures. Field emission scanning electron microscopy was carried out using a JEOL JSM 7800F revealed the morphological features of the GO structure.The X-ray diffraction (XRD) pattern was obtained using a Bruker D8 Advance X-ray diffractometer . This device is equipped with a Ni filter, and generates monochromated Cu-Kα radiation (λ = 0.154 nm) operated at 40 kV accelerating voltage and 30 mA current. The samples were scanned in step mode with a 2° minThe structural and chemical analysis of sugarcane bagasse is clearly described by previous investigators ,21,22. I−1 is attributed to stretching vibration modes of C=O in carboxylic acid and carbonyl groups. The peak at 1594 cm−1 is assigned to the skeletal vibrations of un-oxidized graphitic domains. The band at 1060 cm−1 is assigned to C–O (epoxy) groups while the band at 1210 cm−1 is usually attributed to C–OH stretching vibrations [−1 can be attributed to the O–H stretching vibrations of the C–OH groups and water [The FT-IR spectrum of GO shown in brations ,26,27. TThe surface morphology of the graphene oxide (GO) produced by sugarcane bagasse oxidation is identified by FESEM. The SEM image of the GO shown in sp3 hybridized carbon atoms may also be introduced during the oxidation process. HRTEM micrographs of GO are shown in −1, which are attributed to the local defects (found at the edges of graphene sheets) and the 2sp graphitized structure, respectively. The G peak formation is due to the double degenerated zone center E2g mode [sp2 plane domains are possibly due to the extensive oxidation of sugarcane bagasse [D and G bands at 1358 cm−1 and 1585 cm−1 [sp2 carbon hybridization in the observed multi-layer stacks [DI/GI peak intensity ratios are assigned to lower defects/disorders. Raman spectra show an intensity ratio of DI/GI at 0.76 for GO which is in line with previous investigations [The Raman spectrum of graphene oxide synthesized from sugarcane bagasse is shown in E2g mode ,35. The  bagasse . The two585 cm−1  indicater stacks . Thus, Iigations ,37,38.A simple and rapid method is shown here to effectively convert solid sugarcane bagasse waste into value-added graphene oxide. The produced graphene oxide presents a well-graphitized structure. In comparison with the commonly used methods, our method is more environment friendly as it avoids toxic gas emission during the synthesis. This simple and low-cost synthesis process could lead to new opportunities for cost-effective production of graphene-based materials for gas sensors, energy storage, and other functional devices.


Commercialization of graphene based applications inevitably requires cost effective mass production. From the early days of research on graphene, direct liquid phase exfoliation (LPE) of graphite has been considered as the most promising strategy to produce high-quality mono or few-layer graphene sheets in solvent dispersion forms. Substantial success has been achieved thus far in the LPE of graphene employing numerous solvent systems and suitable surfactants. This invited review article principally showcase the recent research progress as well as shortcomings of surfactant assisted LPE of graphene. In particular, a comprehensive assessment of the quality and yield of the graphene sheets produced by different categories of the surfactants are summarized. Future direction of LPE methods is also proposed for the eventual success of commercial applications. Later in 2011, Nuvoli et al. reported an unprecedented graphene concentration as high as 5.33 mg/mL by sonicating a commercially available IL 1-hexyl-3-methyl imidazolium hexafluorophosphate (HMIM)  are semi-organic molten salts, composed of ions, which exhibit highly viscous liquid behaviour . ILs genVery recently, Texter and co-workers developed two excellent water stabilizers for graphene viz., triblock (TB) copolymer and copolymer nanolatex (NL)  to facilitate the phase transfer of graphene sheets from water phase to organic phase via simple sonication   (PS-b-P2VP)   , poly as well. Notley, in a similar study, compared pluronic non-ionic surfactants, F108 (molecular weight ~ 14.6 kDa) and F127 (molecular weight ~ 12.5 kDa) with some ionic surfactants such as CTAB (hexadecyltrimethylammonium bromide), TTAB (tetradecyltrimethylammonium bromide), DTAB (dodecyltrimethylammonium bromide) and SDS   (polyPA-b-polyPEG-A)  polymerization and graphite powder mixed with different amounts of aqueous copolymer solutions were sonicated for 6 h at 30 °C to provide upto 78 % graphene yield when the copolymer to graphite ratio reached 40. This amphiphilic polymer design with multi-pyrene groups was proposed to bind at the graphite surface simultaneously via π-π stacking, working like “sucking discs” to drag the bound surface graphene sheet off the graphite precursor with the assistance of sonication. The as-prepared graphene/polymer composite films also exhibited increased tensile strength and tunable conductivity. Direct exfoliation of graphite flakes in the presence of pyrene-labeled single stranded DNAs yielded highly aqueous dispersible mono- and bi-layer graphene sheets with 100 nm to 4 μm flake size [As mentioned above, Pyrene derivatives have been widely investigated as small molecule conjugate stabilizers. Concurrently, a few reports on pyrene-based polymers are also exploited as efficient graphene exfoliant. One typical report is from Zheng et al., wherein supercritical (SC) COsability . Specialake size . SubsequHighly conductive and transparent graphene films were fabricated by Jo et al. from direct exfoliation of graphite using a non-ionic semiconducting polymer quinquethiophene-terminated PEG (5TN-PEG) . Relevant crucial processing factors such as solvents, external forces like ultrasonication/shear and purification methods by centrifugation have been discussed in detail in association with their influences on the exfoliation results. Significantly, most of the solvents used in the initial studies had revealed significant drawbacks, such high toxicity, high boiling point etc. that prompted the re-direction of research into environment benign less toxic solvents like water. Unfortunately, the surface energy of pure water is too high for graphene exfoliation such that a variety of surfactants have been introduced thus far. We categorized the large spectrum of surfactants in accordance to their structural functionalities into aromatic, non-aromatic, ionic liquids and polymers. Innumerable surfactants have been studied in this regard and many of them appeared highly promising LPE results.For the further progress of surfactant assisted LPE, several shortcomings must be overcome: (1) The overall yield of LPE is still low; (2) Good exfoliating solvents are expensive and harmful; (3) Sonication/Shearing commonly lead to the drastic reduction in the size of exfoliated graphene sheets; (4) Residual surfactants are difficult to remove; (5) Typical surfactants are electrically insulators, which may significantly deteriorate the electrical connectivity among graphene layers; (6) All LPE methods produce graphene sheet with a high polydispersity in terms of lateral size as well as thickness. The future of real-life graphene applications strongly depends on how materials scientists address these formidable challenges and establish ideal large-scale LPE process for high quality graphene sheets. It is also highly required to attain more fundamental and systematic understanding of the exfoliation mechanism for innovative design of LPE schemes.