Despite its fleeting existence, H2CO3 plays an important role in the global carbon cycle and in biological carbonate-containing systems. The short lifetime in water and presumed low concentration under all terrestrial conditions has stifled study of this fundamental species. Here, we have examined CO2/H2O mixtures under conditions of high pressure and high temperature to explore the potential for reaction to H2CO3 inside celestial bodies. We present a novel method to prepare solid H2CO3 by heating CO2/H2O mixtures at high pressure with a CO2 laser. Furthermore, we found that, contrary to present understanding, neutral H2CO3 is a significant component in aqueous CO2 solutions above 2.4 GPa and 110 °C as identified by IR-absorption and Raman spectroscopy. This is highly significant for speciation of deep C–O–H fluids with potential consequences for fluid-carbonate-bearing rock interactions. As conditions inside subduction zones on Earth appear to be most favorable for production of aqueous H2CO3, a role in subduction related phenomena is inferred.Carbonic acid (H Yet, the exact crystal structure of β-H2CO3 is still unknown. The FT-IR and Raman spectra of solid H2CO3 below 220 K at low pressure are now well established and several theoretical studies have reported on the structure and stability of H2CO3 with respect to decomposition to CO2 and H2O as well as on the relative stability of its isomers and on their vibrational characteristics78131412CO3 at elevated pressure has been predicted by ab initio molecular dynamic simulations2CO3 (aq) have been found up to 1.94 GPaCarbon dioxide CO, when diacid H2CO, but theon2 ref. ; and dir2O/CO2 mixtures experimentally at high pressure and temperatures in a diamond anvil cell (DAC), looking for evidence of increased stability of molecular H2CO3 in aqueous solution at elevated pressure. Within the Earth, at depths corresponding to pressures of up to 4.6 GPa , temperatures are heterogeneously distributed2O/CO2 mixtures were compressed to the desired pressure and then heated by irradiation with a CO2 laser. The laser was focused to irradiate only a fraction of the sample. In this way, the behavior of the system at high T can be investigated in the center of the heated area while phenomena of the transition down to room temperature can be investigated on the hot spot periphery. A solid product was synthesized during laser heating and the pressure-dependence of the vibrational modes of this product was investigated up to 9.1 GPa at ambient temperature. Additionally, the stability of the solid product with respect to pressure was investigated by compression to 25 GPa after synthesis at 4.3 GPa. In the second set of experiments, the sample was heated resistively to up to 280 °C after compression to the desired pressure. During resistive heating, the evolution of the system was monitored in situ by IR absorption spectroscopy. In both sets of heating experiments, the pressure range from 1.5 to 4.6 GPa was covered.In this work, we studied H2/H2O mixtures with a wide range of pressures  up to 1500 °C by CO2 laser heating. The measurement on a CO2/H2O (1:1.4) mixture at 3.5 GPa shall serve as an example of our results. When the directly illuminated sample area was heated above the melting temperature of the components, only a small part around this area remains liquid due to the steep thermal gradients encountered in laser-heating experiments in a DAC. During laser-heating to about 1500 °C, a dark substance formed around the directly heated area, which disappears again when the laser spot is moved above it. However, the dark substance remained at the trailing end of the scanning movement which enabled us to deposit it in the whole sample area. The directly heated region remained clear after the laser was switched off  and the O-H stretching mode of H2O-Ice VII (3000-3400 cm−1) are greatly reduced with the appearance of these new peaks. The only compounds in the CO2-H2O binary system known to form from a reaction of its endmembers are CO2 clathrate hydrates2 clathrate hydrate can be excluded as its Raman spectrum2O and CO2 and even the recently discovered high-pressure modification was shown to decompose above a pressure of 1 GPa2O/CO2 mixtures known to exist as a solid is H2CO3. Indeed, the sharp peak at 1073 cm−1 is coincident with the intense mixed mode of the symmetric stretching vibrations νs(C–O) and νs(C=O) of crystalline H2CO3 , respectivelyip(COH) and ν(C–O) vibration in crystalline H2CO3  vibration of CO2. Also, the IR absorption spectra of both the dark and the light sample region at room temperature  revealed, that the dark product could be observed at any investigated composition  is found at slightly lower wavenumbers, the skeletal bending mode, δip(COO), δoop(COH), νas(C–O)/νas(C=O), and δip(COH) are found at higher wavenumbers than the bands from the reference spectra while νas(C=O), and νas(C(OH)2) appear essentially at the same frequency. We are confident that the position of IR absorption bands in H2CO3 is not very sensitive to temperature as the published IR absorption bands of H2CO3 from Kohl et al.et al.−1 Raman mode is sensitive to sample temperature we first synthesized the dark product as described above from a CO2/H2O (1:1.8) mixture at 4.0 GPa and then turned on the laser again at lower power to heat the center of the directly illuminated the sample area to below 700 °C. A video taken during laser heating  displays a red shift, δoop(COH), and νas(C–O)/νas(C=O), and δip(COH) blue-shift while νas(C=O) and νas(C(OH)2) do not shift significantly. Extrapolating to the low pressure at which the reference spectra were recorded, all modes observed in our experiment shift towards the published spectral features of H2CO3  initially held at 4.3 GPa as described above. After laser heating, the pressure increased to 5.2 GPa, consistent with earlier observations. The sample was then compressed to 25 GPa while the intense Raman band at 1073 cm−1 was recorded  along the thermal gradient at the edge of the directly heated area. To investigate this, a 1:1 mixture of CO2 and H2O at 2.4 GPa was resistively heated to 280 °C with a rate of 5 °C/min and the spectral evolution was monitored in situ by IR absorption spectroscopy  and νas(CO2) mode of HCO3− , no evidence for formation of aqueous H2CO3 appears to be an integral component of CO2/H2O mixtures at and above pressures of 2.4 GPa at elevated temperature. This finding can be rationalized on the following grounds. The relative abundance of H2CO3 and its dissociation products HCO3− and CO32− in an aqueous solution of CO2 is largely determined by the dielectric constant (ε) and the autodissociation constant Kw of its host solvent H2O, and both are complex functions of pressure (p) and temperature (T)23p favors increased ionization, i.e. an increase in Kw of water. As a result of this, in the carbonate system with increasing p, more ionic species (HCO3− and CO32−) are created from neutral, dissolved CO2(aq) due to the larger abundance of H+. Isobaric heating of water affects Kw and thereby the extent of ion pairing of the dissolved members of the carbonate system3− and CO32− in absence of metal ions can only mean pairing with H+, the free acid H2CO3 will be produced. Indeed, it has been observed that both ionization constants of aqueous H2CO3 decrease upon heating252SO4 was observed to increase from 3.2 to almost neutral pH when heated to 400 °C at 0.4 GPa and similar associative behavior upon heating is observed in aqueous solutions of HCl and H3PO4  and H2CO3 which is produced by the novel high-pressure technique presented herein in the investigated pressure range are likely identical.It has been shown here that H−1 Raman mode in the liquid area around the directly heated part of the sample means that solid H2CO3 does not dissolve under the high-pressure conditions of our experiment during laser-heating, even in presence of liquid water. This seems to contradict the observation that solid H2CO3 (produced during laser heating) disappears as soon as liquid water is present (during whole-cell heating). From the latter observation combined with the absence of re-precipitation of solid H2CO3 during slow cooling after whole-cell heating it can be inferred that solid H2CO3 is not thermodynamically stable in our experiment in presence of liquid water even at elevated pressure (>2.4 GPa). The formation and metastability of solid H2CO3 during laser heating must therefore be the result of a non-equilibrium process. Based on the observation of apparent of H2CO3 molecules at elevated temperatures during whole-cell heating, we suggest the following model for this non-equilibrium process. The concentration of H2CO3 molecules increases with temperature in CO2/H2O fluids above 2.4 GPa. By laser heating, a convective motion is initiated in the cell which transports mass away from the heated area, following the steep thermal gradient at its edges. In pure H2O, a polar molecule like H2CO3 would be expected to be miscible at all ratios. However, when CO2 is added to aqueous solutions, the ability to dissolve polar/ionic species is decreased due to the lower dielectric constant (ε) relative to pure H2O at the same conditions2/H2O mixture show an immiscibility gap for high concentrations of CO2 and solute with decreasing temperature while the same solute would be entirely soluble in pure H2O under the same conditions2CO3 molecules is transported to the cooler edges of the heated area faster than thermodynamic equilibrium can be re-established, H2CO3 concentrations could be high enough to make solid H2CO3 a stable phase in the CO2/H2O/H2CO3 ternary system. When the laser beam is moved across the sample, solid H2CO3 that precipitated from the hot fluid at the cooler edges of the heated area is then stabilized by the rapidly solidified CO2/H2O/H2CO3 matrix at the trailing end of the heated spot before it can dissolve by reaction with liquid water at lower temperatures. The steep thermal gradients that are apparently needed to produce solid H2CO3 in a DAC together with the apparent need for stabilization in a solid matrix make it highly unlikely that solid H2CO3 occurs as an abundant solid inside the Earth. However, the necessity of steep thermal gradients for the synthesis of solid H2CO3 also emphasizes the large effect rising temperature has on the abundance of dissolved molecular H2CO3 in deep fluids.The observation of the 1073 cm2CO3 has never been considered significant in models of COH fluids inside the Earth2CO3 is a stable species in aqueous fluids above 2.4 GPa (~ 85 km depth), and at least up to 280 °C at 3.5 GPa, and its overall stability field in aqueous fluids is far from being mapped. Some constraints can be put on the formation of H2CO3 from our experimental results, though. As a classic Brønstedt acid, its stability will depend strongly on pH with high H+ concentrations promoting H2CO3 formation. Its stability will also depend on the redox-state of the environment and is probably limited to regions where CO2 is stable, too. Furthermore, our experiments indicate that fluids present at higher temperature will contain more H2CO3 than cooler fluids.Due to its instability at ambient pressure, H2CO3 will be the simultaneous availability of free CO2 and H2O. In subduction zones, which probably show the most favorable conditions for formation of H2CO3 on Earth in terms of pressure, and temperature312 and H2O, aqueous carbon bearing fluids are created by devolatilization of the subducting slab2 with the expected peak concentration above 750 °C2 concentration in the fluid is well above the minimum pressure of 2.4 GPa identified by us for the stability of aqueous H2CO3. Production of aqueous H2CO3 could therefore be connected to subduction related volcanism.A limiting factor on the provenance of H2CO3 produced, CO2 and H2O could be significantly deprived from hot fluids through H2CO3 formation which has consequences on the geochemistry of these fluids. An apparent “masking” of H2O and CO2 in fluids at pressure above 2.4 GPa can have an influence on numerous properties of deep fluids like activity of its constituents, electrical conductivity, viscosity and density2CO3 below 2.4 GPa would be equivalent to a sudden release of CO2 and H2O which should be accounted for in models of deep fluids in this pressure region. If H2CO3 formation happens at the expense of ionic species of the carbonate equilibrium, increased solubility of carbonate minerals can be expected as compared to models neglecting H2CO3 formation2CO3 molecules in aqueous fluids above 2.4 GPa should concentrate on a quantitative description of the global carbonate equilibrium as all the above mentioned effects will strongly depend on the equilibrium concentration of H2CO3 under the respective conditions. These results will also be relevant for the geochemical modeling of large, water-rich exoplanets.Depending on the amounts of H2 (purity > 99.99%) was loaded into IIa type diamond anvil cell (DAC) containing ultrapure H2O (>18 MΩ) with the aid of a gas loader at 500 bar. The CO2/H2O ratio was determined from the relative volumes occupied by the then solid CO2 and H2O, considering the molar volumes at the respective pressure. Typically we used a gasket made of Rhenium or Platinum. The 632.8 nm line of a He-Ne-laser was used to excite the Raman spectra. Infrared (IR) measurements were conducted using a Bruker IFS-66V FTIR spectrometer equipped with a KBr beam splitter and a globar mid-infrared source. The IR beam size was adjusted according to the measured area. Spectra of the dark and the light region were recorded by shading off the part to be excluded from the light source of the spectrometer. High temperatures were achieved either by irradiation with a CO2-laser or by means of whole-cell external heating. In external heating experiments, the temperature was determined with the help of a thermocouple glued directly on one of the diamond anvils. In laser-heated experiments, temperature was determined by the glow color in the center of the heated area. While temperature estimation from the color of glowing may produce a big uncertainty, our experiments showed that crystalline H2CO3 can be synthesized in a wide temperature range as apparent from no visible glowing to a white glowing sample area. This means, that this big uncertainty in temperature has no significant effect on the validity of the results of this study. A ruby chip was inserted in the sample and the pressure determined from the peak wavelength of the R1 ruby fluorescence band.COHow to cite this article: Wang, H. et al. Stable solid and aqueous H2CO3 from CO2 and H2O at high pressure and high temperature. Sci. Rep.6, 19902; doi: 10.1038/srep19902 (2016).


Spin polarization and stacking are interesting effects in complex molecular systems and are both presented in graphene-based materials. Their possible combination may provide a new perspective in understanding the intermolecular force. The nanoscale graphene structures with zigzag edges could possess spin-polarized ground states. However, the mechanical effect of spin polarization in stacking of graphene nanofragments is not clear. Here we demonstrate the displacement between two stacked rhombic graphene nanofragments induced by spin polarization, using first-principles density-functional methods. We found that, in stacking of two rhombic graphene nanofragments, a spin-polarized stacked conformation with zero total spin is energetically more favorable than the closed-shell stacking. The spin-polarized conformation gives a further horizontal interlayer displacement within 1 angstrom compared with the closed-shell structure. This result highlights that, besides the well-known phenomenologically interpreted van der Waals forces, a specific mechanism dependent on the monomeric spin polarization may lead to obvious mechanical effects in some intermolecular interactions. The interactions between magnetic objects and the earth through the Earth’s magnetic field brought about the birth of compass. Analogizing this macroscopic magnetic induction, we expect to find a similar induced behavior between two molecules with a separation beyond bonding distance. So far, the magnetism of carbon-based materials has been confirmed in experiments13789The inference can be appropriately demonstrated by stacking of graphene nanofragments. Stacking is acknowledged as a typical structural balance held by intermolecular force, which is sensitive to monomeric electronic structure such as the π-electron delocalization in graphene. Realizations about this phenomenon can be traced back to the studies of structures of graphite. The concept of AB stacking for bilayer graphene originated from the Bernal structure for graphite determined in 1924131517S = 0 spin-polarized ground state. Then, the stacking systems of freestanding bilayer graphene nanofragments were constructed.According to studies and discussions above, we chose the rhombic graphene nanofragment with hydrogen passivated edge atoms to be the stacking monomer in this work, which possesses four zigzag edges and an S = 0 spin-polarized conformation has the lowest total energy which is about 0.42 eV lower than that of the closed-shell one. Furthermore, a horizontal displacement about 0.82 Å is presented between two fragments of the spin-polarized structure, compared with the closed-shell stacking. Thus, the slippage in stacking of graphene nanofragments induced by spin polarization is clearly demonstrated. The displacement is clearly shown in After structural optimizations, both spin-polarized and closed-shell stacked conformations of bilayer graphene nanofragments were obtained. The ones most stable in two states respectively are shown in S = 0 spin-polarized ground state of the single rhombic graphene nanofragment is stable when the size increases. And it’s suggested that the mechanical effect induced by spin-polarization would occur when the size of the system increases in a small range. Meanwhile, twisted stacking didn’t give a lower total energy than the optimal slipped conformation in our calculations. Comparison of the total energy among different states of the single rhombic fragment in different sizes can be found in In addition, the Spin-polarized stacked conformations different from the AA and AB stacking have been reported in bilayer zigzag graphene nanoribbons17201922This work, as a typical demonstration, pointed out the bridge between spin polarizations of nanoscale graphene structures and their complex structural conformations in intermolecular interactions. Meanwhile, as an example of intermolecular behaviors response to the monomeric spin polarization, we anticipate this finding to offer an inspiration for further explorations and designs.Simulations were implemented by first-principles calculations in Gaussian 0925How to cite this article: Lei, Y. et al. Slippage in stacking of graphene nanofragments induced by spin polarization. Sci. Rep.5, 10985; doi: 10.1038/srep10985 (2015).


M = 2000), N-methyldiethanolamine, and formic acid. It was found that addition of approx. 2 wt.% of graphene causes the loss of volume resistivity by three orders of magnitude and percolation threshold is already set at approx. 1 wt.%. The frequency characteristic of a real part of permittivity ε′ and imaginary part of permittivity ε″ were measured for the tested films. On the base of Havriliak–Negami equation, parameters of relaxation functions in frequency domain were estimated for samples containing various contents of graphene. The influence of the cationomer phase structure on observed changes of dielectric losses coefficient tgδ in the full-measuring frequency spectrum was discussed.The research was planned to test electrical properties of polymer films made from polyurethane cationomers with 0–2 wt.% graphene admixture. The cationomers were synthetized in the reaction of 4,4′-methylenebis(phenyl isocyanate), polycaprolactone diol ( It was found that the presence of graphene results in increased thermal and mechanical strength of received polymer films and contributes to the increase in hydrophobicity of generally hydrophilic coatings prepared from waterborne polyurethane cationomers [In the part I of the paper  synthesiionomers . Meanwhi3O4 particles modified by oleic acid, which were dispersed in PPG glycol under ultrasonic bath. The addition of 0–3.5 wt.% of Fe3O4 caused the loss of the volume resistivity of obtained polymer foils from the value of 1.04 × 1014 to 5.96 × 104 Ω cm and surface resistivity from the value 1013 to 105 Ω cm−2 [ε′) from the value of approx. 8 to 50 and dielectric loss constant (ε″) from the value of 0.1 to 0.9 measured by the frequency of 0.01 Hz. The measurements conducted in conditions of slow increase of frequency up to 106 Hz in the case of these nanocomposites containing above 1.5 % GO showed nonlinear loss of constants values ε′ and ε″, while these constants in the case of PU without admixtures had values 5 and below 0.1 and changed with frequency insignificantly [So far, there have been many descriptions of modifications of polymer electric properties, for typical dielectrics after admixture introduction of metal nanoparticles like Au, Ag, polyaniline, carbon black, or specially prepared multi-walled carbon nanotubes (MWCNT) –4. Limitficantly , electroN-metyldiethanoloamine. In order to obtain nanocomposites, graphene was previously noncovalent functionalized in THF in the field of ultrasound. The reference films were prepared by covering poly(tetrafluoroethylene) (PTFE) or glass plates by the polyurethane cationomers water dispersions. Next, the films were placed in a vacuum drier, at 80 °C, for over 6 h, and the process of film formation was completed by exposure to ambient air during 10 days. In this way, polymer films containing respectively 0 (PU-0 sample), 0.1 (PU-0.1), 0.5 (PU-0.5), 1 (PU-1), 1.5 (PU-1.5), or 2 wt.% (PU-2) of the graphene were received [The research included the polymer films prepared from polyurethane cationomer produced from polyurethane cationomers with added graphene. Polyurethane cationomer as polymer matrix for 0–2 wt.% graphene was synthesized in the reaction of MDI, polycaprolactone diol, and IEC 93:1980 Methods of test for volume resistivity and surface resistivity of solid electrical insulating materials.ASTM D 257-99 Standard test methods for dc resistance or conductance of insulating materials.The measurements of volume resistivity were taken in accordance with the fopllowing standards:There was a measuring setup used based on three electrodes, presented in Fig. −4–5 × 103 Hz. The relaxation processes and the basic dielectric parameters were defined on the basis of frequency domain spectroscopy (FDS). The system DIRANA of OMICRON was used to conduct measurements. The software Origin v.8.0 and WinFit of Novocontrol was used to present and analyze the results of the measurements. The generic complex equation Havriliak–Negami (H-N) equation was applied to set basic relaxation functions’ parameters in frequency, which described the changes of dielectric permittivity in frequency function [kk = 1 for low-frequency component HN1 and k = 2 for high-frequency component HN2);is the total number of partial  and optical high-frequency permeability is the polarizability, the difference between static permittivity (low frequency)  and 2,2-bis(hydroxymethyl)propionic acid (DMPA) had the volume resistivity of 1011 Ω m [Tg1 = −49.7 °C) and hard segments (Tg2 = −23.9 °C), that give it good mechanical properties and considerable polarity with high surface energy of 45.3 × 10−3 J m−2. In the chain of anionomers analyzed in the paper [8 Ω m for the pure material to 3.4 × 105 Ω m. The surface of approximately 1 % of the graphene admixture where the sudden decrease of resistivity is probably caused by the exceed of percolation threshold is noticeable. It should be noticed that a little smaller amount of percolation threshold (0.5–1 %) was observed in polyurethane composites modified by multi-walled carbon nanotubes (MWCNT), where the significant decrease in resistivity from 2.5 × 1012 Ω m to 5–6 orders of magnitude was observed [Regarding data presented Table 1011 Ω m . The volhe paper , there wSignificantly more cross-linked structure polyurethane/hyperbranched poly(urea-uretane) doped the similar amount of MWCNTs did not show any significant changes in resistivity . It can ε′ (capacitance) and imaginary part of electric permittivity ε″ (dielectric losses) over the whole measurement range of frequency. The obtained parameters ε′ and ε″ for the polyurethane cationomers which have not been doped are approximately 1–2 orders of magnitude higher than the ones registered in the case of the polyurethane anionomers [Having analyzed the obtained FDS characteristics Fig. a, b, it ionomers  as well ionomers  that conα-relaxation occurring in the area of hard urethane segments, which is especially visible in the DSC and DMTA research in the temperature Tg2 in the extent of 25–40 °C. While the low-frequency relaxation β  is relal–Wagner  occurrinσ0, and there was an analysis of observed relaxation processes conducted in tested samples.The parameters of FDS characteristics, estimated with H-N equation for tested polyurethane films are given in Table N = 1 occurring in the equation H-N allows to conclude that in the tested films the ionic conductivity prevails. The increase of the graphene concentration in the range from 0.1 to 2 % results in logarithmic increase of AC conductivity parameter σ0 (conductivity) from 1e−12 Ω−1 m−1 for the undoped material to 4.5e−11 Ω−1 m−1 for 2 % of admixture. It is the process related to the increase of the concentration of ions and the change of structure as well. The admixture of graphene significantly influences the structural relaxation of the material which is observed in prolongation of the relaxation time and the rapid increase of the parameter value τ when amount of admixture exceeds 1 % is visible  for various concentration of graphene in tested polyurethane cationomers. The increase of admixture amount causes the parameter α1 to reach the value of 1, which confirms Debye character of relaxation  and imaginary part of permittivity ε″ (dielectric losses), which can be explained by the high polarity and the phase structure. In the relaxation phenomena in the tested material, there are probably dominant interactions of the dipole–dipole type; therefore, a strong frequency dispersion of constants ε′ and ε″ especially in the range of 10−4–10−1 Hz and significant increase of relaxation time (with the increase of graphene contents) especially at low frequency are observed. The admixture of graphene in the amount 0.5–2 wt.% causes the increase of conductivity and percolation threshold is set on the level of approx. 1 wt.%. The values of constants ε′ and ε″ clearly grow with the increase of graphene amount with the similar shape of frequency dispersion curves as for PU without admixture. There has been the large decrease of PU resistivity in the presence of graphene, which was noncovalent functionalization in THF solvent in the field of ultrasound gives a significant advantage if compared to nonconducting polyurethane compounds obtained with oxide graphene.The research showed that polymer film made from waterborne cationomer polyurethane is characterized with lower volume resistivity, on the level 6.0 × 10


Programmed death 1  and programmed death ligand 1  are involved in generating tumor-associated immunosuppression by suppression of T-cell proliferation and interleukin 2 (IL-2) production and immune checkpoint inhibitors targeting these molecules are showing compelling activity against a variety of human cancers. PD-L1 expression has shown a positive association with response to PD-1 inhibition in non-central nervous system (CNS) tumors, e.g., melanoma or non-small cell lung cancer, and is discussed as a potential predictive biomarker for patient selection in these tumor types. This review summarizes current knowledge and potential clinical implications of PD-L1 expression in glioblastoma. At present, the following conclusions are drawn: (a) functional data support a role for PD-1/PD-L1 in tumor-associated immunosuppression in glioblastoma; (b) the incidence of PD-L1-expressing glioblastomas seems to be relatively high in comparison to other tumor types, however, the reported rates of glioblastomas with PD-L1 protein expression vary and range from 61 to 88%; (c) there is considerable variability in the methodology of PD-L1 assessment in glioblastoma across studies with heterogeneity in utilized antibodies, tissue sampling strategies, immunohistochemical staining protocols, cut-off definitions, and evaluated staining patterns; (d) there are conflicting data on the prognostic role and so far no data on the predictive role of PD-L1 gene and protein expression in glioblastoma. In summary, the ongoing clinical studies evaluating the activity of PD-1/PD-L1 inhibitors in glioblastoma need to be complemented with well designed and stringently executed studies to understand the influence of PD-1/PD-L1 expression on therapy response or failure and to develop robust means of PD-L1 assessment for meaningful biomarker development.  As early as 1863, Rudolf Virchow reported inflammatory infiltrates in tumor tissues and suggested an important link between cancer and the immune system . Cancer-PTEN) gene, gene rearrangements of the PD-L1 gene CD274 or mutations of the 3’-untranslated region (UTR) of the CD274 mRNA and other molecular alterations , 1210, 1Rate of PD-L1-expressing glioblastomas The reported rates of patients with glioblastomas with any PD-L1 protein expression on tumor cells were 61%  in the study by Nduom et al. , 12 and PD-L1 protein expression patterns We found membrane-bound PD-L1 expression on interspersed epithelioid glioblastoma cells in 37.6% of newly diagnosed and 16.7% of recurrent glioblastoma cases  10]. In. In10]. Prognostic role of PD-L1 gene expression CD274) gene of 446 patients using level 2 Agilent microarray gene expression data and found no correlation with patient outcome. In contrast, Nduom et al. , 1210, 1eviously . Overalleviously , 46. Prognostic role of PD-L1 protein levels MGMT promoter methylation status showed the expected separation of prognostic cohorts in this series, thus showing the principal validity of this patient cohort for exploratory survival analyses. There was no correlation with overall survival times for presence or absence of diffuse/fibrillary or membranous PD-L1 expression. Of note, PD-L1 expression on neurons in the infiltrated cortex did not show a correlation with patient outcome, which is in contrast with a prior small study of 17 cases [There are conflicting reports on the prognostic role of PD-L1 protein levels. Berghoff et al.  investig17 cases , 48. MGMT promoter methylation status were not reported. Using the median of 2.77% PD-L1 positive glioblastoma cells they did not find a significant correlation with patient outcome (p = 0.066). However, using a 5% cut-off in a secondary analysis, patients with high PD-L1 expression had significantly shorter survival times (p = 0.086). Nduom et al.  performeThe considerable differences in applied methodology between the studies  and theiPredictive role of PD-L1 expression So far, no clinical trials with PD-1 or PD-L1 inhibitors have been completed in glioblastoma and no data on the predictive role of PD-L1 expression for response to these drugs are available . TestingEmerging clinical studies document compelling antineoplastic activity of immune checkpoint inhibitors including those targeting PD-1/PD-L1 across several tumor types . So far,Antibody 5H1 was kindly provided by Dr. Lieping Chen, Yale University. MP has received honoraria and research support from Bristol-Myers Squibb, Merck Sharp & Dohme, Boehringer Ingelheim, GlaxoSmithKline, Mundipharma, and Roche. ASB has received honoraria and travel support from Bristol-Myer Squibb, Amgen, and Roche. WW has received research grants from Apogenix, Boehringer Ingelheim, Eli Lilly, immatics, MSD, and Roche as well as honoraria for lectures or advisory board participation from MSD and Roche. MW has received research grants from Acceleron, Actelion, Alpinia Institute, Bayer, Isarna, MSD, Merck & Co, Novocure, PIQUR, and Roche and honoraria for lectures or advisory board participation or consulting from Celldex, Immunocellular Therapeutics, Isarna, Magforce, MSD, Merck & Co, Northwest Biotherapeutics, Novocure, Pfizer, Roche, and Teva.


Magnetic domain structure and spin-dependent reflectivity measurements on cobalt thin films intercalated at the graphene/Ir(111) interface are investigated using spin-polarised low-energy electron microscopy. We find that graphene-covered cobalt films have surprising magnetic properties. Vectorial imaging of magnetic domains reveals an unusually gradual thickness-dependent spin reorientation transition, in which magnetisation rotates from out-of-the-film plane to the in-plane direction by less than 10° per cobalt monolayer. During this transition, cobalt films have a meandering spin texture, characterised by a complex, three-dimensional, wavy magnetisation pattern. In addition, spectroscopy measurements suggest that the electronic band structure of the unoccupied states is essentially spin-independent already a few electron-Volts above the vacuum level. These properties strikingly differ from those of pristine cobalt films and could open new prospects in surface magnetism. Just a decade after being isolated, graphene has attracted considerable attention in the scientific community. Studies on this material allowed the discovery of rich and fascinating phenomena12371317181920With its weak spin-orbit coupling and hyperfine interaction, graphene also offers new opportunities in nanomagnetism2526272829353637pz and Co 3d orbitals60 molecules are deposited on top of a Co thin film43If magnetism can be induced in graphene when contacted to a ferromagnet17181920Using spin-polarised low-energy electron microscopy and spectroscopy, we investigate in this work the in-plane to out-of-plane spin reorientation transition (SRT) that occurs upon Co intercalation between graphene and Ir(111), with a special focus on the determination of the surface magnetic anisotropy. The most striking result of our study is the observation of an unusually gradual thickness-dependent SRT: the canting angle the magnetisation vector makes with the surface normal is found to change by less than 10 degrees per additional Co monolayer, meaning that about 10 Co atomic layers are required to drag magnetisation into the surface plane. Analysis of this reorientation transition allows estimation of the second and fourth-order surface anisotropy terms, which both differ substantially from the values determined in pristine cobalt films. In particular, the second-order term is found to be unusually large, confirming that graphene favors perpendicular magnetic anisotropy. Besides, measurement of all three Cartesian components of the magnetisation vector during the SRT reveals that in-plane and out-of-plane magnetic domain patterns are characterised by different length scales that evolve differently when the cobalt thickness is increased. While in-plane domains show no change throughout the spin reorientation transition, meandering out-of-plane domains with well-defined, slowly decaying periodicity are observed when the cobalt film gets thicker. A complex three-dimensional spin texture is deduced from sets of magnetic images in graphene-covered cobalt films, in sharp contrast with what is found in pristine surfaces. Finally, spin-dependent reflectivity measurements suggest that spin-scattering asymmetry is suppressed when the energy of the incoming electrons becomes larger than a certain threshold value, regardless the thickness of the cobalt film.in situ, in the spin-polarised low-energy electron microscope (SPLEEM) available at the National Centre for Electron Microscopy of the Lawrence Berkeley National Lab. In a SPLEEM, an electron beam is directed at the sample in normal incidence and electrons that are backscattered from the surface are used for imaging. Because the energy of the incoming electrons is low, typically a few eV above the Fermi level of the material under investigation, the technique is surface sensitive and a few atomic layers are usually probed. The electron beam being spin-polarised, the technique is also sensitive to the surface magnetisation. The main strength of the technique is the capability to orient in any space direction the spin polarisation of the incident electron beam, thus allowing to probe unknown magnetic configurations. Moreover, since the energy of the spin-polarised electron beam can be tuned continuously, reflectivity measurements can provide useful information on the spin-split band structure of unoccupied electronic states4647All measurements were done −11 mbar range). An Ir(111) single crystal was used as a substrate and its surface was cleaned in a preparation chamber attached to the microscope, following a procedure based on repeated cycles of Ar+ ion sputtering and high temperature (1200 °C) flashes under oxygen (10−8 mbar). A last temperature flash (1200 °C) was done in the microscope chamber to remove the oxide layer. Graphene was grown by chemical vapor deposition, following a well-established procedure−8 mbar, typically for several tens of minutes), while keeping the substrate at about 600 °C, leads to the formation of a single layer of graphene, covering >99% of the Ir(111) surface, thanks to the self-limited mechanism involved in the ethylene decomposition4043404043504043515253The samples studied in this work were fabricated and imaged under ultra-high vacuum conditions /(I+ + I−), where I± is the reflected intensity for a spin polarisation ±P0, is a function of the incident energy. In most cases, the spin asymmetry changes in sign and amplitude depending on details of the unoccupied band structure of the material. Examples of energy-dependence of SPLEEM contrast can be found in previous literature55When a low-energy, spin-polarised electron beam is directed at a metal surface, part of the incident electrons enters the crystal and part of the incident intensity is backscattered. If the metal is ferromagnetic, the backscattered intensity depends on the relative orientation of the incoming spin polarisation and the direction of the local magnetisation within the material. This mechanism leads to a magnetic contrast in SPLEEM, which is used to image magnetic domain patterns. For common ferromagnetic metals, such as Fe, Co and Ni, the spin asymmetry When the same surface is graphene-terminated, the spin asymmetry is drastically modified. In particular, it shows a peculiar feature: while the spin asymmetry first increases as the incident energy becomes larger than the surface work function, it quickly decreases to become negligible above 5 eV, and up to at least 20 eV . Within this energy window, the graphene-covered Co surface seems non magnetic. Although the Co surface is of course ferromagnetic, above a certain threshold energy, the incident spin-polarised electrons do not suffer substantial spin-dependent elastic or inelastic collisions.This suppression of the spin asymmetry above a few electron-Volts is thickness-independent. Indeed, comparing the spin asymmetry of 3 and 9 ML-thick Co film intercalated between graphene and Ir does not show clear differences see . Our expIn the following, we study the spin reorientation transition (SRT) that occurs when the amount of intercalated cobalt is increased, and we compare it to the SRT observed in pristine cobalt. For thin-enough films, magnetisation is out-of-plane, while it is in-plane for thicker films. To investigate how magnetisation rotates, we image magnetic domain patterns for different cobalt thicknesses in the three Cartesian coordinates. From the set of SPLEEM images, we deduce the local direction of the magnetisation vector as a function of Co thickness. SPLEEM measurements for Co films intercalated between graphene and Ir(111) are reported in θ and φ, as illustrated in the inset of M thus continuously rotates from θ = 0° to θ = 90°. In the canted phase, the θ values can be extracted from 59The magnetisation vector can be represented in terms of the two spherical angles The SRT of our Co films can be described by the anisotropy flow model, in which the influence of both the surface and the interface on the magnetisation is accounted for by their respective phenomenological anisotropy constantsnd-order and the shape magnetic anisotropies, and K4 is the 4th-order term of the magnetocrystalline anisotropy. Minimising E with respect to θ gives three possible equilibrium states: θ = 0°, θ = 90° and θ dependence of the Co film thickness t, we express K2 and K4 by the phenomenological ansatzwhere Ks2 and Ks4 are the sum of two terms associated with the Co/Ir(111) and graphene/Co interfaces. In this expression, Kb2 and Kb4 stand for the bulk anisotropy values. Hence, the SRT is delimited by two critical thicknesses:Note that the interface anisotropy constants tc1 < tc2, then the θ dependence of the Co thickness reads:If tc2 − tc1 and where Δ = Ms = 1.44 × 106 A/m, Kb2 = 5.0 × 105 J/m3 and Kb4 = 1.25 × 105 J/m3)Ks2 and Ks4. For bare Co, we find Ks2 = 0.64 mJ/m2 and Ks4 = −0.03 mJ/m2, which are comparable to the values found for Co films on Au(111) or Pd(111)58Ks2 = 2.20 mJ/m2 and Ks4 = 0.16 mJ/m2. Considering that nd-order anisotropy term, the 4th-order term (Ks4) is found to be positive, while it is usually negative in the case of bare Co film in contact with Ir(111), Au(111), Pd(111) or Pt(111)58Ks4 determines the upper thickness limit tc2 of the SRT, its positive value in graphene-covered Co is associated to this exceptionally large thickness range.Fitting our data with We note that in-plane and out-of-plane magnetic domains show distinct patterns: while in-plane domains are large with complex symmetry see , out-of-θ averaged over a set of SPLEEM images. In the following, we measure θ, pixel by pixel, to get a local estimate of the canting angle and to determine how it evolves within a given field of view. 6667In the previous section, we determined the canting angle A is the exchange stiffness constant. The limit case of uniform (non-twisted) magnetisation corresponds to ρ ≈ 0 or tan (ρ)/ρ ≈ 1. For the graphene/Co interface, ρ ≈ 0.28 giving tan ρ/ρ ≈ 1.03 M can be assumed to be uniform across the Co film.We could wonder whether the magnetisation is uniform within the film thickness or if it also curls in the direction perpendicular to the surface. In fact, spin-twisted configuration may arise in some systems in which the spins at the outermost atomic layer feel a strong anisotropy and remain aligned along the surface normal, while those in the middle of the film are tilted towards the film plane to minimise the magnetostatic energy6969in situ grown graphene/Co/Ir(111) heterostructures. Compared to bare Co/Ir(111) films, we found that the graphene capping layer drastically affects the spin texture and the spin-dependent band structure of the intercalated Co film. Similarly to what has been reported recently when PcCo molecules are deposited on a ferromagnetic surfaceUsing the capabilities of SPLEEM microscopy to probe the band structure of unoccupied electronic states and to resolve spin configurations in all space directions, we investigated the magnetic properties of How to cite this article: Vu, A. D. et al. Unconventional magnetisation texture in graphene/cobalt hybrids. Sci. Rep.6, 24783; doi: 10.1038/srep24783 (2016).


We therefore propose that surface detachment of membrane invaginations results from an ability of CADs to interfere with PIP2 interactions with cytoskeletal or BAR domain proteins. This establishes a detubulating action of a large class of pharmaceutical compounds.Several cell types develop extensive plasma membrane invaginations to serve a specific physiological function. For example, the megakaryocyte demarcation membrane system (DMS) provides a membrane reserve for platelet production and muscle transverse (T) tubules facilitate excitation:contraction coupling. Using impermeant fluorescent indicators, capacitance measurements and electron microscopy, we show that multiple cationic amphiphilic drugs (CADs) cause complete separation of the DMS from the surface membrane in rat megakaryocytes. This includes the calmodulin inhibitor W-7, the phospholipase-C inhibitor U73122, and anti-psychotic phenothiazines. CADs also caused loss of T tubules in rat cardiac ventricular myocytes and the open canalicular system of human platelets. Anionic amphiphiles, U73343  and a range of kinase inhibitors were without effect on the DMS. CADs are known to accumulate in the inner leaflet of the cell membrane where they bind to anionic lipids, especially PIP It is not surprising therefore that altered T tubule structure is emerging as a likely contributory factor to heart failure in several disease models1011Adaptation of cellular membrane morphology allows specialized cells to perform dedicated functions. For example, photoreceptors develop surface membrane invaginations or internal membrane disks to increase the surface area for expression of opsins and other proteins, thereby increasing sensitivity to light1245612 circulating platelets, which have a limited lifespan of 5–9 days192122Another important surface invagination system is the demarcation membrane system (DMS) of megakaryocytes, large polyloid marrow cells responsible for producing blood platelets13141516Despite their importance, the control and stability of plasma membrane invagination systems is poorly understood. Previous measurements of the megakaryocyte DMS have mainly relied on EM studies which are time-consuming, difficult to quantify and require measurements in fixed tissues192+ signalling, most of which had no effect on the percentage dye entering the megakaryocyte DMS  within the surface-connected tubules of this membrane invagination system see . When ex15cyte DMS . Howevercyte DMS . This efcyte DMS . W-7 waste DMS 26. Moreovete DMS 26. Taken t2 after 150 μM W-7 , likely reflects a small amount of surface membrane blebbing or micovilli14In untreated megakaryocytes, the impermeant, reversible membrane indicator FM 1–43 stained the DMS extensively through the extranuclear volume, as shown previously for the irreversible styryl lipophilic dye di-8-ANEPPS252+ (ionomycin or BAPTA)  . TogetheH-pyrrole-2,5-dione) (33β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione) (2) turnoverAnother inhibitor within our screen that prevented extracellular HPTS access to the DMS tubules is the phospholipase-C (PLC) inhibitor, U73122 (1-amino]hexyl]-15-dione) . A prope5-dione) . In contdione) 33. FurtherWe were also interested in whether reagents that collapse the DMS exert similar effects on specialised membrane invagination systems in other cell types. Although cardiac myocyte T tubules have a similar or slightly larger diameter than the DMS815212 is a major target for CAD binding in the membrane392, and PIP3, however these are less abundant compared to PIP2 under resting conditions2 plays widespread roles in recruiting proteins to the membrane via electrostatic docking interactions and pleckstrin homology (PH) domains, including scaffolding molecules that link the plasma membrane to the cytoskeleton402 through polycationic regions and have diverse roles in cellular architecture and function through their ability to curve membranes40422 in DMS formation2 is the submembranous cytoskeletal protein spectrin, which can bind through its PH domain and is also important for DMS formation2 which interferes with the ability of this phosphoinositide to interact directly or indirectly with the cytoskeleton in manner that is regulated by BAR domain proteins. This can explain the W-7-induced movement of the peripheral plasma membrane away from the underlying DMS, along with DMS collapse P2. Future studies using megakaryocytes derived in vitro will also need to ensure that the properties of the DMS within cultured cells match those of the native tissue. In our initial studies of standard megakaryocyte culture systems, membrane capacitance measurements indicated considerably lower levels of DMS in cultured megakaryocytes compared to similar size or even smaller cells obtained directly from the marrow .Cationic amphiphilic drugs (CADs) represent one of the largest classes of pharmaceutical compounds, which are used as anti-depressants, anti-malarial, anti-bacterial, anti-arrhythmic and cholesterol-lowering reagentscollapse . Collapss U73122 . Further2 in DMS formation, the importance of this lipid in T tubule formation is unclear. However, in myotonic dystrophy, altered skeletal muscle T tubule formation results from expression of a mispliced variant of Bin1 that loses its ability to bind to phosphoinositides2 shows a periodicity that coincides with T tubule location2 depletion leads to loss of T tubules2 contributes to the function of this scaffolding protein in the heart, including tubule morphology and optimal positioning of voltage-gated Ca2+ channels near ryanodine receptors4552In contrast to the direct suggestion of a role for PIP2 by such drugs may also interfere with other cellular roles dependent upon this phosphoinositide such as endocytosis, exocytosis and regulation of ion channels and transporters406364662122Phenothiazines such as trifluoperazine and chlorpromazine are used as anti-psychotics on the basis of their action at D2 receptors303155565761In conclusion, this study provides evidence for a key role of negatively charged lipids in the stability of specialised membrane invagination systems such as muscle T tubules and megakaryocyte demarcation membranes. The ability of cationic amphiphiles to detach T tubules and the DMS from the cell surface provides a useful tool for the study of these surface invagination systems, however it should also be considered in the design of CADs as therapeutic treatments.U73343 and U73122 were from Tocris Bioscience . Staurosporine was from Enzo Life Sciences . KT 5720 was from Santa Cruz Biotechnology . Jasplakinolide and LIM kinase inhibitor 1 LIMKi3 were from Calbiochem . FM 1-43 was from Invitrogen . Glutaraldehyde, 25% EM Grade was from Agar Scientific . All other reagents, including HPTS were from Sigma-Aldrich .egakaryocytes and ventricular myocytes were isolated from adult male Wistar rats (300-400 g) following euthanasia in accordance with the UK Animals Scientific Procedures Act. Cells were used within 12 hours of dispersal from the native tissue. Femoral and tibial marrow cells were extracted as described in detail elsewhere2, 1 mM CaCl2, 10 mM HEPES, 10 mM glucose, pH 7.35 with NaOH. Ventricular myocytes were prepared using retrograde perfusion via a Langendorff cannula as described in detail elsewhere2+-free Tyrode’s solution  and rapidly cannulated via the aorta. After perfusion for 6 minutes with 0CaTS, the perfusate was switched to 0CaTS with an enzyme mix  and then exchanged for 2 mM Ca2+ Tyrode’s solution (2CaTS) on identification of rod-shaped cardiomyocytes. The heart was then cut down, and cardiomyocytes isolated from the tissue in a shaking water bath. Cells were resuspended in fresh 2CaTS, filtered to remove undigested tissue and resuspended in fresh 2CaTS. For confocal recordings, myocytes were immersed into NPS with 1 mM EGTA that had an estimated free Ca2+ of 50 nM  to avoid spontaneous contractions, prior to addition of FM 1–43 (8 μM). Washed human platelets were prepared using a previously established method that minimizes spontaneous activationg for 5 minutes to obtain platelet-rich plasma (PRP) which was treated with apyrase (0.32 U/ml) and aspirin (100 μM) to reduce spontaneous activation by released nucleotides and thromboxaneA2, respectively. Washed platelet suspensions were prepared by centrifugation of PRP at 350 g for 20 minutes and resuspension in nominally Ca2+-free NPS containing 0.32 U/ml apyrase but without aspirin. Human erythroleukaemia (HEL) cells were sourced and cultured as described elsewhereMhttp://imagej.nih.gov/ij/, 1997–2012). In order to avoid uptake of fluorescent indicators by endocytosis, all experiments were conducted at room temperature and measurements made within 5 min of exposure to the fluorophore. For wash experiments, the reagents were removed by multiple exchange of the extracellular medium and indicators added after 2 or 120 minutes. HPTS was excited at 488 nm and emission collected at 500–600 nm. FM 1–43 (8 μM) was excited at 488 nm and emission collected at 550–650 nm.Confocal fluorescence imaging was conducted on an Olympus IX81 inverted microscope equipped with a FluoView1000 laser scanning module . All experiments used a 60x oil immersion lens  and a confocal slice thickness of 1.25 μm. Confocal images were visualized and analysed using ImageJ software .We used standard confocal fluorescence imaging to measure entry of an extracellular impermeant fluorescence indicator  into tubules of the DMS as a means of quanitifying this plasma membrane invagination system. This approach represents an extension of previous work with 2-photon fluorescence microscopy2, 1 mM MgCl2, pH 7.35 with HCl), washed 3 times and postfixed for 90 minutes in 1% aqueous osmium tetroxide with 1.5% potassium ferricyanide. After further washing, the samples were stained for 60 minutes in 1% aqueous uranyl acetate, washed, pelleted, and embedded in 3% agar gel. Once set, the agar pellets were cut into 1 mm cubes, serially dehydrated by ethanol, and then transferred through a series of pure ethanol mixed with an increasing concentration of Spurr’s modified resin . After several exchanges in pure resin, the samples were polymerised for 16 hours at 60 °C. Sections of approximately 90 nm thickness were cut using a Reichert Ultracut E ultramicrotome , collected onto copper mesh grids and counterstained for 2 min in Reynold’s Lead citrate. Sections were viewed on a JEM-1400 TEM (JEOL UK) with an accelerating voltage of 80 kV and images captured using a Megaview III digital camera with iTEM software . The OCS was quantified as the percentage cytoplasmic area occupied by all vacuolar regions within an individual platelet.After treatment with reagent or vehicle control, suspensions of marrow cells or human platelets were fixed for 60 minutes in 1.25% glutaraldehyde in 0.1 M sodium cacodylate  under the control of Clampex v6.0 electrophysiological software (Molecular Devices). Currents were low-pass filtered (5 kHz) and acquired at a rate of 50 kHz. Capacitative current transients were recorded in response to 60 ms duration −10 mV voltage steps from a holding potential of −80 mV and membrane capacitance calculated from the integral of the current as described in detail elsewhereP > 0.05; ns), or significant at P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) and P < 0.0001 (****).Data were presented as the mean ± SEM and significance was determined using one way ANOVA with Bonferroni post-testing, with the exception of changes in platelet OCS area which was assessed using Student’s unpaired T test with Welch’s correction (GraphPad Prism 6). The level of significance is indicated as not significant .


Graphene and its bilayer structure are the two-dimensional crystalline form of carbon, whose extraordinary electron mobility and other unique features hold great promise for nanoscale electronics and photonics. Their realistic applications in emerging nanoelectronics usually call for thermal transport manipulation in a controllable and precise manner. In this paper we systematically studied the effect of interlayer covalent bonding, in particular different interlay bonding arrangement, on the thermal conductivity of bilayer graphene using equilibrium molecular dynamics simulations. It is revealed that, the thermal conductivity of randomly bonded bilayer graphene decreases monotonically with the increase of interlayer bonding density, however, for the regularly bonded bilayer graphene structure the thermal conductivity possesses unexpectedly non-monotonic dependence on the interlayer bonding density. The results suggest that the thermal conductivity of bilayer graphene depends not only on the interlayer bonding density, but also on the detailed topological configuration of the interlayer bonding. The underlying mechanism for this abnormal phenomenon is identified by means of phonon spectral energy density, participation ratio and mode weight factor analysis. The large tunability of thermal conductivity of bilayer graphene through rational interlayer bonding arrangement paves the way to achieve other desired properties for potential nanoelectronics applications involving graphene layers. Graphene, the first two-dimensional atomic crystal available to us, has attracted great attentions due to its supreme mechanical4ab initio total energy calculations have identified that the interlayer sp3 bonds in graphite can be formed by femtosecond-laser excitation3 bonds can lead to monotonic reduction of the thermal conductivity of bilayer graphene as a function of interlayer sp3 bond concentration. However, in fact, interlayer bonding  simulations, we investigated the effect of interlayer sp3 bonded bilayer graphene structures, we first investigated the size effect on the thermal conductivity of regularly bonded bilayer graphene with interlayer sp3 bonding density of 6.25%, as shown in 3 bonded bilayer graphene structures we studied. Note that the thermal conductivities are different for armchair and zigzag directions, which should be due to the difference of the detailed topological arrangement of the interfacial bonding in armchair and zigzag directions. The thermal conductivity of the regularly bonded bilayer graphene with interlayer sp3 bonding density of 6.25% converges for simulation box size of ~5 nm and 10 nm for armchair and zigzag direction, respectively. This is comparable with the convergence size of 10 nm for single layer graphene from our separate simulation run. Note that the results shown in To get a converged size for calculating the thermal conductivity of the interlayer sp3 bonding, the thermal conductivity of randomly bonded bilayer graphene decreases monotonically. However, the thermal conductivity of regularly bonded bilayer graphene changes non-monotonically. Especially, there are two obvious peaks at the interlayer bonding densities of 6.25% and 12.5%. The results suggest that the thermal conductivity of sp3 bonded bilayer graphene depends on not only the bonding density, but also the detailed topological arrangement of the interfacial bonding. The thermal conductivity of sp3 bonded bilayer graphene falls in a broad range between several tens and a few hundred W/mK, indicating that the interlayer bonding is an effective way to manipulate the thermal transport properties of bilayer graphene, which could be very beneficial for varieties of potential applications.The thermal conductivity of bilayer graphene as a function of interlayer bonding density is presented in Researchers have shown that covalent bonds will influence the thermal conductivity disregard whether there is a second layerIn order to explore the mechanisms for the non-monotonic thermal conductivity results of the regularly bonded bilayer graphene and the generally higher thermal conductivity as compared with the randomly bonded structure, we investigated the phonon mode dependent thermal conductivity of bilayer graphene by performing spectral energy density (SED) analysis. First, the phonon normal modes are obtained by16where the eigenvector and the phonon lifetime is obtained by fitting the Lorentzian function2021ν is the phonon branch, κα denotes the thermal conductivity in α direction, cph is phonon volumetric specific heat calculated by kB is the Boltzmann constant, V is the system volume, υg,α is α-component of the phonon group velocity, τ is the phonon lifetime. Note that the above phonon SED method has been successfully used by us to perform the phonon mode analysis for a wide variety of nanostructures, e.g., two-dimensional silicenewhere The frequency dependent phonon lifetimes of bilayer graphene (interlayer bonding density of 6.25%) with randomly and regularly bonded structures are compared in 3 bonded bilayer graphene (not shown for brevity) show the obviously different feature between the randomly and regularly bonded bilayer graphene. To identify the phonon frequency dependent relative contributions of the overall thermal conductivity, we also calculated the accumulated thermal conductivity percentage as a function of frequency for both the randomly and regularly bonded bilayer graphene, as shown in 2627283 bonds would lead to dramatic reduction in phonon MFP. Combining Using the above definition for thermal conductivity, we obtained four results of the thermal conductivity of bilayer graphene with interlayer bonding density of 6.25%: We show in Furthermore, the comparison of the frequency dependent phonon lifetime and square of phonon group velocity of regularly bonded bilayer graphene between interlayer bonding density of 6.25% and 15% is shown in pλ, defined for each mode λ as3031323334Furthermore, we analyzed the phonon mode localization by calculating the participation ratio i sums over all the atoms studied, α is a Cartesian direction and sums over x, y, and z, εiα,λ is the vibrational eigenvector component corresponding to the λth normal mode, and N is the total number of atoms. The participation ratio describes the fraction of atoms participating in a particular phonon mode and, hence, it varies between O(1) for delocalized states to O(1/N) for localized states. 3 bonded bilayer graphene between random and regular bonding configuration with the same interlayer bonding density of 6.25%. It can be seen that when the phonon frequency is lower than 2.5 THz, the participation ratio for the randomly and regularly bonded bilayer graphene are nearly the same, while for the higher frequency, the regularly bonded bilayer graphene has a significantly larger participation ratio than the randomly bonded structure throughout the whole frequency range, suggesting that the high frequency phonon modes dominate the enhancement of the thermal conductivity of regularly bonded bilayer graphene compared to the randomly bonded bilayer graphene. This is consistent with results of 3233where j (atoms) and is alternatively restricted to the sp2 bonded C atoms and the sp3 bonded C atoms. Consequently the sum of the mode weight factors in the whole bilayer graphene structure should be equal to unity, i.e.,2 bonded C atoms and vice versa. 3 bonded bilayer graphene between random and regular bonding configuration with the same interlayer bonding density of 6.25%. Unlike the phonon participation ratio, there is no obvious difference for the mode weight factors of sp2 and sp3 bonded C atoms, suggesting that the phonon mode weight factor may only depend on the interlayer bonding density, not on the detailed interfacial topological arrangements.where the prime denotes that sum over 3 bonded C atoms is larger than that of 6.25% interlayer bonding density. The above mode weight factor results combining with that of To analyze the mechanisms for the non-monotonic interlayer bonding density dependent thermal conductivity of the regularly bonded bilayer graphene, we further calculated the phonon participation ratio and mode weight factor for the regularly bonded bilayer graphene with different bonding density. The comparison of the phonon participation ratio between bonding density of 5%, 6.25%, 8.75%, 12.5% and 15% is shown in 3 bonding density on the thermal conductivity of bilayer graphene, especially the results of the randomly and regularly bonded bilayer graphene structures were compared in detail. The thermal conductivity of randomly bonded bilayer graphene decreases monotonically with the increase of interlayer bonding density, which follows the same trend as that obtained in literature. However, for the regularly bonded bilayer graphene structure, we observed the unexpectedly non-monotonic interlayer bonding density dependence of thermal conductivity. The phonon spectral energy density, participation ratio and mode weight factor analysis were performed to explore the underlying mechanism for this counter-intuitive phenomenon. We found that the lifetimes of low-frequency (<5 THz) phonons for randomly and regularly bonded bilayer graphene are nearly the same, which is consistent with the general knowledge that the low-frequency phonons are not sensitive to the detailed interfacial bonding configuration, considering that the characteristic size of the covalent bonding is on the order of only a few angstroms, which is much shorter than the typical wavelength of the low frequency phonons. In contrast, for medium-frequency (5–30 THz) and high-frequency (30–50 THz) phonons the lifetimes in the regularly bonded bilayer graphene are greatly larger than that of randomly bonded structure, especially for the medium-frequency phonon modes which have a rather large contribution to the higher thermal conductivity of the regularly bonded bilayer graphene than the randomly bonded structure. By evaluating the relative importance of the contributions of phonon lifetime and square of phonon group velocity, we conclude that, compared with random bonding, the much higher thermal conductivity of the regularly bonded bilayer graphene is mainly due to the large enhancement in the group velocity of the phonon modes in the medium-frequency range (5–30 THz). In addition, we observe that the thermal conductivity of bilayer graphene depends on not only the interlayer bonding density, but also the detailed topological arrangement of the interlayer sp3 bonds. We find that, comparing the regularly bonded bilayer graphene between different bonding densities, the change of phonon lifetime, instead of the change of group velocity, is the main reason for the non-monotonic thermal conductivity dependence. We also characterize the phonon mode localization by calculating the participation ratio and provide evidence that the participation ratio at different bonding density resembles the non-monotonic dependent thermal conductivity. Again this can be correlated with their atomic structure, where the regular bonding density of 6.25% and 12.5% has more homogeneous sp3 bonding than that of other bonding densities, which should be responsible for their different phonon band structure. We believe that our analysis provides a comprehensive understanding of the underlying mechanism and the abnormal phenomenon identified in this study can also be extended to other similar bilayer and multilayer structures, that is, the thermal transport properties of bilayer and multilayer structures can be effectively modulated by tuning the detailed arrangement of the interlayer bonding. Other physical properties like electronic and optical properties may also be tuned in the same way and thus such interfacial bonding engineering could offer a robust approach to target desired properties for potential electronic, thermoelectric, photovoltaic, and opto-electronic applications.To summarize, equilibrium molecular dynamics simulations were performed to investigate the effect of interlayer spn × n supercells with each unit cell constructed by a bilayer honeycomb lattice and composed of 320 carbon atoms, where n is ranging from 1 to 6 in our simulations. The structures of a unit cell of bilayer graphene with different interlayer sp3 bonding are shown in 3 bonding. To form the sp3 bonded bilayer graphene structures shown in NPT (constant pressure and temperature) ensemble for 100000 steps. Thirdly, the fix on the bonded atoms is removed and the entire system is relaxed with NPT ensemble for additional 1000000 steps. Finally, the stable bilayer graphene with inter-layer bonding is achieved. For each model system we have carefully checked the topography and bonding density of the interlayer bonding before and after structure relaxation, and also before and after the later on EMD simulation of calculating thermal conductivity. For all cases we confirm that there is no change for the topological configurations of the interlayer bonds and the interlayer bonding density due to temperature fluctuations. To investigate the effect of the topological configuration of the interlayer bonding on the thermal transport in bilayer graphene, here we constructed both the randomly and regularly distributed bonding structures. In our simulations, we only considered the interlayer bonding density lower than 15% but the thermal conductivity of nanostructures is very sensitive to the component of the structuresOur model system of bilayer graphene sheets consist of x and y directions (in-plane), and a free boundary condition is used in z direction (out-of-plane). In the first stage of the MD simulations, we relaxed the system at constant atmospheric pressure and room temperature (300 K) for 0.1 ns, using the Nosé-Hoover thermostat and barostat45NPT relaxation, we continued to relax the system with NVT (constant volume and temperature) ensemble for 0.1 ns using the Nosé−Hoover thermostat and NVE (constant volume without thermostat) ensemble for additional 0.1 ns.The adaptive intermolecular reactive empirical bond order (AIREBO) potentialFollowing equilibration, the thermal conductivity of bilayer graphene was estimated by the Green-Kubo method47V is volume, kB is the Boltzmann constant, T is temperature, where Ei is the total energy  of atom i, i, i defined as44where withα and β denote the x, y or z direction. The three terms in the definition of Sαβ define the contribution from two-body, three-body and four-body interaction, respectively. The thickness of bilayer graphene was chosen as 0.68 nm in calculating the system volume, which is equal to two times of the van der Waals diameter of the C atomwhere We used a unit cell of bilayer graphene structure with 2.13 nm × 1.97 nm (320 atoms) as the input for the phonon normal mode calculation. And limited by the large unit cell, we only used a 4 × 4 supercell (5120 atoms) for the phonon spectral energy density analysis. To achieve enough frequency range for bilayer graphene, we sampled the atomic velocities with a time interval of 9 fs, and the max phonon frequency we can achieve is about 55.56 THz which is a little larger than the max frequency of bilayer graphene. It is worth pointing out that, strictly speaking, there is no periodicity for the randomly distributed bonding structure and the wavevector-related phonon information cannot be obtained for the phonon spectral energy density calculations, here we just distributed the atoms irregularly in the unit cell to achieve the randomly bonded bilayer graphene structure.How to cite this article: Zhang, X. et al. Robustly Engineering Thermal Conductivity of Bilayer Graphene by Interlayer Bonding. Sci. Rep.6, 22011; doi: 10.1038/srep22011 (2016).


In this paper, COOH+/graphene was prepared by modified Hummers' method and implanted by COOH+ ions. The structure and surface chemical and physical properties of COOH+/graphene were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and contact angle measurement. Systematic evaluation of anticoagulation, including in vitro platelet adhesion assays and hemolytic assays, proved that COOH+/graphene has significant anticoagulation. In addition, at the dose of 5 × 1017 ions/cm2, COOH+/graphene responded best on platelet adhesion, aggregation, and platelet activation.Graphene may have attractive properties for some biomedical applications, but its potential adverse biological effects, in particular, possible modulation when it comes in contact with blood, require further investigation. Little is known about the influence of exposure to COOH Graphene, the thinnest two-dimensional 2D) allotrope of carbon, is a novel nanomaterial with a single sheet of carbon atoms packed in a perfect honeycomb structure ,2. It isD allotro+ ions were implanted into graphene, instead of MWCNTs, to achieve a better anticoagulant material. Except toxicological and ecological risks, anticoagulation is a prerequisite for the use of graphene in biological or medical applications. It is necessary to evaluate the anticoagulant risks of graphene [Enhancement of the surface properties of medical implants for improved integration into their biological environment is a major focus of current biomaterial research . Ion impgraphene . Rabbit + implantation-MWCNTs when compared with pristine MWCNTs [+ implantation introduced functional carboxyl groups into the surfaces of materials. The existence of hydroxy and other oxygen-containing functions was thought to be able to induce the hydrophilicity of MWCNTs [+ implantation could also enable it to exhibit attractive anticoagulation [+/graphene shows better anticoagulation than pristine graphene for platelets and red blood cells. In addition, the content of carboxyl group in COOH+/graphene affected its anticoagulation. Compared with our earlier works, the ion implantation in this study is in great dose and high purity. The findings presented in this study also throw light into that COOH+/graphene may serve as an excellent candidate material for potential biomedical applications in various areas.Some previous investigation demonstrated that the anticoagulation of MWCNTs was greatly influenced by surface functional groups. In our previous work, we focused on cell and blood compatibility of MWCNTs by different functional groups -20. The e MWCNTs ,16. The f MWCNTs ,21. To ggulation . In the +/graphene were as follows: in the first step, graphene was ultrasonically dispersed in an organic solvent called 1-methyl-2-pyrrolidinone (NMP) for several hours to make it dissolved evenly. The concentration of graphene was 0.5 mg/cm2. Then, the graphene solution was sprayed by airbrush with Ar flow on the heated (100°C) SiO2 wafers which were used as support materials for graphene. Next, baking was done for 3 h (250°C) under argon protection to remove the organic solvent completely [+ ions were implanted on graphene using a Kaufman-100 implanter . The COOH+ was generated from methanoic acid, which would be ionized by heating in water bath and bombardment of accelerated electrons in ion source [2 [−2 Pa. The different doses of COOH+/graphene  were obtained by adjusting the irradiation time.The graphene powders were produced according to modified Hummers' method -26. Detampletely ,27. Finan source ,11,13. Tn source . The beaource [2 ,29, and +/graphene was analyzed with sessile drop method. Deionized water was dropped onto the sample surface, and images were recorded using the CAM KSV021733 optical contact-angle inclinometer . The process lasted for ten times, and the results came from the average of these data. Scanning electron microscope (SEM) imaging was performed using a field-emission scanning electron microscope . X-ray photoelectron spectroscopic (XPS) measurements were performed on a Kratos Axis Ultra Al  X-ray photoelectron spectrometer .The surface hydrophilicity of graphene and COOHPlatelet-rich plasma (PRP) was prepared by centrifuging the fresh blood with anticoagulant at 1,200 rpm for about 15 min. The fresh blood was withdrawn from a healthy rat. Additionally, PRP was put immediately into 24-well culture plate which all the samples were placed in them. The 24-well culture plates were placed immediately in the incubator at 37°C for 30 min. The samples were then fixed in glutaraldehyde, dried with critical point drying, and plated for examination of SEM.+/graphene in vitro, we measured the absorption of free hemoglobin (Hb) at 545 nm in the plasma. The samples were immersed in the physiological salt water and placed in the water bath at 37°C for 30 min. The diluted blood  was successively added into the solution of all groups. After incubation at 37°C for 60 min, the blended liquid was centrifuged at 1,000 rpm for 5 min. The supernatants were transferred into the cuvettes, and the optical density (OD) values were measured at 545 nm with a spectrophotometer. Hemolysis ratio was calculated by the formula in Figure A, B, and C are the average absorbance of samples, negative controls, and positive controls, respectively [In order to investigate the hemolysis of pristine graphene and COOH+/graphene. The transparent nature of the folded crystalline layers is clearly shown in this figure. Graphene and COOH+/graphene exhibit typical wrinkled structure, indicating the high surface/volume ratio and two-dimensional structure of graphene. And COOH+/graphene show more wrinkles with the COOH+ dose increasing. The most remarkable effect of carboxyl ion implantation also can be seen in the contact angle measurements as shown in Figure 17 ions/cm2 leaves a contact angle of 20° ± 2°. COOH+/graphene with 5 × 1017 ions/cm2 leaves a contact angle of 4° ± 2°. COOH+/graphene with 1 × 1018 ions/cm2 leaves a contact angle of 16° ± 2°. But the droplet on pristine graphene's surface remains highly spherical with the angle of 70° ± 2°, which implies that the pristine graphene is more hydrophobic than COOH+/graphene. As we know, graphene as well as other carbon nanostructures are naturally hydrophobic, so surface wettability is highly improved by COOH+ ion implantation. And the materials' surfaces are rich in oxygen after carboxyl ion implantation. Surface hydrophilia may have an effect on anticoagulation property because the initial phase of connection affects the contacts between blood and materials through various forces and adsorbing sundry proteins [+/graphene is an oxygen-containing graphene derivative with partial breakage of sp2-sp2 bonds into sp3-sp3 bonds for inserting some pendent groups . These functional groups facilitate the interaction between the host solution and implanted samples. It gives us much inspiration to realize that COOH+ ion implantation may bring great advantages for biomaterials to improve their anticoagulation properties [Figure proteins . COOH+/goperties .Figure 2+/graphene are presented in Figure +/graphene are different as displayed in the figure. COOH+/graphene shows distinguished COOH+ signal. The oxygen-related functional groups such as hydroxyl (C-O), carbonyl (C = O), and hydroxy (C-OH) were present in COOH+/graphene, as O content increases from 6.97 atm.% for pristine graphene  to 10.In vitro platelet adhesion testing is carried out to study the quantity, morphology, aggregation, and pseudopodium of the adherent platelets [+/graphene are shown in Figure +/graphene with 1 × 1017 ions/cm2 > COOH+/graphene with 1 × 1018 ions/cm2 > COOH+/graphene with 5 × 1017 ions/cm2. It is clear that the number of platelets on the COOH+/graphene is lower than that on the pristine graphene, and the number of adhered platelets decreases by COOH+ ion implantation in graphene, which performs preferable anti-adhesive capacity for COOH+/graphene. The value of COOH+/graphene with 5 × 1017 ions/cm2 is even lower than that of COOH+/graphene with 1 × 1018 ions/cm2. Comprehensively considering the results of both platelet adhesion and water contact angle assays, the platelet numbers adhering to the surface reduce obviously as surface wettability increases. The enhanced wettability of both functionalized graphene results in the reduction of platelet adhesion, which may lead to the improvement in anticoagulation. Figure +/graphene, as shown in Figure +/graphene has much better anticoagulation than pristine graphene. Nevertheless, COOH+/graphene show slightly thrombogenicity at a fluence of 1 × 1018 ions/cm2. Thrombogenicity occurring on the material surface in contact with blood is a complicated process. The surface free energy has an important effect on the process, particularly for some hydrophilic polymers that show less platelet activation. Compared with pristine graphene, COOH+/graphene with 1 × 1017 ions/cm2, 1 × 1018 ions/cm2, and 5 × 1017 ions/cm2 shows the lowest platelet aggregation and activation, although some platelets exhibit the slight activation  cann than 5% . The des than 5% . Slight  than 5% . And aft+ ion implantation while microstructural aspects, microstructure, and valence bond were systematically investigated. The interaction of red blood cells and platelets with pristine graphene and COOH+/graphene were investigated and compared detailedly. Three kinds of COOH+/graphene were shown to exhibit lower platelet adhesion, aggregation, and platelet activation than pristine graphene under the same conditions, especially for the COOH+/graphene with 5 × 1017 ions/cm2. No significant toxicity effects could be found on COOH+/graphene. The COOH+/graphene showed an observable improvement in anticoagulation as the COOH+ ion was implanted. This study demonstrated superior anticoagulation of COOH+/graphene with 5 × 1017 ions/cm2 as promising and effective biomedical material for biomaterial industry.We have shown here that functionalized graphene was successfully acquired by COOH


Moreover, the sensor is capable of detecting MB over broad concentration ranges from 1 nM to 100 μM. Therefore, AgNPs/GF is a highly promising SERS substrate for detection of chemical substances with ultra-low concentrations.In this work, a novel platform for surface-enhanced Raman spectroscopy (SERS)-based chemical sensors utilizing three-dimensional microporous graphene foam (GF) decorated with silver nanoparticles (AgNPs) is developed and applied for methylene blue (MB) detection. The results demonstrate that silver nanoparticles significantly enhance cascaded amplification of SERS effect on multilayer graphene foam (GF). The enhancement factor of AgNPs/GF sensor is found to be four orders of magnitude larger than that of AgNPs/Si substrate. In addition, the sensitivity of the sensor could be tuned by controlling the size of silver nanoparticles. The highest SERS enhancement factor of ∼5 × 10 The LSP wavelength compression ratio (λ/λsp whereλsp = LSP wavelength) due to 2D confinement of LSP on graphene surface could be in the range of 40 ~ 70The rise of graphene has opened up many promising applications in various fields due to intriguing relativistic properties of graphene such as massless fermion dispersion, high electron mobility, high conductivity, etc. Recently, there are numerous articles claiming that SERS on graphene could be greatly enhanced in comparison with other substrates. Wang 2 detection at very low concentration67Graphene is also a promising material for chemical sensing due to its excellent physical, chemical and electrical properties including large effective surface area, high electron transfer capability and good chemical stability. For example, electrochemical sensors based on microporous three-dimensional 3D) graphene foam (GF) has demonstrated excellent chemical sensing performances with high sensitivity, low detection limit and good stabilityD graphenAccording to Kneipp9ε0 and ε are vacuum and metallic-nanomaterial dielectric constants, respectively, d is the distance from analyte to silver nanoparticle surface, r is the radius of the metallic nanoparticle, E0 are outgoing (enhanced) and incoming electric field intensity, respectively.where PG, the electric field on graphene (EG) will be given byThe EM enhancement will be analyzed using the LSP model on graphene surface where plasmonically metallic sphere is placed onto the graphene surface as depicted in ω = induced dipole frequency and c = light velocity. Assuming that the graphene response latency is negligible, εG = the dielectric constant of graphene. Putting PG into (2), it can be shown thatwhere where λ0 = the wavelength of incident light, εr = the dielectric constant of medium, α = fine constant and EF = Fermi energy of graphene. The enhancement factor (EF) depends strongly on the dimensionless ratio, whereTherefore, the enhancement factor of ∼100 could be achieved on flat graphene.There are two most common types of chemical sensors: electronic and optical. Optical chemical sensors have recently earned much attention due to short detection time, high selectivity, wide dynamic range and non-contact measurement capability. Among various optical sensing platforms, SERS is becoming increasingly popular due to its extremely-high sensitivity with the detection limit as low as a single molecule and high specificity at ultra-low analyte concentrations. Recently, many chemical sensors using a flat graphene sheet as a SERS substrate have been reported16H18N3SCl) detection. In addition, SERS performances are optimized by varying the Ag nanoparticle size via the control of sputtering time.Nevertheless, the performances of flat graphene platform should be further improved by employing 3D graphene structure2) that appears like a thin black sponge. A portion of graphene foam examined by field-emission scanning electron microscopy (FE-SEM) is illustrated in To obtain the most effective surface enhanced Raman scattering, the SERS substrate should be excited at its LSPR peak. The typical LSPR peak of AgNPs-GF measured by UV-Vis absorption spectroscopy is demonstrated in −1 are enhanced substantially as the sputtering time increases from 20 to 30 seconds. In contrast, MB peaks, background signal and noise peaks are greatly diminished when the sputtering time increases further to 40 s. Thus, the optimal sputtering time of AgNPs on GF for the highest MB signal is 30 s. However, the magnitudes of background signal and noise peaks from contaminations also increase considerably compared with the signal peaks. The impurity peaks are also amplified due possibly to the multiple-cascaded amplification process in graphene foam structure and very broad LSPR peak of AgNPs/GF hybrid. Thus, there will be a trade-off between sensitivity and specificity of the SERS sensor based on the AgNPs/GF structure. A practical solution to this problem is to apply a suitable noise filtering system, which can effectively eliminate the majority of noise peaks.−1 can be clearly observed in the SERS spectra for 100 μM and 1 μM MB concentrations while SERS spectra for 1 nM and 1 pM MB concentrations shows only four  and one (446 cm−1) MB Raman peaks, respectively. In addition, SERS intensity at a given MB peak increases monotonically as the MB concentration increases from 1 pM to 100 μM. The detection limit for MB is taken to be 1 nm as it is the lowest concentration that four MB peaks can still be observed. The results demonstrate the possibility of using GF platform for detection of trace-level molecules or low concentration of biochemical analytes.Silver-decorated graphene foam with the average particle size of 50 nm (30 s sputtering time) was used as SERS substrate to detect MB with varying concentration from 1 pM to 100 μM as demonstrated by SERS spectra in 4.From overall results, graphene foam decorated with silver nanoparticles has shown high SERS enhancement factor and excellent MB detection sensitivity. In addition, the Ag sputtering time of 30 s, which produces the average Ag particle size of 50 nm, yields optimal sensitivity for MB detection with a low detection limit of 1 nM. With a short sputtering time (10 s), there is very low density of small Ag nanoparticles. The large distance between small AgNPs will exhibit low local field enhancement and hence low SERS sensitivity. The increase of sputtering time from 10 to 30 s will result in the larger particle size and higher particle density, leading to enhancement of local electric field and LSP. However, AgNPs will become too dense and approaches a thin-film structure as the sputtering time becomes longer than 30 s, resulting in lower surface enhancement capability. In addition, the large particle size will cause non-radiative multimode excitation of LSPIn the multiple cascaded amplification process as schematically depicted in According to multiple cascaded amplification scheme, the electromagnetic enhancement factor (EF) can be expressed asAssuming thatThus,Am(νl)2 = SERS enhancement factor of silver, typically 104–105, Am(ν) = SERS electromagnetic gain of a noble metal (m), ν = light frequency, νl and νs are incident and scattering light frequencies, respectively, and Ag(ν)2 = SERS enhancement factor of graphene4.where 0, which is given byIn the absence of graphene, EF is defined as EFDividing 4 which is in the same order of magnitude as the estimated value from the experiment of 5 × 104. However, the other amplification mechanism, the collective excitation model, will also be considered since the first scheme still exhibits considerable discrepancy.Therefore, the theoretical ratio of enhancement factor for AgNPs substrates with and without GF is 10The collective excitation model as depicted in DefineandIG) and graphene foam (IGF) can be computed asThus, the amplification gains of graphene  could be estimated as 1 mm/0.1 mm ~ 10 layers. Thus, the enhancement factor of AgNps/GF substrate based on collective excitation scheme could be ~102 or 100, which is much lower than the enhancement factor from the experimental data of ~5 × 104. Therefore, the multiple cascaded amplification process, which yields a high enhancement factor of 104, is the more likely mechanism for the explanation of large SERS enhancement of AgNPs/GF substrate.For the AgNPs/GF substrate in this work, GF thickness is 1 mm and inter-layer distance between graphene layers is roughly 0.1 mm. Therefore, the average number of graphene layers  and hydrogen (H2) was used as the carbon precursor and reducing gas carrier for graphene synthesis. C2H2/ H2 (3/24) mixture were flowed through Ni foam located at the center of a horizontal tube furnace under a vacuum pressure of 0.2 Torr at 700 °C for 3 minutes and then cool down rapidly at a rate of more than 10 °C per minutes under hydrogen flow at a pressure of 1 Torr. Carbon atoms from C2H2 were dissolved on Ni surface and then segregated as graphene layer on Ni scaffold during cooling process. The resulting grahene coated Ni foam was etched in 3 M HCl solution at 60 °C for 30 minutes in order to remove the Ni scaffold. The GF was then decorated with silver nanoparticles (AgNPs) using DC magnetron sputtering with varying sputtering time from 10 to 40 s. The sputtering was conducted in argon at a vacuum pressure of 3 × 10−3 mbar with a DC current of 0.2 A.Graphene was grown on commercial Ni foams, which were simultaneously utilized as catalyst and scaffold, by chemical vapor deposition (CVD). Acetylene . The surface morphology of Ag-decorated graphene foam was examined using field emission scanning electron microscopy . The optical absorption spectrum of AgNPs-GF was measured by UV-Vis absorption spectrometer .SERS measurement was conducted using a portable Raman spectrometer (Enwave Optronics Field-type EZRaman-M) with the laser wavelength of 785 nm (red) and reflection detection at zero degree. Before MB detection, Raman spectrum of SERS substrate was measured in order to determine the baseline signal. Afterwards, MB solution was dropped onto a SERS substrate and then exposed to the laser in order to take Raman spectrum. Firstly, SERS measurement was carried out at a fixed MB concentration of 1 μm on AgNPs/GF substrates prepared with different Ag sputtering times (10–40 s) in order to determine the optimal Ag deposition time. Next, the SERS measurements were repeatedly performed on each substrate with varying MB concentration from 1 pM to 100 μM to obtain calibration curve and sensitivity. Lastly, SERS experiments were done on different SERS substrates including AgNPs/GF, AgNPs/Si, Si, and graphene at a fixed MB concentration of 1 μM in order to compare the performances of AgNPs/GF substrates and determine the enhancement factor derived from GF structure. It should be noted that the graphene substrate used for the comparison was a planar CVD graphene layer grown on a copper foil.How to cite this article: Srichan, C. et al. Highly-Sensitive Surface-Enhanced Raman Spectroscopy (SERS)-based Chemical Sensor using 3D Graphene Foam Decorated with Silver Nanoparticles as SERS substrate. Sci. Rep.6, 23733; doi: 10.1038/srep23733 (2016).