In this work graphene-based aerogel anodes and graphene/stainless steel cathodes have been optimised as platinum-free electrodes in Rhodopseudomonas palustris microbial fuel cells, achieving a maximum power output of ∼3.5 W m–3. Rhodopseudomonas palustris CGA009. We achieve a volumetric peak power output (PV) of up to 3.51 ± 0.50 W m–3 using graphene based aerogel anodes with a surface area of 8.2 m2 g–1. We demonstrate that enhanced MFC output arises from the interplay of the improved surface area, enhanced conductivity, and catalytic surface groups of the graphene based electrode. In addition, we show a 500-fold increase in PV to 1.3 ± 0.23 W m–3 when using a graphene coated stainless steel (SS) air cathode, compared to an uncoated SS cathode, demonstrating the feasibility of a platinum-free, graphene catalysed MFCs. Finally, we show a direct application for microwatt-consuming electronics by connecting several of these coin sized devices in series to power a digital clock.Microbial fuel cells (MFCs) exploit the ability of microorganisms to generate electrical power during metabolism of substrates. However, the low efficiency of extracellular electron transfer from cells to the anode and the use of expensive rare metals as catalysts, such as platinum, limit their application and scalability. In this study we investigate the use of pristine graphene based electrodes at both the anode and the cathode of a MFC for efficient electrical energy production from the metabolically versatile bacterium  Global population expansion and economic development result in increasing demand for energy and clean water, leading to a pressing need for innovative renewable energy sources and more efficient and sustainable waste treatment technologies. Microbial fuel cell (MFC) technology may satisfy both requirements by tapping into the significant chemical energy in wastewater, exploiting the electrogenic nature of various microorganisms that oxidize organic substrates and donate electrons to an external electron acceptor. Although examples of carbon-based anodes and platinum (Pt) cathodes exist, there remains major scope for improving the performance of electrodes for MFCs. Enhanced understanding of the parameters determining electrode performance will help in the development of environmentally-friendly, abundant catalytic cathode materials, and highly electron-accepting anode materials. Here we identify key parameters of a range of MFC electrodes and characterise the performance of a set of novel, environmentally-friendly low-cost graphene-based anodes and cathodes.via direct extracellular electron transfer (DEET) from anodic biofilms2O formation and evaporation via ORR at the air exposed cathode.11Power output from a single chamber MFC is primarily limited by the efficiency of extracellular electron transfer (EET) from the cell to the anode,i.e. their resistance to corrosion in an aqueous environment), surface area (∼0.5 m2 g–1) and electrical conductivity.2 g–1 ,via an electron shuttling processes, and potentially providing an alternative and cheaper cathodic catalyst to commonly used Pt–1). MFC anodes using chemically modified GO have been shown to enhance power densities to 2.67 W m–2 (∼18-fold) for stainless steel mesh,–3 (∼3-fold) from carbon cloth,–3 (∼19-fold) for nickel oxide foams.–3 . Their low density and high surface area, together with high conductivity, establish aerogels based on GO as high performance MFC anodes.2 g–1) structures.e.g. methane) for CVD,2O.4 S m–1), and positively charged backbone that may interact electrostatically with negatively charged cells to facilitate cell–anode interactions and biofilm formation.44Cost-effective carbon-based materials such as carbon felt,Rhodopseudomonas palustris  CGA009  from MFCs using carbon foam anodes was doubled to 265 ± 12.1 mW m–3 by coating with a pristine graphene/PD (Gr–PD) based ink, and the enhanced surface from composite Gr–PD aerogel anodes increased PV 13-fold to 3.51 ± 0.50 W m–3, closer to our benchmark provided by carbon fibre as an anode material (5.37 ± 1.16 W m–3). As a practical application, we show that a circuit of 10 single chamber MFC devices operated with Gr–PD aerogel anodes with a total volume of 1.32 cm3 generated 4.19 μW of power, sufficient to run a digital clock. In addition, we show that a Gr–PD ink coating is able to impart catalytic activity onto a standard marine grade stainless steel mesh (SS), showing feasibility as a cost-effective Pt-free air cathode. Finally, we demonstrate a fully pristine graphene-enabled MFC by integrating our pristine graphene-based anode aerogel and air cathode in a single chamber MFC, paving the way to a cost-effective, environmentally friendly energy source.Pristine graphene can be produced sustainably by liquid phase exfoliation (LPE) or cracking of methane biogas (derived from food waste and other renewable sources) both of which we used to produce low-cost, scalable, and environmentally friendly electrodes for MFCs. In the anodic chamber we use the metabolically diverse, purple non-sulphur α-proteobacterium, ) CGA009 . R. paluWe prepared low density, highly porous aerogel anodes using a biocompatible and biodegradable non-conductive polymer, carboxymethylcellulose sodium salt (CMC) as a scaffold material.–1 (PD1) and ∼1508 cm–1 (PD2), are assigned to the asymmetric Cα = Cβ stretching and symmetric Cα = Cβ (–O) stretching vibrations respectively.2g phonon at the Brillouin zone centre in graphene, while the D peak  is due to the breathing modes of carbon sp2 atoms and requires a defect for its activation.–1 nm–1) for each of the aerogels with graphene flakes  indicates that the D peak originated from defects in the basal plane of the graphene in addition to defects along the flake edges.R. palustris cells were used to determine the charge transfer resistance (Rct) of each aerogel. Nyquist plots are generated by plotting the imaginary impedance Im(Z) versus the real impedance Re(Z) for each aerogel, and show a semicircle at high frequencies, where the system is under kinetic control , followed by a straight line at low frequencies, where the system is under a diffusive controlled regime .Rs) combines the ionic resistance of the electrolyte, the intrinsic substrate resistance and the contact resistance, and it is defined as the value where the semicircle intercepts the real impedance (Re(Z)) axis.Cd), and the charge transfer resistance (Rct). The diffusive regime is modelled by the Warburg resistance (ZW), which describes the frequency dependence of the ion transport to the electrode. In cases where the Rct is sufficiently high and the diffusive regime is not reached, ZW is set to zeroRct is ∼101 kΩ and ∼176 kΩ respectively, with the single semicircle indicating that the electrochemical reaction is kinetically controlled, hindering the process of electron transfer and indicating a resistive behaviour of the materials.Rct for A-CMC–Gr (∼46.2 Ω) (red curve) and A-CMC–Gr–PD (∼21.0 Ω) (blue curve) suggests that the addition of Gr flakes is primarily responsible for decrease in Rct, while PD likely helps to bridge between the conductive graphene flakes, improving Rct further. On the other hand, we noticed the absence of a diffusion element in the A-CMC–PD electrode, which indicates a slower ion transfer process. Therefore we can conclude that the addition of graphene flakes can improve electron transfer in the anode aerogels.The Nyquist plots of the electrochemical impedance spectra (EIS) before the addition of Sa) was 3.9 m2 g–1 for the A-CMC–PD aerogel and 7.1 m2 g–1 for A-CMC, while Sa increased to 20.2 m2 g–1 and 8.2 m2 g–1 for A-CMC–Gr and A-CMC–Gr–PD respectively, with the addition of graphene flakes. We suspect that the surface areas are indeed much higher (i.e. 50–100 m2 g–1) than those calculated. However, a collapse of the aerogels due to increasingly high mercury pressure (∼400 psi) is known to affect soft foams analysed with mercury porosimetryThe pore size distribution of the aerogels was estimated by mercury porosimetry . 2 g–1) carbon foam (CF) coated with a graphene–PD ink (Gr–PD–IPA) formulated by liquid phase exfoliation (LPE) . Isopropyl alcohol (IPA) was used as the solvent for this ink as the low surface tension (∼29 mN m–1) helps to transport the graphene flakes by capillary action around the porous structure. Additionally, to investigate the catalytic activity of a graphene–PD coating as a Pt-free air cathode we coated a standard SS mesh with a graphene–PD ink (Gr–PD–W), by vacuum filtration , to make a graphene coated stainless steel (SS–Gr–PD) cathode. Water was chosen as the solvent for this ink as organic solvents such as IPA will dissolve the nitrocellulose membrane used in the vacuum filtration. The optical absorption spectra of the Gr–PD–W and Gr–PD–IPA inks  statistics showed a thickness of ∼6 nm , surface tension (γ), and density (ρ) for the two inks showed that ηGr–PD–W ∼ 0.89 mPa s, γGr–PD–W ∼ 70 mN m–1, ρGr–PD–W ∼ 1.02 g cm–3; ηGr–PD–IPA ∼ 2.5 mPa s, γGr–PD–IPA ∼ 27 mN m–1, ρGr–PD–IPA ∼ 0.785 g cm–3, consistent with previous reports.In order to establish the effect of enhancing anode conductivity and surface area on MFC performance, we compared the aerogel anodes with a conductive graphene coated carbon foam (CF-Gr–PD) . This was prepared using a low surface area  and CF-Gr–PD anode (red curve) have the typical D, G and 2D peaks of graphene as described in the previous section which are in line with the spectra of Gr–PD–IPA flakes (green curve). The blue curve showed a combination of both Gr–PD and PD spectra, while the red curve brings additional features to the CF anode (black curve) where the absence of a distinct 2D peak and the G peak position Pos(G) ∼ 1600 cm–1 indicated the more defective nature of the CF.–1 nm–1.–1 nm–1) lower than that expected for disordered carbon.49Raman spectroscopy was also used to characterize the quality of the cathode and anodes. Rct of CF-Gr–PD anode decreased with respect to that of the CF anode from 41.4 kΩ to 0.930 kΩ.  and CF-Gr–PD (2 m2 g–1) anodes. SEM of the CF and CF-Gr–PD anodes showed average pore sizes of ∼2 μm  OD660 = 3.0 without stirring, to encourage cells to form an electroactive biofilm on the anode surface  Rsur , and has similar surface resistance (15.3 ± 1.21 Ω m–2), however the higher OCV of CFi (605 ± 70.3 mV) may have facilitated higher current (39.7 ± 4.86 A m–2) due to the unbroken conductive CFi connection, as opposed to discontinuous pristine graphene flakes.We used linear sweep voltammetry (LSV) to calculate polarization and power curves of the aerogel anodes by applying a linear sweep potential from the open circuit voltage (OCV) to 0 V. The OCV , surfaceve) Rsur , maximumRsur .71R. palustris taken at 1 mV s–1 between –900 mV and 900 mV after 72 hours in the devices. A broad oxidation–reduction peak pair at ∼360 mV and ∼–200 mV from A-CMC are most likely a result of the interaction between cells and the stainless steel anode connector,R. palustris on carbon paper,via interaction with excreted mediators.Shewanella oneidensis.R. palustris homologs to OmcA have low genetic and structural similarityR. palustris is known to have other important mechanisms of both oxidising and reducing its surroundings such as the phototrophic iron oxidation (Pio) pathway.R. palustris in a MFC. However, further work is required to elucidate the molecular basis of the precise redox active mechanisms acting in synergy with graphene.Cyclic voltammetry (CV) can be used to gain a qualitative insight into the redox mechanisms used to transfer electrons between the cell and the anode and thus give information on pristine graphene's catalytic properties  and 138 ± 28.2 mW m–3 (p = 0.007) respectively  CF-Gr–PD anodes to 3.51 ± 0.504 W m–3 from the A-CMC–Gr–PD aerogel anodes with a surface area of ∼10 to 20 m2 g–1. This clearly indicates that increasing anodic surface area is a key factor to improve MFC performances.We assessed the influence of surface area on the MFC anodes by acquiring the polarisation and power curves from linear sweep voltammetry from MFC devices equipped with CF anodes and the SS anode connector alone (used as control), as done for the aerogels. The n curves  and powen curves  of the CR. palustris biofilms on the SS, CF, and CF-Gr–PD anodes after operations, the anodes were removed from the MFC and prepared for SEM imaging after performing the bioelectrochemical measurements. The SS anode connector  of pristine graphene with the SS–Gr–PD electrode. ID and PV of 0.172 ± 0.0378 A m–2 and 1.04 ± 0.252 W m–3 respectively, which are 500-fold greater than those achieved with SS (black curve) at the cathode (0.000373 ± 0.000103 A m–2 and 0.00228 ± 0.000552 W m–3) (p = 0.011) and is only slightly lower than what was achieved when using a CP-Pt cathode (red curve) (ID ∼ 0.672 ± 0.152 A m–2 and PV ∼ 5.01 ± 0.302 W m–3). We attribute the improved performance of the SS–Gr–PD to the catalytic properties of pristine graphene flakes on the SS mesh facilitating the ORR.ID of 0.0753 ± 0.739 A m–2  at the cathode.39 A m–2  and PV o90 W m–3 , demonstID and PV decreasing from 8.3 to 6.5 μA and from 4.19 to 2.76 μW, respectively. This application demonstrates how graphene based MFCs are able to produce sufficient power to run commercial electronic devices such as those that may be found in wearable technology or low powered sensors.We tested the feasibility of graphene based MFCs to power small electrical devices, such as a digital clock. A circuit of 10 MFC chambers using A-CMC–Gr–PD anodes and Nafion® carbon–Pt cathodes was connected, with two series of five MFCs in parallel . This coIn this study we demonstrated the three-fold advantage of graphene based aerogels towards enhancing the efficiency of MFC electrodes. First, we demonstrated the effectiveness of unmodified, pristine graphene composite aerogels. Second, we showed that a pristine graphene coating can enhance EET compared to standard carbon anodes. Third, we reveal that pristine graphene can catalyse the cathodic ORR as well as anodic EET. We provide a direct demonstration of graphene enabled MFCs powering a commercial digital clock, illustrating the possibility of MFC in power demanding applications such as micro-electronics, beyond the already existing application ranges of sustainable electricity production, treatment of municipal waste water streams and biosensors.An isopropanol (IPA) based ink (Gr–PD–IPA) was prepared by ultrasonication  of graphite flakes (Sigma Aldrich) for 9 hours in IPA with PD (1% v/v). Additionally, a water based graphene ink (Gr–PD–W) was prepared by ultrasonication of graphite flakes for 9 hours in deionized water with PD (1% v/v) (Sigma 739316) to produce graphene flakes. In both ink preparations, the small amount of PD acts as a stabilisation agent and conductivity enhancer. The stabilization mechanism is due firstly to the π–π interaction between the graphene sheets and the backbone of the PEDOT and secondly the electrostatic repulsion between the negatively charged PSS.via the Beer–Lambert law expressed in the formula A = αcl, where α [L g–1 m–1] is the absorption coefficient, c [g L–1] is the concentration and l [m] is with the beam path length. The Gr–PD–W and Gr–PD–IPA inks were diluted 1 : 10 with water–PD and IPA–PD respectively. Assuming α ∼ 1390 L g–1 m–1 .The surface tension of the inks was measured using the pendent drop method (FTA1000B). The shape of the drop results from the relationship between the surface tension and gravity. The surface tension is then calculated from the shadow image of a pendent drop using drop shape analysis. A parallel plate rotational rheometer (DHR rheometer TA instruments) was used to evaluate the viscosity as a function of shear rate and the infinite-rate viscosities were determined for the Gr–PD–W and Gr–PD–IPA inks. Ink density was evaluated from a (Sartorius ME5) microbalance where the density is the mass per unit volume (–1) of the Gr–PD–IPA ink transported the graphene flakes by capillary action around the porous structure. For the aerogel anode fabrication a CMC/water precursor for each aerogel was created by adding 5 mg ml–1 CMC  to deionised water. Graphene powder (10 g L–1) (Cambridge Nanosystems) produced by cracking methane and carbon dioxide in a plasma torch was then added to the CMC/water solution for the A-CMC–Gr–PD and A-CMC–Gr aerogels while PD (10% v/v) (Sigma 739316) was added to the CMC/water solution for the A-CMC–Gr–PD and A-CMC–PD aerogels. Each sample solution (1 ml) was pipetted into an aluminium container and frozen at –10 °C allowing the growth of ice crystals to shapes the pore geometry of each structure. The samples were then placed into a freeze dryer (Telstar LyoQuest) to remove the ice crystals by sublimation under vacuum.A graphene coated carbon foam CF-Gr–PD anode was created by drop casting 3 ml of Gr–PD–IPA ink onto a conductive carbon foam anode . The low surface tension  of each cathode was determined with a Jandel probe head in a 4-point probe configuration. The Rs of a Gr–PD film transferred onto glass is 3.7 kΩ □–1, significantly higher than the Rs of the SS cathode (<10 Ω □–1).The cathodes were prepared by vacuum filtration transfer. First (∼4 ml) of the Gr–PD–W ink was diluted with deionized water at a ratio of 1 : 9 respectively and passed through a nitrocellulose membrane (100 nm pore size), hastened with the use of a Büchner flask attached to a vacuum pump. The graphene/PD film on the membrane was then transferred onto a stainless steel mesh with 0.2 mm spacing. After oven annealing (∼80 °C) the sample was placed in an acetone bath overnight in order to dissolve the nitrocellulose membrane and leave behind a graphene/PD film of thickness 635 nm (Bruker Dektak Stylus Profilometer) on the metal mesh (cathode SS–Gr–PD). SS–Gr–PD was then cleaned in acetone and isopropanol baths sequentially and then died in a nitrogen flux. The sheet resistance (P) and the pore diameter (D) can be found through the Washburn equation assuming the pores are cylindrical,D = –4γ cos(θ)/P where γ is the surface tension of mercury (485 mN m–1), θ is the contact angle (130°) acting along the parameter of the pore. The pore size diameter is plotted as a function of the differential intrusion (ml g–1 Å–1) which is found by dividing the incremental intrusion (ml g–1) by the difference in pore diameter (Å). The specific pore area (A) is then calculated (A = 4V/D) assuming a cylinder pore volume (V = πD2h/4) and open cylinder pore area (A = πDh). For each specific surface area measurement the contribution from the macro (∼75 μm) and meso (∼1 μm) pores while the contribution from the micropores (∼10 nm) could not be determined as the foams collapsed at the higher pressures (∼400 psia), a known problem when examining soft foams with mercury porosimetry.The pore size distribution and specific surface area of the anodes were determined using a mercury intrusion technique (Micromeritics AutoPore IV 9500). Mercury was pushed into the sample from 6.9 kPa (1 psia) to a maximum pressure of 206 843 kPa (30 000 psia). The relationship between this pressure  and pressed together between PTFE blocks. The cell was immersed in 1.0 M tetraethyl ammonium tetrafluoroborate (TEABF)/propylene carbonate (Sigma-Aldrich) non-aqueous electrolyte. EIS experiments were conducted using a BioLogic VSP-300 potentiostat, using an AC voltage of 0.2 V with 5 mV amplitude over a frequency range of 10 mHz to 10 kHz. All experiments were performed at room temperature.40Ø: 13 mm, height 30 mm), between which were clamped in descending order: a marine grade stainless steel  anodic electrode connector, a dialysis membrane of 10 kDa pore size (Thermo), a Nafion® proton exchange membrane (Dupont) with a conductive and catalytic carbon–Pt coating facing into the air exposed cathodic chamber, and finally a second SS mesh. The top and bottom SS mesh layers served as contacts for the anode and cathode respectively. Carbon fibre and aerogel electrodes were placed over the anodic SS mesh with wet thickness of 4 mm and 1 mm respectively, and total geometrical area of 1.33 cm2. No electron shuttle mediator was used. 4 ml of adjusted cell culture was injected into the top anodic chamber and cells were allowed to settle and form a biofilm on the anode. To characterize the SS–Gr–PD cathodes, the MFC device was modified with a secondary channel (Ø: 6 mm) drilled perpendicular and intersecting midway to the anodic channel. The SS–Gr–PD cathode was placed at the end of this channel, with a total geometrical area of 0.283 cm2, and 2 g of carbon fibre  was used in the anodic chamber. Platinum coated carbon paper (CP-Pt) was used as a benchmark (hydrogen electrode/reformate cathode Alfa Aesar 45452). Bioelectrochemical characterisation was carried out with a PALM-SENS MultiEmstat 8-channel potentiostat. Following stabilisation at the open circuit potential, polarisation and power curves were calculated by Ohm's law via linear voltage sweeps scanning the applied voltage from its open circuit potential to 0 volts with a slow scan rate of 1 mV s–1 to avoid overpotential effects.–1 between –900 mV and 900 mV after incubation for 3 days in the MFC.The MFCs used in this study were based around the design shown in λL, where λL is the laser excitation wavelength. Raman spectra for the Gr–PD–IPA and Gr flakes were acquired on Si/SiO2 substrate while all other spectra were acquired on the anodes and cathodes directly.Raman spectra for the cathode and anodes were acquired with a Renishaw 1000 InVia micro-Raman spectrometer at 457, 514.5, and 633 nm and a ×20 objective, with an incident power of below ∼1 mW.Scanning electron microscopy images were taken with a high resolution Magellan 400L scanning electron microscope (SEM). The field emission gun was operated at an accelerating voltage of 5 keV and gun current of 6.3 pA. Images were obtained in secondary electron detection mode using an immersion lens and TLD detector. The biological samples were taken on a FEI Verios 460 scanning electron microscope. Samples were washed in distilled water, fixed in liquid ethane, and mounted on a liquid nitrogen cooled plate before freeze drying overnight and coating in 14 nm of iridium.R. palustris and prepared cell cultures, T. P. C. and P. B. designed and constructed the MFCs and performed electrochemical measurements. T. P. C. undertook SEM on biological samples while P. H. preformed EIS on the aerogel and carbon foam samples with the assistance of T. C. The manuscript was written by T. C., T. P. C., F. T. and C. J. H., in close consultation with other authors.T. C. and T. P. C. conceived and designed the experiments. T. C., T. P. C., P. B., F. T. and C. J. H. analysed the experimental data and interpreted the results. T. C. developed the inks and composites and performed UV-vis, rheometry, AFM, Raman and pendant drop analysis on the inks and conducted mercury porosimetry, SEM, optical microscopy on the carbon foam and aerogel composites. T. P. C. and D. L. S. curated The authors have no conflict of interest.Supplementary informationClick here for additional data file.


ROS). Herein, we used a homology modeling approach to search the substrates for the oxidase activity of MsrA. We found that dimethyl sulfide (DMS), a main metabolite that produced by marine algae, emerged as a good substrate for MsrA‐catalytic antioxidation. MsrA bounds to DMS and promoted its antioxidant capacity via facilitating the reaction of DMS with ROS through a sulfonium intermediate at residues Cys72, Tyr103, and Glu115, followed by the release of dimethyl sulfoxide (DMSO). DMS reduced the antimycin A‐induced ROS generation in cultured PC12 cells and alleviated oxidative stress. Supplement of DMS exhibited cytoprotection and extended longevity in both Caenorhabditis elegans and Drosophila. MsrA knockdown abolished the cytoprotective effect and the longevity benefits of DMS. Furthermore, we found that the level of physiologic DMS was at the low micromolar range in different tissues of mammals and its level decreased after aging. This study opened a new window to elucidate the biological role of DMS and other low‐molecular sulfides in the cytoprotection and aging.Methionine (Met) sulfoxide reductase A (MsrA) is a key endogenous antioxidative enzyme with longevity benefits in animals. Only very few approaches have been reported to enhance MsrA function. Recent reports have indicated that the antioxidant capability of MsrA may involve a Met oxidase activity that facilities the reaction of Met with reactive oxygen species ( O2−·), hydrogen peroxide (H2O2), and hydroxyl radical, is important cellular toxicants that generated as products or by‐products in cells. A series of antioxidant mechanisms maintain intracellular redox homeostasis. When they deficit, overproduction of ROS causes DNA mutations and protein dysfunction, which eventually leads to cell injury and aging. The thiomethyl group that exists on the surface exposed methionine (Met) residues has been recognized as a key endogenous antioxidant defense , including superoxide . In previous studies, it has been shown that in the first step of MsrA‐catalytic oxidation, a sulfenic acid residue on Cys72 of MsrA forms by ROS attacking (MsrA‐CYO). Thus, we analyzed the orientation of different MsrA substrates in the active site of MsrA‐CYO . We found DMS adopts a preferred orientation with the distance of O‐S 3.8 Å (Table S1). In the presence of DMS binding, the distances of donor and acceptor atoms (D‐H···A) between the O atom from CYO72, the H atom from Tyr103‐OH, and Glu115‐COOH were 3.6 and 1.6 Å, respectively. Another question is whether the preferred orientation of DMS in the active site formed easily. Thus, energy barrier was used to evaluate the difficulty of orientation. Steering molecular dynamics (SMD) can operate single molecules to move closer or apart just like the working mode of atomic force microscopy. For oxidative reaction catalyzed by MsrA, substrates including DMS were pulled 16 Å away from MsrA and then pulled to the active site of MsrA. Curves of works showed how much energy was required to pull substrates to the active site as close as possible . It was obvious that much less energy was required for pulling DMS than Met substrates, indicating that the formation of sulfonium intermediate on DMS occurs more easily. As shown in Fig. S2C, DMS fitted properly in the catalytic pocket of MsrA, which also indicated DMS was a dominant substrate of MsrA.m) was incubated with different concentrations of DMS, and its binding was measured by fluorescence anisotropy. As shown in Fig. m), indicating a direct binding of DMS to MsrA.The DMS conformation from docking experiment indicated that DMS could easily bind to the catalytic pocket of MsrA Fig. A, which et al., 2O2‐induced luminescence was not inhibited by 0.1 mm DMS . Neither MsrA nor nonactive MsrA alone (1 μm) had a perceptible effect on the luminescence intensity . Meanwhile, cotreated with 0.1 mm DMS and MsrA (1 μm), but not nonactive MsrA, remarkably reduced the luminescence signal intensity to 52 ± 5% . In electron paramagnetic resonance (EPR) assay, hydroxyl radical was induced by Fenton reaction and trapped by DMPO to form a stable spin adduct DMPO‐OH. The signal intensity of DMPO‐OH adducts was not changed by MsrA or nonactive MsrA alone . However, cotreatment with 0.1 mm DMS and MsrA (1 μm) decreased the DMPO‐OH signals to 73 ± 1% when compared to the group cotreated with 0.1 mm DMS and nonactive MsrA .Previous studies indicate that DMS exhibits antioxidant function in marine algae (Sunda P < 0.01), respectively, indicating that Cys72 is the most critical one among these three residues. Furthermore, it was found that the predicted key residues  were conservative in various organisms , indicating that this catalytic mechanism may exist extensively.As predicated, Tyr103 and Glu115 facilitated the transfer of oxygen atom to DMS via the formation of hydrogen bond . The Phe73 and Trp74 increased the binding of DMS to the pocket via the hydrophobic force. To validate this model, three single site mutants  were built. The peroxidase activity was measured by the ability to scavenge hydroxyl radicals in EPR tests  was used to evaluate total ROS. Application of AA (100 μg mL−1) for 0.5 h induced an increase of the total ROS level to 256 ± 15% of control . Pretreatment of PC12 cells with DMS (1 and 5 μm) significantly decreased the ROS level to 125 ± 14% and 100 ± 18% of control, respectively  and high concentrations (10 and 100 μm), DMS significantly attenuated H2O2‐induced lipid peroxidation from 159 ± 7% to 131 ± 4%, 123 ± 6% and 120 ± 8% in the brain, respectively , and this was reduced to 90 ± 15% of basal level by 5 μm DMS (P < 0.01).To further investigate the biological function of DMS, we observed the effect of DMS on cell lines. ROS production in PC12 cells was stimulated by antimycin A (AA), a mitochondrial respiratory complex III inhibitor. AA increased the levels of Drosophila, the lipid oxidation product MDA decreased significantly with the treatment of DMS (10 μm) in aged Drosophila from 15.2 ± 0.5 nmol mg−1 protein to 11.3 ± 0.6 nmol mg−1 protein . We further tested whether DMS exerted lifespan‐extending properties in Drosophila. The mean survival time of control group was 48.25 ± 0.91 day. Meanwhile, the mean survival time was extended to 54.80 ± 1.06 day (10 μm), 59.95 ± 1.11 day (100 μm), and 55.99 ± 0.58 day (1000 μm) with DMS exposure . However, DMS did not prolong the lifespan of Drosophila at low concentrations .MsrA has longevity benefits in different organisms. As a substrate of the peroxidase activity of MsrA, DMS may exert longevity benefits. In C. elegans. MitoSOX red was used to detect the superoxide contents in aged C. elegans . Treatment with 10 μm DMS remarkably decreased superoxide content to 228 ± 26% (P < 0.01). Worms were fed with DMS medium at three different concentrations: 1, 5 and 10 μm. The mean survival time of DMS‐treated worms was extended from 21.00 ± 0.52 day to 22.77 ± 0.59 day (1 μm), 25.23 ± 0.72 day (5 μm), and 26.11 ± 0.71 day (10 μm), respectively . The MDA content decreased significantly with DMS (5 μm) treatment in aged worms from 161 ± 10% to 125 ± 13% of control . These results support the hypothesis that DMS mediates longevity via alleviating the accumulated oxidative damage in aged animals.A similar effect of DMS was observed in ans Fig. C,D. The 2O2 (150 μm) was used to induce oxidative stress on SH‐SY5Y cells. After treatment for 12 h, the cell viability of SH‐SY5Y cells decreased to 59 ± 7% of control group. DMS increased the cell viability of SH‐SY5Y cells under oxidative stress to 81 ± 2% at 6.25 μm, 90 ± 3% at 12.5 μm, 88 ± 3% at 25 μm, 82 ± 4% at 50 μm, and 76 ± 4% at 100 μm , the cell viability in DMS‐treated groups increased to 76 ± 2% (6.2 μm), 79 ± 3% (12.5 μm), 82 ± 3% (25 μm), 84 ± 3% (50 μm), and 84 ± 5% , 62 ± 5% (500 μm), and 70 ± 6% (1000 μm) of control . As shown in Fig. P < 0.01). However, in MsrA shRNA group, the effect of DMS (10 μm) on AA‐induced superoxide production was abolished  on cell viability against H2O2 was also abolished. With control shRNA, the DMS increased the cell viability of PC12 cells from 68 ± 2% in H2O2 group to 93 ± 3% in DMS group . With MsrA shRNA, DMS showed little effects on the cell viability under oxidative stress (70 ± 4% in H2O2 model group vs. 78 ± 4% in DMS group). The protective effect of DMS on lipid peroxidation was also abolished, which resembled the findings in the H2O2‐induced cell death. As shown in Fig. 2O2 model group (160 ± 7%).We then analyzed how DMS at low micromolar range scavenged ROS in PC12 cells. Considering the intrinsic catalytic function of MsrA in the oxidation of thiomethyl groups, we tested whether the antioxidation effect of DMS is dependent on MsrA. After treatment with lentiviral‐expressed specific short hairpin RNAs (shRNA) against MsrA , the expression level of MsrA decreased to 22 ± 6% (msra‐1 expression to 10 ± 5% of control group (L4440 plasmid) in C. elegans . In msra‐1 RNAi group, lifespan was significantly shortened from 22.5 ± 0.8 day to 17.8 ± 0.6 day, which suggested that MsrA plays a critical role. DMS (10 μm) could not extend lifespan in msra‐1 RNAi group . As shown in Fig. 2O2 was used to introduce oxidative stress to test the resistance of worms. The survival rates of worms in each group were evaluated after 3 h in S‐basal buffer containing H2O2 (10 mm). The survival rate of DMS‐treated raised from 44 ± 2% (control RNAi) to 62 ± 4% . However, DMS failed to elevate the survival rate of worms treated with RNAi plasmid. The survival rate of msra‐1 RNAi group (28 ± 2%) showed no significant difference with that of msra‐1 RNAi with DMS group (33 ± 6%).In Fig. 6 cell count. As shown in Fig. 2O2‐treated groups raised significantly to 184 ± 16% and 281 ± 20% of control, respectively . The endogenous cellular DMSO was at 5.73 nmol per 5 × 10et al., et al., −1 wet tissue (at about 1 μm) in rat brain, 2.42 ± 0.09 nmol g−1 wet tissue (at about 2 μm) in rat heart, and 1.09 ± 0.11 nmol g−1 wet tissue (at about 1 μm) in mice brain  and from 2.53 ± 0.12 to 1.44 ± 0.07 nmol g−1 (heart), respectively . MitoSox red was purchased from Invitrogen . DMEM/F12, RPMI1640 and fetal bovine serum were purchased from HyClone . NaBH4 was obtained from Sinopharm chemical reagent . SPME holder and 75‐μm carboxen‐PDMS SPME fiber was purchased from Supelco . Ni‐Trap nickel‐chelating column was obtained from Qiagen . Thrombin kit was purchased from New England Biolabs . Fast Mutagenesis System was obtained from Transgene . MDA and cell survival rate kits were obtained from Jiancheng Biotech .AA, dithiothreitol (DTT), H2DCF, HC. elegans N2 worms were grown on E. coli OP50 using standard techniques on 20 °C . The Drosophila strain w1118 was used in all control crosses and as the background for generation of transgenic lines. All Drosophila stocks were maintained, and all experiments were conducted at 25 °C on a 12‐h: 12‐h light: dark cycle at constant humidity using standard sugar/yeast/agar (SYA) media. Adult male Sprague Dawley rats and C57BL/6J mice were obtained from the Experimental Animals of Tongji Medical College, Huazhong University of Science and Technology. The rats/mice were housed on a controlled 12‐h: 12‐h light cycle at a constant temperature (24 ± 1 °C) with free access to water and food and allowed to acclimate a week. The use of animals for experimental procedures was conducted in accordance with the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. The experimental procedures were approved by the Animal Welfare Committee of Huazhong University of Science & Technology.The wild‐type Brenner, . The RNAet al., 4 and analyzed by SPME‐GC‐MS. Headspace sampling of DMS was carried out with a SPME holder equipped with a 75‐μm carboxen‐PDMS SPME fiber. Samples were incubated for 5 min at 35 °C and then extracted for 10 min at the same temperature. Then, the GC/MS analysis was performed with GC‐MS analysis using Agilent 6890N‐5975B GC‐MS system . The injector temperature was 200 °C, and injections/SPME desorptions were made in splitless mode. Helium was used as a carrier gas with linear velocity 40 cm s−1. The MS analyses were carried out in a SIM scan mode, with a scan of m/z 47 and 62. Electron ionization was used at energy of 70 eV. The temperature of the ion source and the transfer line was 190 and 150 °C, respectively. The acquisition of chromatographic data was performed by means of the Chemstation Software (Agilent).Different tissue samples were collected and prepared as described by published report , and MsrA/nonactive MsrA (1 μm) were added before the Fe (II) and H2O2. Samples (20 μL) were loaded into a quartz tube, and the EPR spectra were recorded at room temperature. The EPR microwave power was set to 4.88 mW. The modulation frequency was 9.76 GHz. The time constant was 81.92 ms, and conversion time was 81.92 ms. A sweep time of 41.94 s was used. Each sample was scanned once. A sweep width of 100 G was used for experiments with DMPO. The receiver gain was set to 3.17 × 103. Simulation and fitting of the EPR spectra were performed using the Bruker WinEPR program.Dimethyl pyridine N oxide (DMPO) was used as a free radical trapper. EPR signals were detected with a Bruker e‐scan EPR spectrometer  as previous report . The PC12 cells were cultured in RPMI1640 supplemented with 15% horse serum, 2.5% fetal bovine serum, and 100 U mLA third generation of self‐inactivating lentivirus vector  containing a CMV‐driven GFP reporter and a U6 promoter upstream of the cloning sites (Age I and EcoR I) was used for cloning shRNAs. The target sequence for rat MsrA was 5′‐AGCACGTCAGCTTTGAGGA‐3′ and for control scrambled shRNA was 5′‐TTCTCCGAACGTGTCACGT‐3′. PC12 cells were infected with lentivirus at a multiplicity of infection (MOI) of 20 for 8 h. Then, the medium was replaced with fresh complete medium. After 72 h, cells were observed under fluorescence microscopy to confirm that more than 80% of cells were GFP‐positive.See details in the Supplementary Methods section.See details in the Supplementary Methods section.See details in the Supplementary Methods section.See details in the Supplementary Methods section.See details in the Supplementary Methods section.See details in the Supplementary Methods section.See details in the Supplementary Methods section.See details in the Supplementary Methods section.See details in the Supplementary Methods section.See details in the Supplementary Methods section.See details in the Supplementary Methods section.t‐test or LSD test post hoc ANOVA. The Kaplan–Meier method was used to compare the differences in survival rates between groups. A P‐value < 0.05 was considered statistically significant.Data are expressed as mean ± SEM Significance of differences between groups was determined by Student's This work was supported by grants from the National Basic Research Program of China  and the National Natural Scientific Foundation of China . It was also supported by PCSIRT (No. IRT13016), the National Key Scientific Instrument and Equipment Development Project of China (No. 2013YQ03092306) and Science Fund for Creative Research Groups of the Natural Science Foundation of Hubei Province (2015CFA020) to J.G.C.No competing financial interests exist.Appendix S1 Methods.Fig. S1 DMS serves as a dominant substrate of MsrA.Fig. S2 The catalytic pocket model of DMS‐MsrA interaction.Fig. S3 DMS decreases the luminal chemiluminescence in vitro, while MsrA enhances the OH˙ scavenge property.Fig. S4 Neither MsrA nor non‐active MsrA alone has a perceptible effect on ESR signals.Fig. S5 DMS does not prolong the lifespan of Drosophila in low concentrations.Fig. S6 MsrA shRNA significantly inhibits the expression of MsrA in PC12 cells.Fig. S7 The effects of MsrA RNAi are evaluated by RT‐PCR and RT‐qPCR.Fig. S8 The detection method of DMS is built using GC‐MS.Table S1 Distances (Å) between atoms CYO‐S/CYO‐O and S atom; S from DMS, 1 EMS, MPS, L‐Met, AMN, Tripep, Hexpep.Click here for additional data file.


Aortic graft infection is a potentially lethal complication of open and endovascular repair of aortic aneurysms. Graphene is the only existing two-dimensional material, and its unique structure gives graphene and its derivatives a plethora of original characteristics. Among other characteristics, graphene demonstrates bacteriostatic and bactericidal effects that could potentially resolve the problem of graft infection in the future. Data already exist in literature supporting this antibacterial effect of graphene oxide and reduced graphene oxide. Combining these materials with other substances enhances the antibacterial effect. Additionally, it looks feasible to expect antibiotic-delivering graphene-based graft materials in the future. Based on already published data, we could conclude that regarding graphene and its derivatives, the blessing of bactericidal effect comes with the curse of human cells toxicity. Therefore, it is important to find a fine balance between the desired antibacterial and the adverse cytotoxic effect before graphene is used in graft materials for humans. Prosthetic graft infection is a grave and frequently lethal complication of both open and endovascular repair of aortic aneurysms (AA). Despite its relatively low overall incidence, the absolute numbers of graft infection are rising, as the use of synthetic grafts  has become an everyday occurrence in both elective and emergency AA repair, with a reported graft infection incidence of 0.2 to as high as 6% in EVAR cases –4. Overain situ reconstruction of the aorta using either cryopreserved allografts or autologous veins is a similarly disabling method of aortic reconstruction after infected graft excision , and epitaxial growth . FurtherS. aureus and E. coli bacteria (In existing literature, pristine graphene sheets have already been reported to have bacteriostatic and bactericidal properties. These two properties are of great interest for bioengineers committed to improve the graft materials of today to become less prone to bacterial infection. In a recent detailed review of graphene’s future in the grafts materials engineering, it seems that despite existing data supporting these beneficial properties of graphene, not all publications agree to the extent and efficacy of these . In thisbacteria –20. The bacteria . At presbacteria –24.N-carbazole, lactoferrin, diazonium salt, poly-l-lysine, and polyhexamethylene guanidine hydrochloride are a few of the substances used to increase the bactericidal and bacteriostatic effects of graphene sheets (S. aureus and S. epidermidis, although secondary hematogenic contamination with Gram-negative bacteria is also possible. rGO shows the strongest antibacterial effect toward both Gram-positive and Gram-negative bacteria, but in order to achieve the best possible antibacterial effect against the usual bacteria involved, further studies have to examine various combinations of graphene and other substances, although some evidence already exist (In addition to the nature of the material surface, binding other molecules to graphene can also affect its bactericidal or bacteriostatic effect. Silver particles, platinum particles, chitosan, polyvinyl-e sheets , 25–27. dy exist .Graphene has been used as a delivery agent for medication since 2008 . Since tInfection-resistant graphene-based graft materials could be constructed today with the already existing knowledge and technology. In the near future, these same graft materials could go a step further and evolve into infection fighting antibiotic-delivery agents. Unfortunately, before we see these steps ahead of current bioengineering, the question of biocompatibility and biotoxicity should be resolved . Graphenin vivo studies by a multidisciplinary team of bioengineers and vascular surgeons are necessary in order to research the potential role of graphene and its derivatives in building infection-resistant aortic grafts and provide the necessary data on patency, biotoxicity, and biocompatibility. These future materials would further reduce the incidence of graft infection that remains a lethal complication of open and endovascular aortic repair.Graphene and its derivatives could potentially lead the way to new and truly infection-resistant or bactericidal graft materials that can be primarily used in aortic or other arterial repair in order to avoid the occurrence of graft infection, not fighting an already-occurring infection. A proper animal model and NP—conception, data collection, literature review, manuscript writing, and critical appraisal. DS—data collection and critical appraisal. TL—conception and critical appraisal. CK—conception, manuscript writing, and critical appraisal. All the authors have accepted the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Supplemental Digital Content is available in the text P values < 0.001. Using 96.5 pg/mL as the cutoff, patients with lower baseline sPD-L1 level had longer OS than those with higher sPD-L1 level . Using multivariate analysis, the following factors were significantly associated with longer OS: female, adenocarcinoma, higher TRT dose, and lower baseline sPD-L1 level. Patients with both characteristics of lower baseline sPD-L1 level and higher TRT dose (BED10 ≥84 Gy) had the longest OS. To conclude, the lower baseline sPD-L1 level was significantly associated with longer OS in NSCLC patients treated with TRT, which may serve as an independent biomarker and needs further clinical study.Immune-checkpoint signaling plays an important role in immunosuppression of tumors. We aimed to investigate the association of soluble programmed death-ligand 1 (sPD-L1) level in plasma with overall survival (OS) in locally advanced or inoperable nonsmall-cell lung cancer (NSCLC) patients treated with thoracic radiotherapy (TRT). We used ELISA to evaluate the sPD-L1 levels at diagnosis and during TRT in 126 clinically inoperable NSCLC patients. OS rates were followed up and recorded. SPSS software and GraphPad Prism 5 were used for statistics. In this study, the average sPD-L1 levels at baseline, week 2, and week 4 during TRT and post-TRT were 107.2, 51.3, 65.4, and 111.1 pg/mL, respectively. Levels of sPD-L1 at week 2 and week 4 were significantly less than at baseline, with both  In Zeng animal study involving mice with glioblastoma multiforme, improved survival was demonstrated with a combination of anti-PD-1 therapy and stereotactic body radiation therapy, compared with either modality alone.Radiation therapy (RT) is the mainstay treatment for nonsmall-cell lung cancer (NSCLC) patients. Based on current knowledge, a higher radiation dose and concurrent chemoradiotherapy may improve the survival of patients, as has been demonstrated in previous trials.,7 There is evidence that tumor cells can express PD-L1 on the cell membrane by activated T-cells.,9 This has been investigated in metastatic renal cell carcinoma (RCC), suggesting that primary RCC tumors with PD-L1 positivity—either on tumor cell membranes or inflammatory cells—will have a better response to PD-1/PD-L1-targeting therapies.Programmed death 1 (PD-1) is a transmembrane surface glycoprotein encoded by the CD274 gene located on chromosome 9. As the main ligand for PD-1, PD-L1 induces a coinhibitory signal in activated T-cells and promotes T-cell apoptosis, anergy, and functional exhaustion. From the ASCO 2015 Annual Meeting, treatments targeting PD-1/PD-L1 pathway for NSCLC patients were widely reported. According to Tiffany's study, PD-L1 overexpression was significantly associated with increased CD8+ TILs and KRAS mutations in resected lung adenocarcinomas. It was reported that the PD-1 blockade demonstrated durable manageability as a first-line therapy for PD-L1+ metastatic NSCLC.,14 Moreover, PD-L1 level in cell supernatants and staining slides could be used as a predictive factor of survival.–18 A survival model established by Jiang et al showed that PD-L1 expression was predictive to OS in patients with squamous NSCLC. However, some other studies did not found such significant correlations.,20For immunotherapies, many candidate biomarkers such as IFN-γ and TGF-β are under investigation.–23 However, data were insufficient to describe the changes in the PD-1/PD-L1 level during thoracic radiotherapy (TRT). In this study, we aimed to investigate the changes of plasma sPD-L1 level in NSCLC patients receiving TRT, and to find the association between sPD-L1 level and overall survival (OS) in those patients.Soluble PD-L1 (sPD-L1) was easily detectable in human plasma using a commercial ELISA kit. Although the function and mechanism of release is debated, sPD-L1 could be alternative for clinical use, especially for patients without enough tumor tissue to test, which has been demonstrated by lots of previous studies.2The procedures in this study were in accordance with the ethical standards of the responsible Committee on Human Experimentation of Tongji Hospital and with the Helsinki Declaration.2.118FDG PET/CT or CT scan for staging, including full visualization of the liver and adrenal glands. A radionuclide bone scan and an MRI scan of the brain were required. Routine blood work, including complete blood count, and liver and renal function tests were also necessary. We obtained written informed consent from all patients before enrolling them onto the trials, in accordance with the procedures of the Institutional Review Board.This is a prospective study (between 2009 and 2013). NSCLC Patients with locally advanced stage were eligible. Patient was required to have at least 1 measurable or assessable lesion, and prior TRT were not permitted. An Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 2 was eligible. Initial evaluation consisted of a history and physical examination, 2.2Patients were treated definitively with TRT alone or with concurrent chemoradiotherapy. The gross tumor volume (GTV) was restricted to the primary tumor, any hilar or mediastinal lymph nodes with a short-axis diameter of at least 1 cm on CT, and any abnormal findings detected on bronchoscopy or mediastinoscopy. The clinical target volume (CTV) was routinely created by expanding the GTV by 0.5 cm. Clinically uninvolved hilar, mediastinal, and supraclavicular nodal regions were not purposely included in the CTV. The planning target volume (PTV) was defined as the CTV, plus 1.0 cm anterior, posterior, medial, and lateral margins and 1.0 to 2.0 cm superior and inferior margins, when necessary to account for respiratory motion.The radiation dose was prescribed to isocenter and accounted for tissue heterogeneity. Radiation planning required that 100% of the PTV be encompassed by 95% of the isodose. Typically, a single-treatment plan was used, which consisted of a median of three noncoplanar static fields . All patients received a daily treatment using 2.0 to 3.8 Gy fractions with beams ranging in energy from 6 to 25 MV.2) were started on week 1 of TRT and continued weekly for 6 weeks.Chemotherapy was administered weekly, concurrent with radiation on the same day each week. Carboplatin (AUC = 2) and Paclitaxel , 2 and 4 weeks after the first TRT treatment day (during TRT), and in 3 months of the last TRT treatment day (post-TRT). Plasma samples were collected into antiproteasic BD P100 blood-collection tubes , and centrifuged and stored at −80°C.2.4Soluble PD-L1 in plasma was measured using an enzyme-linked immunosorbent assay , according to the manufacturer's instructions. The minimum detectable dose of sPD-L1 was 57 pg/mL. Each sample was analyzed in duplicate. Protein levels were calculated according to standard curves. The intraassay and interassay coefficients of variation were below 20%. The same assay and procedure were used to measure sPD-L1 levels for all studied cohorts.2.5Follow-up procedures included the patients first being examined at the end of therapy, then every 3 months for 2 years, then every 6 months for 2 years, and yearly thereafter. Patients underwent a CT scan of the chest at every visit. At the time of progression, restaging procedures included CTs of the chest and abdomen and an MRI of the brain.2.6P-value < 0.05 was considered statistically significant.The primary endpoint is OS, which was calculated from treatment initiation, and estimated using the Kaplan–Meier method. OS was defined as the interval between the date of treatment initiation and the date of death, or last follow-up for patients still alive. Age, gender, smoking status, ECOG performance status, histology, concurrent chemotherapy, and radiation dose were evaluated for their association with each survival endpoint in multivariate analysis. Results are presented as median and 95% confidence interval (CI), unless otherwise specified. Analyses were carried out using SPSS 18.0 and GraphPad Prism 5 software. All tests were 2-sided, and a 3Table 3.1P < 0.001), as were the levels of week 4 compared with the baseline (P < 0.001). However, there was no significant difference between the baseline and post-TRT levels (P = 0.62) . The median OS (95% CI) in patients with lower sPD-L1 (<96.5 pg/mL) was 27.8 (19.7–36.0) months, compared with 15.5 (8.2–22.9) months in patients with higher sPD-L1 level (P = 0.005)   OS were 29.0 18.8–39.1) and 18.8 (13.8–23.90) months, respectively  . For example, IFN-γ produced by inflammatory cells could act as a potent PD-L1 upregulator. This is thought to be important for coinhibition during the T-cell initiation of an immune response. CD8+ T-cells play a major role in cellular responses, including in antitumor immune defense, while TILs contribute to good clinical outcomes in many types of cancer. Muenst et al found expression of PD-L1 was significantly associated with an increase in the number of TILs in breast cancer. It might be an indication that as the tumor progresses, PD-L1 is increasingly produced by tumor cells in order to evade the mounting antitumor immune response. Ghebeh et al also found PD-L1 expression by TILs in 41% of their specimens, predominantly on CD4+ T-cells. Moreover, expression of PD-L1 on TILs might inhibit T-cell function by binding to PD-1 expressed on other TILs, as demonstrated by Seo et al. The capacity of the PD-1/PD-L1 interaction to downregulate a CD3/CD28-stimulated response shows that PD-1 engagement results in the delivery of a strong inhibitory signal.The upregulation of PD-L1 is a common phenomenon in cancer patients, Additionally, the PI3K/AKT inhibitors could reduce the PD-1/PD-L1's induced resistance to antitumor agents. The clinical data have shown that PD-L1 is important for immune evasion by tumor cells. It has been reported that a doubling of sPD-L1 levels was associated with a 41% increased risk of death (P = 0.01). Azuma et al reported that high expression of sPD-L1 was associated with the presence of epidermal growth factor receptor (EGFR) mutations in surgically resected NSCLC and was an independent negative prognostic factor for this disease. In Rossille's study, elevated sPD-L1 was correlated with a poorer prognosis for patients with B-cell lymphoma treated with R-CHOP chemotherapy (P < 0.001), using a cutoff of 1.52 ng/mL for PD-L1 level. ASCO 2015 Annual Meeting reported that NSCLC patients with PD-L1-negative tumors showed longer progression-free and OS than those with PD-L1-positive . Increasing data indicate that the PD-1/PD-L1 signaling pathway is related to immune suppression and disease progression, such as melanoma, renal, urothelial, gastric, lung, and colorectal cancer.–39 Further studies are needed to identify how PD1/PD-L1 signaling impacts prognosis.The mechanisms by which elevated sPD-L1 levels contribute to poor prognosis are not clear. PD-1 was observed to induce drug resistance via the AKT signaling pathway in myeloma cells. Kong et al have reported the dose effects of 237 patients with stage III NSCLC, showing that BED was a significant prognostic factor associated with the risk of death . In a randomized trial from China, stage III patients treated with a total dose of 68 to 74 Gy had significantly better 5-year tumor local control and 2-year OS than those treated with 60 to 64 Gy . A high radiation dose was considered to induce an anti-tumor immune function including increasing inflammatory cells and effective T-lymphocyte invasion. However, it is usually difficult to clarify the relationship between radiation and the immune effect, which may largely depend on the biological equivalent radiation dose as well as immune cell types.,43 Preclinical studies have indicated the critical role of radiation with appropriate dose and fractions inducing tumor immunogenic antigen release during antitumor immune response, in which new peptides were generated in complex antigenic environment. For example in Sharabi et al's study using B16-OVA melanoma cells and 4T1-HA breast carcinoma cells, it was observed that radiation could enhance tumor-specific antigen-MHC complexes, increase T-cell infiltration into tumor cells, and upregulate antigen cross-presentation in the draining lymph nodes. These results are expected to be demonstrated in future clinical trials.A high TRT dose has been observed to be a significant predictive factor for OS. Investigators from Memorial Sloan-Kettering Cancer Center reported that patients who received ≥64 Gy had better survival than those who received <60 Gy in stage III patients receiving chemoradiotherapy.,46 As far as we know, TRT together with concurrent chemotherapy in this study induced tumor deaths in most of the patients, which contributed to the low PD-L1 level. However, whether the change of PD-L1 expression on tumors affected the level of soluble PD-L1 in plasma is unclear up to now. Secondly, it is interesting that the PD-L1 level recovered to the baseline after RT, which may be associated with the increase in inflammation; however, no such data have been achieved. In Deng et al's animal study, the synergistical effects of TRT and anti-PD-L1 treatment were first observed, which encourage us to perform more clinical trials in the future.There are some limitations to this study. Firstly, we only test the soluble PD-L1 level in the blood and we observed a decreased trend in plasma level during TRT, which was opposite to the results in mice.To conclude, the baseline plasma sPD-L1 level before TRT could be an independent prognostic biomarker for NSCLC patients. Patients with lower baseline sPD-L1 level together with the treatment of high TRT dose (BED10 ≥84 Gy) were significantly correlated with longer OS.


Graphene aerogels with high surface areas, ultra-low densities and thermal conductivities have been prepared to exploit their wide applications from pollution adsorption to energy storage, supercapacitor, and thermal insulation. However, the low mechanical properties, poor thermal stability and electric conductivity restrict these aerogels’ applications. In this paper, we prepared mechanically strong graphene aerogels with large BET surface areas, low thermal conductivities, high thermal stability and electric conductivities via hydrothermal reduction and supercritical ethanol drying. Annealing at 1500 °C resulted in slightly increased thermal conductivity and further improvement in mechanical properties, oxidation temperature and electric conductivity of the graphene aerogel. The large BET surface areas, together with strong mechanical properties, low thermal conductivities, high thermal stability and electrical conductivities made these graphene aerogels feasible candidates for use in a number of fields covering from batteries to sensors, electrodes, lightweight conductor and insulation materials. Compared with those aerogels, graphene aerogels possess larger Brunauer-Emmett-Teller (BET) surface areas (SBET > 500 m2∙g−1)8 and ultra-low densities10 because of the very high SBET of a single graphene sheet 11. Tang et al. synthesized ultra-light graphene aerogels with densities from 1.8 mg∙cm−3 to 27.2 mg∙cm−3 using different starting graphene oxide (GO) concentrations. Despite the good thermal conductivity of single-layer graphene (4.84 × 103–5.30 × 103 W·m−1·K−1)12, the nanoporous structure, low density, and perfect opacity of graphene aerogel depress the gaseous thermal conductivity13, solid thermal conductivity14, and radiant thermal conductivity10, respectively. Moreover, Yue et al.10 demonstrated the defects in graphene, and the relatively small sizes of the graphene sheets further suppressed thermal conductivity. Due to the larger thermal contact resistance at the interfaces between adjacent GO sheets15, Xie et al. prepared graphene aerogels with ultra-low thermal conductivities (as low as 4.7 × 10−3–5.9 × 10−3 W·m−1·K−1) at room temperature approximately 80% lower than that of air (0.0257 W·m−1·K−1 at 20 °C).Inorganic17, highly compressibility (strain>50%)17, recoverability18 and thermal insulation10. The poor mechanical, thermal, and electric properties restrict their applications in energy storage, catalyst supports, supercapacitor, and thermal insulation. Ultralight and ultra-flyweight graphene aerogels have highly compressibility and recoverability at maximum strain ≥50%, however, the compression strengths were in the order of KPa19. Several studies have promoted the mechanical properties of graphene aerogels. Zhang et al.7 created mechanically strong graphene aerogels which were dried by supercritical CO2 and with L-ascorbic acid as the reducing agent. The yield strengths and Young’s moduli of these aerogels ranged from 0.04 MPa to 0.66 MPa and from 1.2 MPa to 6.2 MPa, respectively, with densities in the range of 12–96 mg∙cm−3. Huang et al.20 have prepared PEI-crosslinked graphene aerogel with yield strength and Young’s modulus of 1 MPa and 20 MPa. The thermal stability of graphene aerogels was depended on reduction level. High-temperature annealing of graphene aerogels results in enhanced thermal stability22 and an increase in Young’s moduli by one order of magnitude22. Graphene aerogel annealed at 400 °C exhibits significantly improved thermally stability compared with GO and as-prepared graphene aerogel21. With annealing temperature increases from 1050 °C to 2500 °C, the graphene aerogels become more resistant to oxidation in air and exhibit an approximately 200 °C improvement in oxidation temperature22. The bulk electrical conductivity of macroscopic 3D graphene networks was less than 1 S/m24. The electrical conductivity of graphene aerogels increased to >550 S/m after annealing at 2500 °C22. Chen et al.21 have studied the effect of various reducing agents on the electrical conductivities of the graphene aerogels and found that the electrical conductivity of the hydrogel prepared using HI and NaHSO3 have higher values.Taking advantage of larger surface areas or lower thermal conductivities, current researches on graphene aerogels mainly focused on the ultra-light weightIn this paper, we prepared mechanically strong graphene aerogels with large BET surface areas, low thermal conductivities, high thermal stability and electric conductivities via hydrothermal reduction and supercritical ethanol drying. Annealing at 1500 °C resulted in slightly increased thermal conductivity and further improvement in mechanical properties, oxidation temperature and electric conductivity of the graphene aerogel. The large BET surface areas, together with strong mechanical properties, low thermal conductivities, high thermal stability and electrical conductivities made these graphene aerogels feasible candidates for use in a number of fields covering from batteries to sensors, electrodes, lightweight conductor and insulation materials.25. Graphene hydrogels were prepared by hydrothermal reduction of GO dispersions with the assistance of a mild reducing agent NaHSO3. The obtained gels were dried using supercritical ethanol to obtain the graphene aerogels, the resulting samples were referred to as GA-S. The obtained GA-S samples were then heat treated at 1500 °C, the resulting samples were labeled as GA-S-1500C. The GA-S and GA-S-1500C prepared with various concentrations of GO (CGO) were denoted as GA-Sx and GA-Sx-1500C, where x is the concentration of GO dispersion in milligram per milliliter. In addition, we investigated the effect of the drying process by freeze-drying graphene aerogels with GO aqueous dispersion of 5 mg∙mL−1 using the same method as that for GA-S, but deionized water, instead of ethanol, was used to purify the hydrogels. The resulting sample were denoted as GA-F.The synthesis of the mechanically strong graphene aerogels with cylindrical morphology is illustrated in Fig. The removal of a large amount of water from the highly porous network interconnected by partial overlapping or coalescing flexible graphene sheets resulted in the formation of the 3D framework. The graphene aerogels exhibited an interconnecting porous structure of randomly oriented graphene sheets with wrinkled texture. The obtained hydrogels had smaller pore sizes, because of remarkable shrinkage in the volume of GO dispersion during hydrothermal reduction process. The poor microscopic structure of GA-F Fig.  was main26. They both circumvent the destructive differential capillary stresses of conventional evaporative drying either by transferring the solvent into its supercritical state or by replacing the solvent mainly with supercritical CO2, thus suppressing any liquid-vapor interface inside the sol-gel product27. The usually used ethanol was chosen as supercritical drying solvent in our experiments. When ethanol in autoclave was forced to a temperature and pressure above its critical point 28, it transformed into a supercritical fluid. Above the critical point, the various properties of supercritical ethanol are between those of gas and liquid. Although the density of supercritical fluid is similar to that of liquid, the viscosity is similar to that of gas, and its diffusivity is between the two states29. Because the supercritical fluid has lower viscosity and higher diffusion velocity than liquid, it can easily diffuse through the pores of the gel, resulting in higher drying efficiency. Shrinkage of a gel during drying is driven by the capillary pressure, which can be reduced by reducing surface tension of the pore liquid, and supercritical drying eliminates surface tension and keeps the structure of original fine framework of the graphene gels from destroying to obtain aerogels30. Drying process impacted the microstructure of aerogels, and further affected the physical properties of aerogels, such as bulk density, mechanical property, thermal and electrical conductivity. The microscopic morphology of GA-S and GA-S-1500C graphene aerogels prepared with 1, 2, 5, and 10 mg∙mL−1 GO are shown in Fig. GO from 1 mg∙mL−1 to 10 mg∙mL−1 or after high-temperature annealing, the pore structure of graphene aerogels appeared to be much finer and ordered, so, the network architecture, especially those of GA-S10 and GA-S10-1500C, became much “stronger” to support greater external load. The scanning images of graphene aerogels prepared by supercritical drying and freezing drying demonstrated supercritical dried graphene aerogels had more fine, uniform and ordered microscopic morphology. Digital photographs of the GA-S and GA-S-1500C samples are shown in Figs Supercritical drying methods include high temperature supercritical drying from solvents such as methanol, ethanol and acetone, and low temperature supercritical drying from carbon dioxide3-type hysteresis loop at high relative pressure, which was typical characteristic for aggregates of plate-like particles and open large pores31. The total adsorption volumes and the size of the hysteresis loops of the GA-S samples decreased as CGO increased. Moreover, the pore size distribution of GA-S and GA-S-1500C calculated by the BJH method indicated a large proportion of mesopores in a narrow distribution from 3 nm to 5 nm with peak pore diameter of approximately 3.8 nm , pore volumes, and average pore diameters of GA-S and GA-S-1500C were shown in Table BET of the graphene aerogels were much lower than 2600 m2∙g−1 of the theoretical value for a single graphene sheet11 because of the layering or overlapping of graphene sheets8. This values apparently improved by reducing CGO or enhancing the exfoliation of graphene sheets during the self-assembling process6. The SBET and pore volume of GA-S1 at approximately 600 m2∙g−1 and 4.487 cm3∙g−1, respectively, decreased to corresponding values of 435.6 m2∙g−1 and 2.153 m3∙g−1 for GA-S10 as CGO increased. The dramatic decline in the surface area of the graphene aerogels at high CGO was attributed to the improved sites of partial overlapping or cross-linking of the flexible graphene sheets generated during the self-assembling process to form a 3D network with higher density. The healing or repairing of defects in the graphene sheet reduced the textural mesopores, accounting for the lower SBET of GA-S-1500C than GA-S. However, the SBET of GA-S-1500C increased from 336.5 m2∙g−1 with 1 mg∙mL−1 CGO to 440.8 m2∙g−1 with 10 mg∙mL−1 CGO, this trend was also broadly applicable to the desorption surface area cumulated by BJH method  was attributed to the presence of isolated double bonds that resonate at frequencies higher than that of the G-band of graphite32. The presence of G-bands for GA-F and GA-S at 1586 and 1590 cm−1, respectively, were ascribed to the recovery of the hexagonal network of carbon atoms with defects33. After hydrothermal reduction, the significant increase of ID/IG for GA-F and GA-S indicate a decrease in the average size but an increase in the number of sp2 domains upon reduction of the exfoliated GO32. The reduction effect obviously cannot be considered as the healing or repairing of defects in GO34. Annealing at 1500 °C resulted in the evident decrease in ID/IG, strongly suggesting the removal of a considerable number of defects in the graphene sheets. The intensity of the two-dimensional (2D) peak (2695 cm−1) for GA-S-1500C increased dramatically, revealing better graphitization of graphene sheets after annealing at 1500 °C35.The XRD patterns of GA-F, GA-S, and GA-S-1500C compared with those of GO and graphite are depicted in Fig. 36, C=O , and O–C=O 37 , C–O )37 Fig. . However)37 Fig.  only exh eV Fig.  correspo−3 obtained from purified hydrogel dried by supercritical ethanol. A 75.0 mg graphene aerogel cylinder could support a 2 kg counterpoise, at least 26000 times their own weight  were 5.13 and 0.25 MPa, respectively, whereas those of GA-F (ρ = 48.0 mg∙cm−3) with the same CGO were only 1.05 and 0.06 MPa, respectively 22 for GA-S increased from approximately 625 °C to nearly 705 °C after annealing. This improvement in thermal stability was ascribed to the decrease in the amount of oxygenated functional groups which improved the ability of graphene aerogels to tolerate higher temperature in the air. The GA-S and GA-S-1500C samples exhibited the same tendency in thermal conductivities at room temperature, which both improved with the increase of bulk density  were much higher than those of the graphene aerogels prepared by Tang et al.9 (1.8–27.2 mg∙cm−3), the thermal conductivities  for GA-S shown in Fig. −1∙K−1). This result could be ascribed to the finer pore structure of our graphene aerogels. The thermal conductivity of graphene aerogels can be as low as <0.01 W∙m−1·K−1, but bulk density and mechanical property was very low15. To indicate the thermal stability of graphene aerogels more visually, the GA-S-1500C graphene aerogel was tested in a hot flame of an alcohol burner  also enhanced the electrical conductivity of GA-S-1500C samples. Although, the GA-S10-1500C has higher electrical conductivity than recent reported graphene aerogels43, but lower than the highly crystalline graphene aerogels , hydrogen peroxide , and sodium hydrogen sulfite (NaHSO3) were purchased from Tianjin Bodi Chemical Co., LTD. . Potassium permanganate (KMnO4), concentrated sulfuric acid , and hydrochloric acid  were obtained from local suppliers .Graphite flakes  were used to prepare GO through a modified Hummers’ method. Sodium nitrate  hydrogels. The obtained hydrogels were first purified in a large amount of ethanol for at least 1 week and then dried using supercritical ethanol at 270 °C with a pressure of 8 MPa to obtain the graphene aerogels. The resulting samples were referred to as GA-S. The obtained GA-S samples were then heat treated at 1500 °C for 1 h under N2 atmosphere. The resulting samples were labeled as GA-S-1500C. The GA-S and GA-S-1500C prepared with various concentrations of GO (CGO) were denoted as GA-Sx and GA-Sx-1500C, where x is the concentration of GO dispersion in milligram per milliliter. In addition, we investigated the effect of the drying process by freeze-drying graphene aerogels with GO aqueous dispersion of 5 mg∙mL−1 using the same method as that for GA-S, but deionized water, instead of ethanol, was used to purify the hydrogels. The resulting sample were denoted as GA-F.GO was prepared from natural graphite using the improved Hummers methoders Fig. . The lar−1 under air atmosphere. The bulk electrical conductivities of cylindrical graphene aerogels were measured by four-probe method with metal electrodes attached to the ends of samples.Atomic force microscopy  in tapping mode at a scan rate of 0.999 Hz was performed to obtain AFM images of the GO sheets. The bulk densities of the resulting aerogels have been calculated by the mass of the graphene aerogel divided by its volume. The microscopic morphology of the graphene aerogel was investigated via a scanning electron microscope . Nitrogen adsorption–desorption measurements were performed using a Micromeritics 2020 ASAP instrument at 77 K. Surface areas were calculated using the BET (Brunauer-Emmett-Teller) method, and the pore size distribution was calculated by the Barrete-Joynere-Halenda (BJH) method. XPS (X-ray photoelectron spectroscopy) analysis of GO, GA-F, GA-S, and GA-S-1500C was performed on an ESCALAB 250Xi photoelectron spectrometer . X-ray diffraction (XRD) patterns of GO, graphene aerogels, and natural graphite were analyzed by a D/max-RB XRD . Raman spectra were measured by irradiation with laser light at 532 nm in a Renishaw spectrometer. Compression tests were conducted using an electronic universal material testing machine  with loading capacity of 1000 N at a constant loading speed of 2 mm/min. Thermal conductivity was measured by hot wire technique using a thermal conductivity measuring instrument . Thermal stability of the graphene aerogels was investigated by thermogravimetric analysis  with a heating rate of 15 °C∙minSupplementary Information


Sarcoidosis is a multisystemic granulomatous disease of unknown origin. Chronic cutaneous lupus erythematosus (CCLE) is an autoimmune disease that is associated with autoantibody production and T-cell dysfunction. Cutaneous manifestations of sarcoidosis may mimic CCLE and vice versa making it difficult to reach a diagnosis clinically.We present a case of a 57-year-old woman with long-standing sarcoidosis who presented to clinic with diffuse painful plaques that were very distinct and suggestive of CCLE. She had a family history of both sarcoidosis and CCLE. The patient was immediately started on topical corticosteroids and oral hydroxychloroquine. Skin biopsy and the absence of direct immunofluorescence confirmed a skin manifestation of her previously diagnosed sarcoidosis, despite the clinical morphology favoring classic CCLE.Sarcoidosis may have diverse manifestations and may mimic other disease processes. A detailed history along with a low threshold for biopsy is important for determining a diagnosis. Sarcoidosis is a multisystemic granulomatous disease of unknown origin. Cutaneous manifestations are present in 30 % of patients with sarcoidosis and may be the first presenting sign . On physA 57-year-old African-American female presented with hyperpigmented plaques on the face, scalp, arms, hands, buttocks, legs, and feet. She has had similar lesions over the past fifteen years and described them as burning, itchy, and painful.There were dermal papules on the perinasal and periocular areas. The ears displayed plaques with adherent scale and follicular plugging in both conchal bowls and preauricular regions Fig. . The antShe had been diagnosed with systemic sarcoidosis twelve years prior as well as hypertension and congestive heart failure eight years earlier. Her medications included aspirin, metoprolol, and ibuprofen as needed for pain. The patient reported a history of lupus (unknown subtype) in her maternal aunt and sarcoidosis in her maternal sister and two uncles.Laboratory studies included complete blood count with differential, complete metabolic panel, brain natriuretic peptide, erythrocyte sedimentation rate (ESR), and angiotensin-converting-factor (ACE). The only abnormal values were an ESR of 41 mm/h (reference range 0-30 mm/h) and ACE of 73 nmol/mL/min (reference range 9–67 nmol/mL/min).Given the patient’s history of long-standing lesions; photodistribution; and the presentation of plaques with dyschromia, atrophy, follicular plugging, and raised borders, a diagnosis of CCLE was favored. While the patient had a past medical history of sarcoidosis, her current lesions appeared classic for CCLE and both conditions were present in her family history. Given the information and clinical presentation, a biopsy proved necessary as a purely clinical diagnosis was not adequate.Two punch biopsies were taken from a plaque on the left arm and sent for histopathology and direct immunofluorescence. Pathology exhibited non-caseating granulomas Figs.  and 5 wiOur patient represents a case of sarcoidosis with skin manifestations that closely imitate those of CCLE. The lesions seen in sarcoidosis are divided into specific and nonspecific subgroups based on histologic findings. Specific lesions occur in 9-15 % of patients and exhibit noncaseating granulomas . The mosIn sarcoidosis, noncaseating granulomas can be found in tissues and organs such as the skin, lungs, and lymph nodes. These granulomas consist of aggregates of epithelioid histiocytes, giant cells, and mature macrophages . They arCutaneous manifestations of CCLE include erythematous to violaceous scaly plaques with prominent follicular plugging that often results in scarring and atrophy. They are found most commonly on the face, scalp, ears, and less commonly on the mucosal surfaces, conjunctivae, and genital mucosa. Plaques usually appear in a photodistributed pattern, although not always necessary. Skin morphology includes dyspigmentation in longstanding lesions with hypopigmentation in the central region and hyperpigmentation at the periphery . HypertrHistologically, CCLE plaques are found to have inflammatory infiltrates in the superficial and deep dermis as well as the surrounding adnexal structures. Keratinocyte damage and hyperkeratosis are present in the epidermis with possible prominence of amorphous and eosinophilic colloid bodies in the epidermis and/or upper dermis. Melanin deposits within macrophages are located in the dermis. Lymphohistiocytic cellular infiltrates that are often pronounced are located in the dermis at perivascular and periadnexal locations. If histopathology is equivocal for CCLE, direct immunofluorescence may be helpful. In cutaneous lupus, antibody deposition is seen at the dermal-epidermal junction and around hair follicles . While mWithout a biopsy, one might be quick to diagnose this individual with known sarcoidosis with superimposed CCLE based on the clinical morphology. Her plaques had all the characteristic cutaneous findings including dyschromia, atrophy, adherent scales, central hypopigmentation, and hyperpigmentation at the periphery. However, histology did not show follicular plugging or lymphohistiocytic cellular infiltrates. Immunofluorescence studies were also negative. Histology was pathognomonic for sarcoidosis exhibiting non-caseating granulomas, giant cells, and a lymphocytic infiltrate. In the end, strong clinical inclination was not enough and a biopsy was the necessary factor in determining a definitive diagnosis.


Gate modulation of the carrier density is still visible at 150 K, which is lower than the glass transition temperature of most liquid gating electrolytes.We have developed a technique to tune the carrier density in graphene using a lithium-ion-based solid electrolyte. We demonstrate that the solid electrolyte can be used as both a substrate to support graphene and a back gate. It can induce a change in the carrier density as large as 1 × 10 The liquid gating technique has been successfully applied in the study of novel field-effect transistors457891012131415162 thin film can be used to make a proton gate18202114 cm−2, which is much larger than that induced by oxide dielectrics and comparable with that induced by liquid electrolytes. Additionally, the LICGC solid electrolyte can tune the carrier density in graphene samples at temperatures as low as 150 K, which is much lower than the glass transition temperature of most ionic liquid/polymer electrolytes.The electronic properties of many materials strongly depend on their carrier density. In recent years, a technique called “liquid gating” has been widely used in materials research and development of flexible electronics123 solution in deionized water. The floating graphene/PMMA layer was then transferred onto the LICGC substrate, and the PMMA layer was removed with acetone. The samples were then annealed in a hydrogen–argon atmosphere for 7 h. The electrical contacts to graphene were made from indium pellets. The few-layer graphene samples were mechanically exfoliated from highly oriented pyrolytic graphite onto the LICGC substrate using the well-known tape method. The exfoliated few-layer graphene were typically tens of micrometers in size. The standard e-beam lithography technique was used to pattern Ti/Au (5 nm/55 nm) microelectrodes. Single-layer graphene samples were initially grown on copper substrates by the chemical vapour deposition (CVD) technique. The growth details can be found in our previous paper2 substrate. The geometrical capacitance of the EDL is given by Cg = εseε0/d, where εse, ε0, and d are the relative permittivity of the solid electrolyte, the vacuum permittivity, and the spacing of the EDL. Because Cg is inversely proportional to the spacing between the double layer, a large surface roughness may increase the local spacing of the EDL and thus reduce its geometrical capacitance. As we will show below, although not atomically flat, the LICGC substrate can create a considerable gating effect. Solid electrolytes with smaller surface roughness are always preferred. A solid electrolyte with an atomically flat surface is ideal to form a homogenous EDL, and also crucial for direct thin-film growth on solid electrolyte substrates using advanced thin-film growth technique such as molecular beam epitaxy (MBE), because it can give a clean and lattice-matched  interface between the solid electrolyte substrate and the thin-film material.We used an atomic force microscope (AFM) to investigate the surface roughness of the LICGC substrate. Gr, and RSE are the resistance of the graphene sample and the LICGC substrate. Ri is the interfacial resistance between the LICGC substrate and the silver paint, estimated to be greater than hundreds of megohms. By contrast, the resistance between the gold electrode and the liquid electrolyte, estimated from our own experiences, is in the megohm range. The ionic conductivity of LICGC is approximately 0.1 mS cm−1 at room temperature, which is also one order of magnitude lower than that of DEME-BF4 (a widely used ionic liquid)−1). As shown in We first demonstrate the gating effect of the solid electrolyte on single-layer graphene. R) versus gate voltage (Vg) curves. Similar resistance discontinuity has been observed in liquid gating experiments13 cm−2 in the wet transferred single-layer graphene sample, and approximately 1.0 × 1014 cm−2 in the exfoliated few-layer graphene sample. The observed gate tuning capability of LICGC on graphene is similar to the reported value for EMIM-BF4 (1.0 × 1014 cm−2), and slightly smaller than that reported for DEME-TFSI (2.5 × 1014 cm−2); which are both commonly used ionic liquid gating electrolytesTo quantize the gate tuning capability of the solid electrolyte, we measured the carrier density change for a series of single- and few-layer graphene samples. The single-layer graphene covered a large portion of the LICGC substrate. The measurement for these samples gave an average gating effect of the solid electrolyte. The few-layer graphene samples had sizes on the micrometer scale. The gating effect on these small samples was more affected by the local surface roughness of the LICGC substrates. In both situations, we found that the LICGC substrate can realize considerable carrier density tuning. R versus Vg curves for five macroscopic single-layer graphene samples. These samples were cut from neighbouring areas on a large graphene sheet grown on the copper substrate. In these samples, the initial positions of the Dirac point should be similar. Gate modulation of the resistance is visible in all of the samples except for the one measured at 100 K. This suggests that the Li ions are still displaceable at a temperature as low as 150 K, which is lower than the glass transition temperature of most known ionic liquids. We also found that the resistance peak shifted towards higher gate voltage as the temperature decreased, and the width of the resistance peaks became wider. This behaviour can be attributed to two possible reasons. First, because the gate tuning of the carrier density in graphene can be viewed as a charging/discharging process of the EDL capacitor, the RC time constant determines how fast the carrier density change occurs. The ionic conductivity of solid electrolytes follows Arrhenius law and decayed exponentially with temperature, which leads to increased resistance of the solid electrolyte. The interfacial resistance between LICGC substrate and the silver paint can also become greater at low temperature. The longer RC time constant leads to slower carrier density modulation. Second, at low temperatures, a high gate voltage is required to overcome the energy barrier for Li ions to approach the LICGC–graphene interface. Assuming that only the RC time constant affects the gate tuning behaviour, we expect that all of the resistance peaks should appear below a common voltage threshold after a sufficiently long time. However, we found the opposite case in the experiments: at low temperature, the resistance peak did not appear even after a long time unless we moved to a higher gate voltage. We thus believe that the combination of both effects results in the shift and widening of the graphene resistance peak. Detailed experimental evidence is given in the We investigated how the performance of the LICGC solid electrolyte varies with temperature. All liquid electrolytes have a specific glass-transition temperature, below which ion movement in the electrolytes is frozen and the electrostatic gate tuning ability is lost. To make a comparison with the liquid gating technique, we carried out back gate voltage sweeping experiments at various temperatures. The results described above prove the concept of using solid electrolytes to tune the carrier density in thin-film samples. Because solid electrolytes are widely used in the research of Li-ion batteries and fuel cells, there is a long list of available materials that can be used to perform electrostatic gating. In the following paragraph, we will discuss the criteria for selecting a solid electrolyte for electrostatic gating.σ can be expressed as σ = nZeμ, where n is the number of ions per unit volume, Ze is the charge of the ions, and μ is the ion mobility. As previously mentioned, the mobility follows the Arrhenius law and it exponentially decreases with decreasing temperature. Most solid electrolytes used in Li-ion battery research are good candidates for room temperature electrostatic gating, while the solid oxide electrolytes used in fuel cells usually only work at high temperatures. The LICGC substrate used in this study has an ionic conductivity of the order of 10−4 S cm−1 at room temperature. Other electrolytes containing Li ions can reach of the order of 10−3 S cm−1. By contrast, the ionic conductivity of organic liquid electrolytes is generally in the order of 10−2 S cm−1. Materials with higher ionic conductivity are always favorable. Recently, new lithium solid electrolytes with high ionic conductivities have been reported, such as Li10GeP2S12, which exhibits ionic conductivity higher than 10−2 S cm−1 at room temperature. These materials are worthy of further investigation.It is essential to reduce the serial resistance in the RC circuit model to get a smaller RC time constant so that the carrier density change in the sample can keep up with the applied gate voltage. The interfacial resistance between the solid electrolyte and the gate electrode should be reduced, and the solid electrolytes with higher ionic conductivity are highly desired. The interfacial resistance is an active research field in Li-ion battery, which depends strongly on the material choices of solid electrolytes and cathode/anode electrodesThe second factor that needs to be taken into account is the surface roughness. As solid electrolytes are used as the substrate to support thin-film samples, a flat substrate surface is required. LICGC is one of the few commercially available solid electrolytes with a polished surface. To further improve this technique, a solid electrolyte with an atomically flat surface is highly desired. Such solid electrolytes may form a homogeneous EDL with large geometrical capacitance, which can enhance the electrostatic gating effect. Furthermore, a solid electrolyte with an atomically flat surface is an ideal substrate for advanced thin-film growth techniques such as MBE. It can create a much cleaner interface between the solid electrolyte and the thin-film sample compared with the interface made by transferring thin-film samples to the surface of a solid electrolyte. Although most of the solid electrolytes developed for energy storage research are in ceramic or polycrystalline forms, there are some single-crystal solid electrolytes, such as potassium-doped β-alumina. This will be important to advance the solid electrolyte gating technique.2Te3 gated with an ionic liquidSome other factors should be considered. The chemical stability under ambient conditions is important for practical applications. LICGC is a good material in this sense because it is not sensitive to humidity, unlike many ionic liquids. Solid electrolytes with high electrochemical decomposition potentials are also preferable so that large gate voltages can be applied. Using LICGC as the back gate, the gate voltage is limited between −2 and 3.5 V. We also attempted to apply higher gate voltages. As shown in the In summary, we have discussed the advantages of the LICGC solid electrolyte gating technique over the existing liquid gating technique. The LICGC solid electrolyte can serve as both the substrate to support the sample and the back gate. It can achieve a similar carrier density tuning ability to liquid electrolytes, while maintaining an uncovered sample surface. Therefore, it provides the possibility of combining the electrostatic carrier density tuning technique with other surface-probe techniques. The combination of these techniques will be very useful, for example, to monitor evolution of the Fermi level by continuously tuning the carrier density in superconducting materials. LICGC substrates provide a strong and full solid-state gate tuning capability, making them more suitable for practical applications than liquid electrolytes. A sample fabricated on top of a LICGC substrate will also not suffer from problems that may be encountered in liquid gating, such as damage caused by the stress created during the freezing of the liquid electrolyte. All of these advantages make solid electrolyte gating a promising technique for future applications.How to cite this article: Zhao, J. et al. Lithium-ion-based solid electrolyte tuning of the carrier density in graphene. Sci. Rep.6, 34816; doi: 10.1038/srep34816 (2016).