Immune checkpoint inhibitors (CPIs) are associated with a number of immune-related adverse events and low response rates. We provide preclinical evidence for use of a retroviral replicating vector (RRV) selective to cancer cells, to deliver CPI agents that may circumvent such issues and increase efficacy. An RRV, RRV-scFv-PDL1, encoding a secreted single chain variable fragment targeting PD-L1 can effectively compete with PD-1 for PD-L1 occupancy. Cell binding assays showed trans-binding activity on 100% of cells in culture when infection was limited to 5% RRV-scFv-PDL1 infected tumor cells. Further, the ability of scFv PD-L1 to rescue PD-1/PD-L1 mediated immune suppression was demonstrated in a co-culture system consisting of human-derived immune cells and further demonstrated in several syngeneic mouse models including an intracranial tumor model. These tumor models showed that tumors infected with RRV-scFv-PD-L1 conferred robust and durable immune-mediated anti-tumor activity comparable or superior to systemically administered anti-PD-1 or anti PD-L1 monoclonal antibodies. Importantly, the nominal level of scFv-PD-L1 detected in serum is ∼50–150 fold less than reported for systemically administered therapeutic antibodies targeting immune checkpoints. These results support the concept that RRV-scFv-PDL1 CPI strategy may provide an improved safety and efficacy profile compared to systemic monoclonal antibodies of currently approved therapies. A retroviral replicating vector (RRV) platform based on an amphotropic gamma retrovirus that preferentially infects and replicates in tumor cells has been developed to address historical challenges with viral-based treatments for cancer. One example of the RRV approach is Toca 511, (vocimagene amiretrorepvec), an RRV encoding optimized cytosine deaminase derived from yeast) used in combination with an oral prodrug, Toca FC (extended-release 5-fluorocytosine) which has demonstrated durable, complete responses and a favorable safety profile in a Phase 1 trial , 3. FurtImmune checkpoint inhibitors (CPI) such as therapeutic antibodies targeting cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed death-1 (PD-1) and its ligand programmed death-1 ligand 1 (PD-L1) have represented a breakthrough for immunotherapies in treating cancer patients and have specifically made a significant impact in the fight against melanoma, lung, bladder head and neck cancer as well as in Hodgkin lymphoma . HoweverPD-1 is one of the major inhibitory receptors highly expressed on tumor infiltrating lymphocytes where upon its ligation with PD-L1 reduces cytokine production and T-cell proliferation resulting in immune escape from an active anti-tumor T-cell response in the TME . In addiin vitro. This activity was further supported by data from multiple syngeneic mouse models showing that tumors infected with RRV-scFv-PDL1 conferred robust and durable immune-mediated anti-tumor activity comparable or superior to systemically administered anti-PD1 and anti-PD-L1 monoclonal antibodies. Most importantly, our data show that only nominal level of scFv-PD-L1 was found in the circulation in mouse models where significant efficacy was observed. These results support the concept of an RRV-based platform for delivery of an immune CPI for an improved therapeutic window compared to systemic monoclonal antibodies currently approved for clinical use in many cancer types.We have previously reported a new RRV configuration utilizing the “self-cleavage” 2A peptide (RRV-2A) as an alternative of the internal ribosomal entry sequence (IRES) described in Toca 511 , 22. TheWe have previously reported a new RRV configuration utilizing the viral-derived “self-cleavage” 2A peptide for transgene expression  and demoWe have also previously shown that transgenes targeted for different cellular compartments encoded in-frame with the viral envelope (Env) protein in the RRV-2A configuration, are efficiently separated from Env-transgene polyprotein . BecauseWe next evaluated the viral function of both epitope tagged (HA and Flag) and untagged RRV-scFv-PDL1 and RRV-scFvFc-PDL1 vectors, as proper viral Env processing is an important determinant for viral replication . We firsAnother key factor which could influence the therapeutic effect of the transgene is vector stability. We have previously showed that the RRV-2A can tolerate a large transgene(s) insertion up to 1.2 kb . In the D) of scFv PD-L1 for recombinant human PD-L1 and mouse PD-L1 were determined to be 0.426 nM and 4.78 nM, respectively,  followed by anti-HA antibody for detection. The data show that scFvHF PD-L1 and scFvFc-HF PD-L1 bind to PD-L1 expressed on cell surfaces in human and mouse cell lines as indicated by a marked shift in mean fluorescent intensity (MFI) with an anti-HA antibody  and preliminary data demonstrated a failure of anti-PD-1 treatment as a monotherapy to prolong survival  , 31. Desin vitro . In thisin vitro . Furtherin vitro . Moreovein vitro . TogetheSince RRV-scFv-PDL1 is delivered as a virus rather than a therapeutic antibody and that scFv PD-L1 is produced from the infected tumor cells, scFv PD-L1 serum level is anticipated to be significantly lower than the levels reported for therapeutic antibodies delivered intravenously. Given the possibility that scFv PD-L1 may distribute into circulation during tumor lysis, we collected serum from tumor-bearing mice in both subcutaneous and intracranial models that demonstrated anti-tumor activity and measured scFv PD-L1 using a surface plasma resonance method. As expected, our data reveal that the scFv PD-L1 serum concentrations from tumor-bearing mice expressing scFv PD-L1 were comparable to that of negative control groups in both models Figure . AlthougCheckpoint inhibitor blockade has proven to be a promising treatment across many cancer types. However, they are not always effective and are associated with systemic immune toxicities which can lead to discontinuation of treatment. In this study, as a proof of principle, we have demonstrated that PD-1/PD-L1 mediated immune suppression can be rescued by RRV delivery of a secreted scFv targeting PD-L1 in tumors and immune infiltrating cells in the TME resulting in increased survival and a protective anti-tumor memory response associated with minimal serum exposure of scFv PD-L1. Most importantly, in contrast to oncolytic virus, the anti-tumor activity observed in tumor-bearing mice treated with RRV-scFv-PDL1 in our studies is not associated with or primed by innate antiviral immunity as shown by 100% RRV-GFP infected tumor’s growth kinetics (included as one of the control groups rather than naïve tumors).http://ir.cytomx.com/news-releases/news-release-details/cytomx-therapeutics-presents-preliminary-clinical-proof-concept). However and notably, grade 3/4 immune-related toxicity was still identified in some patients.The advantages of using tumor-selective RRV for delivering scFv PD-L1 in the TME are at least two fold. One is the localized and sustained delivery of therapeutic payload in the TME with relatively short half-life compared to recombinant therapeutic antibodies. These features allow for engagement of the checkpoint to promote anti-tumor activity in TMEs that may be difficult to reach with conventional systemic delivery of CPI monoclonal antibodies and consequent efficacy in otherwise poorly responsive tumors as well as avoidance of undesirable binding to healthy tissue leading to systemic immune toxicity . Anotherin vitro can fully occupy PD-L1 on bystander cells which translates to a high level of anti-tumor activity in several mouse tumor models. Also, RRV-scFv-PDL1 efficacy was evaluated in several syngeneic mouse tumor models in pre-transduced setting to evaluate dose effect and via intratumoral vector injection to mimic a potential clinical setting. A dose-dependent anti-tumor activity was observed in all three tumor models tested, indicating that 2% transduction level led to similar therapeutic effect as the anti-PD-1 antibody treated group and that a higher transduction level demonstrated anti-tumor activity that is superior than anti-PD-1 antibody group, suggesting the level of PD-L1 occupancy by anti-PD-1 antibody infusion may be suboptimal at the tumor site.We and others have previously demonstrated PD-1/PD-L1 blockade using the RNAi and aptamer approach –41. NeveD = 0.426 nM vs 0.77 µM) [1/2 = ∼77 min for scFv PD-L1/PD-1 vs. 0.9 sec for human PD-L1/PD-1) [Given that RRV has been demonstrated to be cancer-selective both in preclinical and clinical settings , 42, an 0.77 µM)  as well L1/PD-1)  may provet al., OncoImmunology, https://doi.org/10.1080/2162402X.2018.1501137), their tumorigenic property is not induced by carcinogen and thus are most likely to have low tumor mutational burden which closely resembles human GBM.Immunotherapy studies in brain tumor have been limited by the lack of representative murine models. The anti-tumor activity observed in the Tu-2449 syngeneic orthotopic glioma model brings a second model other than GL-261 reported to respond to checkpoint inhibitor as a monotherapy , 45, 46.RRV-scFv-PDL1 could potentially be applicable in a neoadjuvant setting prior to tumor resection; this type of use with nivolumab, was recently reported to induce expansion of antigen-specific clones in lung cancer . In addiIn conclusion, our study demonstrates that RRV-scFv-PDL1 checkpoint inhibition with a tumor-selective delivery and a high bystander index localized within the TME provides an attractive treatment regimen to target immune checkpoints.Two pairs of two different configurations of single-chain variable fragment (scFv) against PD-L1 were designed. One pair consists of scFv with and without the Fc from human IgG1, designated scFv-PDL1 and scFvFc-PDL1, respectively. The other pair consists of scFv-PDL1 and scFvFc-PDL1 with HA and Flag epitope incorporated at the C-terminus, designated scFv-HF-PDL1 and scFvFc-HF-PDL1. The coding sequence of each configuration contains the 3′ coding sequence of the viral envelope gene followed by the gT2A peptide sequence  and was Human 293T cells , human glioma cell line U87-MG , mouse colorectal cancer cell line CT26 , mouse breast cancer cell line EMT6 , mouse glioma cell line Tu-2449 [Jurkat T cells and Raji B cells overexpressing human PD-1 (Jurkat/PD-1) and PD-L1(Raji/PD-L1), respectively, were generated by transducing the cells with lentiviral vectors coding human PD-1 or PD-L1 at MOI of 1 followed by puromycin selection (1.6 μg/mL) 2 days post transduction. PD-1 expressing Jurkat T cells and PD-L1 expressing Raji B cells were enriched under puromycin selection for 14 days and confirmed by cells surface staining with anti-PD-1 and anti-PD-L1, respectively.Twenty micrograms of plasmid DNA encoding the RRVs were used for transient transfection using the calcium phosphate method as previously described . The virFor viral replication kinetics, 20 μL of collected supernatant from each time points during the course of infection were obtained to extract viral RNA and measure particle titer as described . For vecFor the assessment of the transgene protein expression, separation efficiency of the polyproteins, and viral envelop protein processing, cells lysates were prepared as described .EMT6 cells maximally infected with RRV-scFv-HF-PDL1 (HA-tagged scFv-PD-L1) or RRV-GFP at 90% confluency were treated with recombinant human IFNγ at 250 IU/mL for 24 hours to upregulate PD-L1 expression on the cell surface. One million of IFNγ-treated EMT6 cells maximally infected with RRVscFv-HF-PDL1 or RRV-GFP at indicated ratios were split into 2 sets. One set of cells was stained with Alexa Fluor 647-conjugated anti-HA antibody  and the second set of cells was stained with PE-conjugated anti-human PD-L1 antibody . HA-positive, PD-L1-positive, and GFP-positive cell populations were measured by flow cytometric analysis . HA-positive vs GFP positive cell populations and PD-L1-positive vs GFP-positive cell population were calculated by 2-color compensation for proper gating . Cytometry data analysis was performed using the FlowJo v10 software (FlowJo LCC).Wells in a 96-well microtiter plate were coated with 100 μL of 1.5 µg/mL recombinant human PD-L1-Fc  or mouse PD-L1-Fc  overnight at 4°C, followed by 2 hour blocking at room temperature with blocking buffer (1X PBS + 10%FBS + 0.05% Tween 20) the next day. Subsequently, PD-L1-Fc coated wells were co-incubated with 1 µg/mL His-tagged recombinant human PD-1  or His-tagged recombinant mouse PD-1  in competition with supernatant of undefined protein concentration collected from CT26 cells maximally infected with RRV-scFv-PDL1 or RRV-scFvFc-PDL1 , anti-PDL1 antibody  or purified scFv PDL1 protein (Accelagen Inc.) for 2 hours at room temperature. For interaction between scFv PD-L1 and human PD-L1, bound His-tagged recombinant human PD-1 was detected using an HRP-conjugated anti-6X His tag antibody . For binding interaction between scFv PD-L1 and mouse PD-L1, bound His-tagged recombinant mouse PD-1 was detected by performing an additional signal amplification step using an biotin-conjugated anti-6X His tag antibody ,  followed by HRP-conjugated streptavidin , . Color development was performed by adding 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution  and terminated by TMB STOP solution  after 6–10 minutes incubation. Optical density was read by a plate reader (Molecular Devices iD5) at 450 nm (with 570 nm correction) using the Soft Max Pro software. The percentage of inhibition was calculated with respect to a positive control which supernatant from CT26 maximally infected with RRV-GFP (non-scFv-PD-L1) was used in the competition.5 cells/0.5 mL. Raji B cells were washed and resuspended in serum-free RPMI-1640 medium at 1 × 106 cells/0.5 mL followed by addition of super antigen SEE , (Toxin Technology #ET404) or SEE plus anti-human PD-L1 or SEE plus purified scFv PD-L1 (10 μg/mL) followed by incubation at 37° C for 1 hour. At the completion of incubation, SEE loaded Raji B cells were washed in serum-free RPMI-1640 medium and resuspended in RPMI-1640 medium containing 20% human AB serum  at 5 × 105 cells/0.5 mL. Both rested Jurkat T cells and SEE-loaded Raji B cells were then co-cultured with equal volumes to reach 1 × 106 cells/mL/well in a non-coated 24-well plate and incubated at 37° C for stimulation.Jurkat T cells were washed and rested in serum-free RPMI-1640 medium at 37° C for 3 hours at 5 × 10For IL-2 ELISA, the supernatants of the co-culture cells were collected 20–22 hours after stimulation and separated from the cells by centrifugation at 300 × g for 5 minutes. IL-2 production was measured by using the Quantikine ELISA Kit  according to the manufacturer’s protocol.6 of Tu-2449SC cells maximally infected with RRVscFv-PDL1 or RRV-GFP at indicated ratios were subcutaneously implanted on the right flank of 8-week-old female B6C3F1 mice (Harlan Sprague Dawley Inc.) and assigned to 8 groups (n = 10 per group). Control Group 1 and 2 which are mice implanted with 100% Tu-2449SC/RRV-GFP cells received PBS or anti-PD-1 antibody. Anti-PD-1 antibody (Clone J43) was intraperitoneally (i. p.) administered on day 0 (300 µg per mouse), day 3, day 6 and day 9 (200 µg per mouse). Group 3, 4, and 5 received ratios of 3/97, 30/70 and 100/0 of Tu-2449SC/RRV-scFv-PDL1 and Tu-2449SC/RRV-GFP cells, respectively. Group 6, 7, and 8 received ratios of 3/97, 30/70 and 100/0 of Tu-2449SC/RRV-scFvFc-PDL1 and Tu-2449SC/RRV-GFP cells, respectively. Tumor sizes were monitored 3 times a week. Animals were removed from the study if tumors became necrotic or reached 2000 mm3 in volume. All mice that cleared their initial tumor implant from RRV-scFv-PDL1 or RRV-scFvFc-PDL1 treated groups (n = 15) and naïve group (n = 5) were challenged with 2 × 106 Tu-2449SC on the left side of the flank. Tumor growth and measurement were monitored and recorded over time.All animal protocols and experiments were approved by the IRB/IACUC of Explora BioLabs . For the Tu-2449SC subcutaneous tumor model, 2 × 10n = 10) were intracranially (i.c.) implanted with 1 × 104 of Tu-2449 cells. Survival analysis was monitored for approximately 90 days. Mice in the experimental groups were injected with purified RRV-scFv-PDL1 of 1 × 105 or 1 × 106 transduction unit (TU) on day 4 post tumor implant. Control groups are mice bearing 100% pre-transduced RRV-scFv-PDL1 tumor cells and Tu-2449SC tumor cells treated anti-PD-1 antibody  or isotype control. Survival data were plotted by the Kaplan–Meier method. Statistical significance of survival between mice treated with isotype and 100% pre-transduced scFv-PD-L1 or injection-treated RRV-scFv-PDL1 groups were determined by the Log-rank (Mantel-Cox) test using GraphPad Prism 7 . Mice which had survived from initial tumor implant from RRV scFv-PDL1 treated groups were challenged with 2 × 106 Tu-2449 cells on the right flank. Tumor growth and measurement were monitored and recorded over time.For the orthotopic glioma model, 8-week-old female B6C3F1 mice underwent surgical implantation of the tumor cells by Hamilton syringe. The stereotaxic coordinates were anteroposterior (AP), 0.5 mm; mediolateral (ML), 1.8 mm; and dorso-ventral (DV), 3.5 mm (from bregma). Mice in each group  and single-cell suspensions were obtained using gentleMACS™ C Tubes with gentleMACS™ Dissociator , and enzymatic solution containing 50 µg/mL final Liberase™ TM  and 200 U/mL final Deoxyribonuclease I . After filtration through a 70 µm cell strainer and washing, erythrocytes were removed following incubation with eBioscience 1X RBC Lysis Buffer . Viable cell counts were determined using a Cellometer K2 (Nexcelom Bioscience). For subsequent flow cytometry analyses, cells were first incubated with TruStain FcX antibody  and Zombie UV  before being subjected to surface staining with fluorochrome-conjugated antibodies against mouse CD45 , CD3ɛ , CD4  and CD8α . Acquisition was conducted on an LSRFortessa X-20 flow cytometer and analysis by using FlowJo software (version 10) with flowAI (1.7) plugin as described .20 (Tween 20), and 0.5 mg/mL BSA at 25° C. Purified scFv PD-L1 (>95% purity) at concentrations ranging between 0.01 200 nM (∼0.0028 µg/mL-5.6 µg/mL) was spiked into 1:20 (v/v) diluted naïve serum to generate a standard curve for every 10 regeneration cycles. Experimental samples were also prepared at 1:20 dilution and injected in at a flow rate of 5 µL/min for a 6-minute contact time. Raw data was analyzed in the Scrubber2 software (BioLogic Software). The lower limit of quantification of this assay is determined to be 0.011 µg/mL. The concentration of scFv PD-L1 level in serum was determined using the SoftMax Pro 5.45 software (Molecular Devices) with the standard curves plotted using a 4-parameter fit algorithm.Recombinant human PD-L1 (R&D Systems # 156-B7) was immobilized on the CM5 sensorchip surface standard amine coupling chemistry using a Biacore 3000 instrument. The running buffer comprised 25 mM HEPES, pH 7.5, 150 mM NaCl, 0.005% P


Semiconductor pn junctions are elementary building blocks of many electronic devices such as transistors, solar cells, photodetectors, and integrated circuits. Due to the absence of an energy bandgap and massless Dirac-like behaviour of charge carriers, graphene pn junction with electrical current rectification characteristics is hardly achieved. Here we show a graphene pn junction diode can be made exclusively from carbon materials by laminating two layers of positively and negatively charged graphene oxides. As the interdiffusion of oppositely charged mobile counterions, a built-in potential is created to rectify the current by changing the tunnelling probability of electrons across the junction. This graphene diode is semi-transparent, can perform simple logic operations, and since it has carbon nanotubes electrodes, we demonstrate an all carbon materials pn diode. We expect this graphene diode will expand material choices and provide functionalities (e.g. grafting recognition units on graphene oxides) beyond that of traditional semiconductor pn junctions. Chemically functionalized graphene oxide-based pn junction diodes have potential for future electronic device applications. Here, the authors report an all carbon pn diode with graphene oxide and carbon nanotubes electrodes showing excellent current rectification and efficient logic gates. In contrast to conventional semiconductors, its conduction and valence bands meet at Brillouin zone corners and, therefore, fabricating electronic components from this zero-gap semiconductor should find an unusual way11. Techniques such as cutting graphene into nanoribbons12, placing bias on bilayer graphenes13, and applying mechanical strains14 were developed to open a bandgap and turn off the current of graphene field-effect transistors. In addition, graphene diodes have received particular attention because diodes are the basic building blocks in a wide variety of electronic/photonic systems. Since graphene is metallic at a sufficiently large Fermi energy, Schottky diodes were fabricated by bringing metallic graphenes and semiconductor materials into contact16. With a gate modulation of localized energy levels, tunable light-emitting diode was demonstrated by using a semi-reduced graphene oxide17. Although pn junctions with a planar geometry could be realized by multiple electrostatic gating18, local chemical doping19, and engineering of substrates20, no current rectifying effect was observed owing to the Klein tunnelling of relativistic Dirac fermions21. In the vertical structure pn junctions, current rectifying was achieved by inserting/forming a thin layer of insulating or semiconducting barrier material between p and n graphene layers, showing behaviours of a typical resonant tunnelling diode23. So far, there is still lack of robust technique in making graphene diodes especially the diode as a similar structure to semiconductor pn junction. On the other hand, graphene oxide (GO) contains various reactive oxygen groups , which provide possibility for chemical modification and produce functional materials for a variety of applications25.Graphene, an atomically-thin hexagonal lattice, has currently attracted enormous research interest because of its great promising use for a wide range including electronic devicesHere, the graphene pn junction diode was fabricated by laminating two layers of such chemically functionalized positively (p) and negatively (n) charged graphene oxides on the transparent substrates. As the contact of two layers, mobile counterions diffused across the interface while the anchored charges on graphene oxides frame were immobile. An equilibrium condition was reached in which an internal electric field was established to tune the electron tunnelling probability through the junction. The coupled movement of ions and electrons as well as the current rectification were modelled by using a combination of Nernst–Planck diffusion and Poisson’s equations. Since carbon nanotubes were used as conductive electrodes, we demonstrated an all carbon materials pn diode. To evaluate the utility of these graphene diodes to build the processing units needed for simple computation, in combination with graphene resistors, all carbon materials AND and OR logic gates were constructed.3)4N+OH−, TMAH, pH = 10), while the positively charged GO was synthesized via a two-step procedure27  and O–H (3415 cm−1) stretching peak were greatly suppressed while amide-I band, amide-II band, and amide-III band appeared at 1631 cm−1, 1460 cm−1, and 1384 cm−1, respectively, which were attributed to C=O stretching vibrations in RNH–CO and the coupling of C–N stretching vibration to N–H bending vibration  were seen while these peaks were not observed prior to positive modification  and charged GOs solutions were successively filtered through a mixed cellulose ester membrane . (Note: 1) the CNT electrodes were also chemically functionalized by the similar procedures to the GOs (see Methods) to eliminate possible dipole formation at the interface between CNT and GO layers; (2) the conductivity of charged 20 nm-thick CNT electrodes was over 10 the CNT I–V) characteristics of the graphene pn junction diode was shown in Fig. A typical current–voltage 4N+, see Fig. In the GO layers, the charges anchored onto the GO frames are jammed and restricted to migration, but the counterions (Cl29). This model combined Nernst–Planck diffusion and Poisson’s equations, in which both counterions and electrons could diffuse in response to concentration gradients and migrate due to local electric fields. The interdiffusion of counterions  characteristics cs Figs. .Fig. 3MoTo demonstrate the utility of the graphene pn junction diode to build the processing units needed for simple computation, AND and OR logic gates were constructed Fig. . For theIt must be noted that the cutting-off capability of the AND gate  the interdiffusion happened at the mixing zone rather than the depletion region in semiconductor pn diode, (2) the formation of internal electric field was entropically driven, unlike in semiconductor pn junctions where the voltage arose from equilibration of the electron chemical potential, (3) the internal electric field of graphene diode increased the probability of electron tunnelling/hopping from n to p layer, in contrast to a semiconductor pn diode where the band alignment allowed electrons to flow from n type region to p type region, but not in the reverse direction, and (4) the interdiffusion-created electric field was pointing from p to n layer while in the depletion region of a semiconductor pn diode, this field was reversed from n to p region. On the other hand, diodes based on pure ionic charge transport32 are often labelled as P or N based on the sign of conducting charge carriers: mobile cations are given as P while conducting anions are denoted by N. If positive biases are applied to the P (GO(−) with positive counterions) side of the device—i.e., forward bias—high currents are ordinarily expected. In fact, this bias corresponds to low current in the present diode. Moreover, either the device structure or rectifying principle of the graphene pn junction diode is distinct from other graphene diodes such as Schottky diodes16, light-emitting diode17, and resonant tunnelling diodes23. These differences strongly suggest the use of charged GOs as the electron-carrying phase leads to a different mechanism for diode behaviour than those that have been reported previously. Based on Zeta potential measurements  diffuse/drift across the interface and at the equilibrium condition, a depletion region was created and an internal electric field is established pointing from n region to p region in the absence of an external applied biashowever,  the inteFinally, we wish to emphasize on the relatively low rectification ratio Fig.  of the g33, systems for recording and manipulating tissue behaviours34, and even smart self-powered devices with multi-functionalities35. Also, we wish to integrate functions of GOs  25 into our graphene circuit which is hard to achieve in semiconductor electronics.In summary, we have demonstrated a semi-transparent all carbon materials pn junction diode by laminating two layers of oppositely charged GO layers. This diode is distinct from previous devices where the current rectification happens because an internal field controls the tunnelling and/or hopping probability of conduction electrons. The observation of current rectification in graphene pn junctions greatly expands the class of materials and physical phenomena that can be used to build electronic components. Looking forward, we envision our graphene pn diode being inkjet-printed from aqueous carbon inks on flexible, transparent, and even stretchable substrates in a wide range of applications. These applications could be skin-like electronics to monitor the physiological conditions−1, Graphenea Inc.) under constant stirring. 5 min later, the mixture solution was heated to 35 °C and kept reacting for 3 h36. 63 mL DI water was then poured into the mixture. 15 min later, additional 200 mL DI water and 3.5 mL hydrogen peroxide (30 wt%) were added to stop the reaction. The yellowish product was thoroughly washed with 200 mL dilute hydrochloric acid (3.5 wt%) for 5 times and then re-dispersed in DI water . This further oxidization procedure produces GOs with more –COOH groups .In a typical synthesis, 35 mL sulphuric acid (98 wt%) and 2.60 g potassium permanganate were carefully added into an ice-bath-cooled flask with 50 mL commercially available GO solution , and 60 mg HATU (O–(7-Aza-1H-benzotriazol-1-yl)–N,N,N’,N’-tetramethyluronium hexafluorophosphate) were firstly mixed and reacted at 65 °C for 6 h in 60 mL N,N-Dimethylformamide (DMF). This step was repeated for three times to ensure the majority of –COOH groups were amidated. Next, 500 μL hydrochloric acid (36.5%) and 5.5 g 3-Chloro-1,2-propandiol (98 wt%) were added into an aqueous solution of above amidated GOs . The mixture was heated to 85 °C and kept reacting for 7 h under constantly stirring and refluxing. The grayish products were washed with DI water for 4 times and finally dispersed into DI water (0.25 mg mL−1).A two-step procedure was employed to synthesize positively charged GOs2SO4 and 3 M HNO3 with 3 to 1 volume ratio) and refluxed for 2 h at 80 °C under stirring. This step produces CNTs with more –COOH groups. The products were collected and washed with DI water for 5 times and then re-dispersed into DI water by the assistance of Sodium deoxycholate and sonication . To produce negatively charged CNTs, Tetramethylammonium hydroxide was only used (pH = 10). For the synthesis of positively charged CNTs, the same procedure with GO(+) was employed, while Hexadecyltrimethylammonium bromide (CTAB) was added to improve their dispersity.100 mg carboxyl functionalized CNTs powder  was added into 100 mL mixed acid solution (3 M H−1. 15 mL CNT(−) was firstly vacuum filtered through a mixed cellulose membrane  and thoroughly washed with 20 mL DI water to remove surfactants. 10 mL GO(−) was subsequently added. The MCE-carbons film was peeled off and cut into desired pieces (15 mm × 10 mm) when all the carbons were dried. The MCE was then removed by floating the film onto DMF solvent for approximately 120 min and the negatively charged GO layer was transferred onto the transparent glass slide (20 mm × 14 mm). The layer was finally dried by using nitrogen gas. This produces a layer of approximately 20 nm CNTs and approximately 40 nm GOs  for 1 h. Gradient centrifugation was then used to collect highly dispersed carbon solutions. The concentration of all carbon materials is approximately 0.03 mg mLThe structure of carbon materials was characterized by using scanning electron microscopy  and transmission electron microscopy . The composition of GOs was evaluated by using X-ray photoelectron spectroscopy  and Fourier-transform infra-red spectroscopy . Ultraviolet-visible spectroscopy  was used to evaluate the light transmission capability. Zeta potential is tested on Zetasizer Nano ZS. The surface roughness and thickness of carbon layers were measured by using Dektak XT Stylus Profiling System. The conductivity of carbon layers was characterized by a four-probe technique (MST 4145-B). The measurement of graphene pn diode was conducted under ambient condition with a computer-interfaced high precision electrometer (Keithley 6517). To drive logic circuits, an additional 6517 electrometer was applied.29. For simplicity, we considered one dimensional domain which contain four regions: CNT(−), GO(−), GO(+), and CNT(+). We assumed that the CNT electrode layer (either CNT(−) or CNT(+)) is equipotential37 and the electrons are free to move out or into the layers. The accumulation of mobile counterions in CNT layers was neglected. The flow of electrons as well as the rearrangement of ionic charges throughout the layers produced the electric current J. The dimensionless electric current density (L represents the thickness of GO(+) and GO(−) layers and ni and ne are the number density of mobile ions and conduction electrons respectively, Di and De are the ion and electron diffusivities, Zi is the valence of the mobile ions, e is the elementary charge, kB is the Boltzmann constant, T is the absolute temperature, u is the electrostatic potential, ε0 is the vacuum dielectric constant, and the ε is relative dielectric constant; Di = De = 1.0 × 10−9 cm2 s−1, ε = 4.5. The coupling Eqs.29 were then solved by using a commercial finite element solver (COMSOL 5.1).The graphene pn junction diode was simulated by using a continuum charge-transport model, which combined Nernst-Planck diffusion and Poisson’s equations. The details of this model was presented in referencesmentclass2pt{minimSupplementary Information


We aimed to determine the function of pyrroline-5-carboxylate reductase 1 (PYCR1) on progression of papillary renal cell carcinoma (PRCC) and related mechanism.The TCGA database provided us expression profiles of PYCR1 and overall survival rates. Small interfering RNA (siRNA) was used to knockdown PYCR1; quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were conducted to identify the expression levels of mRNA and protein. The cell counting kit-8 (CCK-8) and colony formation assays were used to explore cell viability in Ketr-3 cells. The migration and invasion of Ketr-3 cells were investigated by transwell assays.We found that PYCR1 was over-expressed in PRCC tissues and cells, causing poor outcomes. Moreover, reduction of PYCR1 played a negative role on cell proliferation, migration and invasion in tumor cells. The important Akt/mTOR pathway proteins, phosphorylated Akt (p-Akt) and phosphorylated mTOR (p-mTOR), also showed lower levels compared with control groups.These findings showed that disordered expression of PYCR1 could modulate PRCC progression through the Akt/mTOR pathway, implying a theoretical basis for PYCR1 as a potential therapeutic target in future clinical PRCC treatment. Complic3) genes . Consideet al. confirm a close relationship between PYCR1 expression and non-small-cell lung carcinoma (NSCLC) progression [PYCR1 plays an important role in proline biosynthesis and conversion of pyrroline-5-carboxylate (P5C) into proline also depends on the involvement of PYCR1 . Prolinegression . Additiogression , lung cagression , 16, andgression , 18.In the present investigation, we first aimed to characterize the function of PYCR1 in PRCC. The expression level of PYCR1 in tumor tissues and cells was assessed, and prognosis relying on its expression level also was analyzed using statistical methods. Cell behaviors were measured using vitro experiments and the underlying mechanism was explored by key protein detection. Taken together, these data may provide new mechanistic insights into the function of PYCR1 for PRCC treatment in future.22.12. Additionally, 10% fetal bovine serum (FBS) , 100 U/mL penicillin and 100 μg/mL streptomycin  were added to RPMI-1640 medium for cell growth.The American Type Culture Collection  provided us with cell lines including HRCE, 786-O, OS-RC-2, Ketr-3 and A498. RPMI-1640 medium  was used to culture these cell lines and environmental conditions were set at 37˚C and 5% CO2.2-ΔΔCt analysis and GAPDH was regarded as standard control.To determine the expression of PYCR1 in all cell lines, we isolated total RNA using Trizol . Then SuperScript III RNase H Reverse Transcriptase  was utilized to reverse transcribe total RNA into cDNA. PCR reactions were carried out on a Bio-Rad Connect Real-Time PCR machine with SYBR Green PCR Master Mix . The PCR procedure was run as follows: 95˚C for 5min, 95˚C for 30s with 40 cycles, 60˚C for 45s and 72˚C for 30min. The primer sequences: PYCR1: F, 5’-TGACCAA-CACTCCAGTCGTG-3’, R, 5’-GTCCAGCTTCACCTTGTCCA-3’; GAPDH: F, 5’-GGAGCGAGATCCCTCCAAAAT-3’, R, 5’-GGCT-GTTGTCATACTTCTCATGG-3’. The relative expression of PYCR1 was assessed by 22.3Lipofectamine2000 transfection Kit  was used to inject si-PYCR1 into cells according to the manufacturer’s instructions. In order to control the unique variables and nonspecific interference, si-con without target was used as internal control. The sequences were as follows: PYCR1 siRNA 1 : F: 5’-CTTCATCCTGGATGAAAT-3’, R: 5’-GAAG-TAGGACCTACTTTA-3’; siRNA 2 : F: 5’-TGCTCATCAACGCT-GTGG-3’, R: 5’-ACGAGTAGTTGCGACACC-3’; si-con: F: 5’-AATTCTCCGAACGTGTCACGT -3’, R: 5’-TTAAGAGGCTTG-CACAGTGCA-3’. Primers were synthesized by GenePharma Co., Ltd .2.4CCK-8 kit  was used to investigate cell vitality. Transfected cells were placed into 96-well plates with 1,000 cells per well and incubated in a carbon dioxide incubator. 10 μL CCK-8 regent was added before cell activity was examined at 0 h, 24 h, 48 h and 72 h. Subsequently, cells were cultured at 37˚C for 1.5 h and then the OD values were read under a microscope at 450 nm.2.52. After washing with 1×PBS, cells were fixed and stained with 4% paraformaldehyde and 0.1% crystal violet respectively. Finally, the clones were counted by naked eye.Colony formation assays were used when the cells entered the log phase and these cells were trypsinized to make into suspensions. Cell suspensions were put into 60 mm dishs which contained 5 mL warmed medium at a density of 1,000 cells/dish and maintained for two weeks until clones appeared on the culture dish at 37˚C with 5% CO2.6Transwell assays were performed to explore cell migration and invasion. For cell invasion, Matrigel was diluted with medium six times after overnight thawing and then put into the upper chamber. Cells (1×105/well) were seeded into the upper chamber pre-coated with Matrigel and to the bottom chamber 500 μL complete medium was added . After 24 h incubation, cells that did not invade were removed by cotton swabs while invasive cells were fixed using 4% paraformaldehyde for 30 min, stained by 0.1% crystal violet for 20min. Next, PBS was used to wash off the floating color and the status was imaged under an optical microscope. Cells were calculated in several random fields. For cell migration, the density was 5, 000 cells per well and the chamber without Matrigel was utilized. The experiment was repeated at least three times and all the results were present as mean ± standard deviation (SD).2.7Proteins were extracted from transfected cells with RIPA buffer and quantified using a BCA kit . Each tank was filled with 20 μg proteins and then 12% SDS-PAGE was used to separate proteins. When proteins were transferred onto PVDF membrane, 5% non-fat milk was utilized to block these membranes for 1 h. Subsequently, PVDF membranes were incubated with primary antibodies  against Akt, p-Akt, mTOR, p-mTOR and GAPDH overnight at 4˚C. After washing with 1×TBST three times, PVDF membranes were cultured with secondary antibodies . Protein images were captured through ECL and the relative expression of protein bands were measured by Quantity One software.2.8Data analyses were performed with SPSS22.0 and Graph-Pad Prism version 5.0. Student’s t-test was conducted to compare the difference in two samples and one-way ANOVA with Dunnett’s post hoc test utilizing in diverse groups. Pearson’s analyses were applied to reveal the association between PYCR1 and PYCR2 or PYCR3. Significant statistical differences were defined as P < 0.05. All these results were expressed as the mean ± standard deviation (SD) in at least three replications. Ethical approval: The conducted research is not related to either human or animal use.33.1To determine PYCR1 expression level and confirm association between PYCR1 expression level and papillary renal cell carcinoma, we first analyzed the expression level of PYCR1 in PRCC tissues and corresponding tumor cell lines. Data about PYCR1 expression in PRCC tissue was downloaded from TCGA database and revealed that compared to normal tissues, PYCR1 showed a higher level in the respect of mRNA (3.2In order to explain the role of PYCR1 accurately, we knocked down PYCR1 using siRNA in Ketr-3 cells. The effective reduction was exhibited at both the mRNA level and protein level, especially si-PYCR1-2 (3.3Next, transwell assays were implemented to explore cell migration and invasion in Ketr-3 cells after PYCR1 knockdown. The experimental results demonstrated that deficiency of PYCR1 inhibited Ketr-3 cell activity in the aspect of invasion while there was no significant difference in the control group . Meanwhi3.4Considering the function of PYCR1 knockdown on PRCC cells, we further studied the underlying molecular mechanism involved in PRCC. We found that the introduction of si-PYCR1 dramatically hindered p-Akt and p-mTOR protein expression in Ketr-3 cells when compared to the si-con group, but that Akt and mTOR implied no difference (4We discovered that PYCR1 was over-expressed in human PRCC tissues compared with controls. Highly regulated PYCR1 expression led to poor prognosis in PRCC patients. All the data suggest that PYCR1 could promote the tumorigenesis of PRCC in the view of cell proliferation, migration and invasion.et al. searched for different genes that were consistent in nineteen cancers and found that PYCR1 is proposed as a novel enzyme [Previously, metabolism is required in tumors and mediates cellular processes, such as proliferation, redox homeostasis and reprogramming . Recent l enzyme . In addil enzyme , 25. Redl enzyme . Moreovel enzyme , 28.in vitro and found that PYCR1 could accelerate the progression of PRCC. There is a study about skin cancer that identified that PYCR2 was abundantly present in melanoma cells and regulated the Akt/mTOR pathway [We performed experiments  pathway . Epiderm pathway , 31.In order to determine how PYCR1 regulates the physiological processes of tumor cells, we first examined the effect of silencing PYCR1 on the Akt/mTOR pathway. The results of western blotting showed p-Akt and p-mTOR were impaired owing to knockdown PYCR1. On the contrary, the normal control exhibited no difference. However, here we did a preliminary investigation about the relationship between PYCR1 and PRCC. To thoroughly understand the underlying mechanism, a series of in-depth studies including in vivo experiments need to be followed up.In total, all the data in this present study confirm that PYCR1 was highly regulated in PRCC and its over-expression could induce poor prognosis in patients with PRCC. Based on the results of the silencing tests, PYCR1 stimulated cell behaviors including proliferation, migration and invasion. The vitality of colony formation was also inhibited because of silencing PYCR1. The key proteins, phosphorylated Akt and mTOR, revealed a downtrend at the level of protein expression level. We predict that PYCR1 might be an independent risk element for patients prognosis in PRCC via regulating Akt/mTOR pathway.


Supercapacitors are a highly promising class of energy storage devices due to their high power density and long life cycle. Conducting polymers (CPs) and organic molecules are potential candidates for improving supercapacitor electrodes due to their low cost, large specific pseudocapacitance and facile synthesis methods. Graphene, with its unique two-dimensional structure, shows high electrical conductivity, large specific surface area and outstanding mechanical properties, which makes it an excellent material for lithium ion batteries, fuel cells and supercapacitors. The combination of CPs and graphene as electrode material is expected to boost the properties of supercapacitors. In this review, we summarize recent reports on three different CP/graphene composites as electrode materials for supercapacitors, discussing synthesis and electrochemical performance. Novel flexible and wearable devices based on CP/graphene composites are introduced and discussed, with an eye to recent developments and challenges for future research directions. The continued use of fossil fuels, combined with limited reserves, has resulted in two serious and urgent issues to be addressed in our daily lives; environmental pollution and concern around continued energy supply. To address these problems, scientists focus on looking for clean and renewable energy sources such as solar energy and wind energy. However, these renewable energy sources are difficult to store, meaning that the development of novel, efficient, and low cost energy storage technology is very significant. In recent years, fuel cells, ionic batteries and electrochemical supercapacitors have been investigated ,2,3. ComBased on the different mechanisms of energy storage, supercapacitors can be broadly classified into two types: electric double layer capacitors (EDLCs) and pseudocapacitive enhanced devices, sometimes referred to as pseudocapacitors ,8,9,10. 2, NiOx, etc.) are typical active materials for pseudocapacitors  and iron (III) chloride (FeCl3), or sodium persulfate (Na2S2O8) and iron (III) sulfate [Fe2(SO4)3] are added as oxidants to catalyze the polymerization . . 113]. F2 hybridization of carbon atoms in the graphene, so it may possess a better conductivity [As mentioned above, most organic molecules can be used to functionalize graphene by non-covalent or covalent methods. Each has its own advantages; the covalent approaches may be easy to control and are stable, but in non-covalent methods organic molecules adsorb on the surface of graphene by π–π stacking interactions . When couctivity . FurtherIn recent years many flexible energy storage devices have been investigated and designed by researchers for applications in flexible displays, smart electronics, medical devices and other flexible devices. Given the advantage of being highly integratable and potentially even wearable, flexible energy storage architechture is considered to be one of the most prominent emerging disruptive-technologies that may completely transform our lives ,123,124.Typical flexible CP/graphene supercapacitors not only have excellent electrochemical properties, but also exhibit favorable mechanical performance. There are five commonly explored methods for fabricating flexible composite electrodes; electrodeposition, in situ polymerization, direct coating, chemical vapor deposition (CVD), and the vacuum filtration technique . −2 at 0.5 A g−1 in 1 M H2SO4. Because of the high electrical conductivity and good chemical stability, graphene papers have been used as a flexible substrate for electrodes in recent years, but it is difficult to fabricate large-scale graphene paper materials via vacuum filtration. Inkjet printing technique address this challenge. Chi et al. used well-controlled full inkjet printing method to fabricate a freestanding graphene paper (GP) supported 3D porous graphene hydrogel-polyaniline (GH-PANI) nanocomposite [−1 at 400.33 W kg−1 combined with remarkable mechanical flexibility.Shu et al. synthesized rGO-PPy hybrid paper for flexible supercapacitors by electropolymerization on a paper-like graphene gel . This hyomposite . GO ink In the traditional fabrication of supercapacitor electrodes, active materials are mixed with carbon black and a binder (such as PVDF or PTFE). However, binder materials are generally non-conducting and therefore decrease the electrical conductivity of electrodes, inhibiting electrochemical performance. For flexible supercapacitors the graphene may act as a flexible provider of mechanical strength, and as an active charge-storing part of composites. Furthermore, PANI or PPy, for example, are able to be polymerized directly on the graphene surface, without any binder, which is a good way to reduce the mass of material in an electrode which is not contributing to the electrochemical performance.−1 and 990 F cm−3 at 1 A g−1 in 1 M H2SO4 and 85% capacitance retention after 60,000 cycles in a three-electrode cell configuration. Out of this 3D-G/PANI composite film, flexible all-solid-state supercapacitors (ASS) were prepared, without the need for any current collectors or supporting substrates  composite electrode . The G/Pbstrates d. The ASbstrates e. Moreovbstrates f. In the−1 and an energy density of 23.2 Wh kg−1 at a power density of 399 W kg−1. Furthermore, in the bent or stretched state, the specific capacitance and capacitance retention are close to the normal state, demonstrating that this wavy shape device can tolerate mechanical deformation and maintain good electrochemical performance at the same time.Xie et al. synthesized graphene/PANI composite film with a wavy shape, able to behave as a stretchable upercapacitance electrode . Porous Recently, Ren et al. combined CVD and chemical interfacial polymerization methods, to fabricate stretchable electrodes based on a graphene foam/PPy . Pure grCompared with the liquid electrolytes commonly used in supercapacitor research, flexible devices often use a PVA based gel impregnated with electrolytes. This provides the possibility of omitting the rigid packaging that energy storage devices generally require to prevent electrolyte leakage. Therefore, the development of flexible leak-proof electrolyte systems is also a critical aspect of producing flexible and/or wearable energy storage, however detailed discussion of such systems is beyond the scope of this review which is centred on electrode materials. Nevertheless, when these components are brought together the uniquely novel mechanical properties of flexible and stretchable supercapacitors show strong potential as a disruptive technology to a wide range of established markets.In the above discussion, flexible supercapacitors are presented as an emerging energy storage device architecture with highly desirable mechanical properties. Wearable energy storage generally refers to devices fabricated within flexible and lightweight fibers or yarns, which are then woven into textile fabrics. To satisfy the needs of wearable energy storage, devices must not only show excellent electrochemical performance and possess all the mentioned flexible behaviors  but must also have added durability in order to resist the destructive forces that worn textiles are exposed to in application. 2@rGO hierarchical structure via electrodeposition [−2 in 1 M Na2SO4 (three-electrode cell), and 411 mF cm−2 in an all-solid-state two-electrode cell with PVA/H3PO4 electrolyte. Mechanical deformation tests were performed, consisting of bending at 90°, knotting, and twisting, each repeated 1000 times. Following each of these tests, the devices had retained capacitances of 80%, 91% and 103%, respectively. In a practical demonstration of the devices’ capabilities, 7 yarn supercapacitors were woven into a 15 × 10 cm2 piece of cloth, connected in series, and then used to light 30 LEDs. This provides clear evidence that yarn-based supercapacitors have the potential to possess excellent electrochemical and mechanical properties, giving them  a bright future in various wearable electronics.Huang et al. synthesized stainless steel yarns coated with a PPy@MnOposition . The proAlthough the electrochemical performance of yarn supercapacitors has been established, it still lags behind the energy requirement for conceivable applications, such as heated clothing, or charging personal devices. In fact the amount of charge stored via the EDL capacitance mechanism is proportional to the surface area of an electrode interfacing with an electrolyte. In the case of yarn supercapacitors, it may not be clear how this can be increased without either longer or thicker yarn. If it were possible to create additional inner interface (i.e. within the core of the yarn), the specific capacitance of yarn supercapacitors could be increased. This is what researchers have set out to do.−2 at 0.08 mA cm−2 and a high energy density of 27.1 μWh cm−2 at a power density of 66.5 μW cm−2. Due to the nematic state of rGO sheets which arrange along the length of a fiber, and π–π interactions between rGO and PEDOT, the fiber showed an outstanding tensile strength of up to 631 MPa. After bending 500 times the CV curve was almost same as the initial curve. It is suggested that the hollow structure not only increases the capacitance but also provides favourable flexibility and stability.Qu et al. fabricated a hollow graphene/CP fiber electrode with a high energy density when tested as a yarn supercapacitor . As show2 modified polyester textile electrodes, as shown in 2 modified polyester fiber (M-PEF) and then PPy is deposited on the substrate of graphene/SnCl2 modified polyester yarn (rGO/M-PEFY). The large-area textile electrodes and ultralong yarn electrodes fabricated by this material showed excellent electrochemical and mechanical properties. This material showed an outstanding areal capacitance of 1117 mF cm−2 at a current density of 1 mA cm−2 in 1 M Na2SO4 (three-electrode cell) and a capacitance retention of 100% after 10,000 cycles at 20 mA cm−2. The textile electrode achieved a capacitance retention of 98.3% when bent from 0° to 180° after 1000 cycles. The polypyrrole/graphene/SnCl2 modified polyester textile (PPy/rGO/M-PEFT) electrodes exhibited outstanding electrochemical performance, which is attributed to the synergistic effect of the pseudocapacitive PPy, and highly conductive electric double layer forming rGO. The results reveal this method is suitable for fabricating textile and yarn electrodes, with both the desirable electrochemical and mechanical behaviors demanded by wearable electronics.High capacity performance and robust mechanical properties are both essential characteristics for wearable supercapacitor electrodes. In some cases, to improve the mechanical properties, flexible textile substrates were chosen ,141. How−3 at the power density of 30.77 mW cm−3. There were no changes in the CV curves under normal, knotted, and twisted states  produced via a self-assembly technique, at room temperature . PANI nad states a. In addd states b,c. Figud states e. The reIn the above studies, graphene and CPs are successfully used to fabricate yarn electrodes and woven textile electrodes for wearable supercapacitors. Graphene not only acts as a highly conductive, electric double-layer capacitance material, but also connects the CPs with a flexible, highly conductive scaffold. On the one hand, the CPs contained in the reviewed composite electrodes contribute pseudocapacitance to the electrochemical performance. On the other hand, they play an important role in the porous structure formation of graphene and may also work as a coating to protect the inner material. The synergistic effect of graphene and CPs effectively improves the electrochemical and mechanical properties of textile based devices for wearable electronics. With continued development of textile energy storage technology, it is anticipated that supercapacitors will be integrated into commercialized wearable electronics within the next few years.As low-cost, high pseudocapacitance materials, CPs have attracted lots of attention in next generation supercapacitor electrode research. While pure CPs show many problems, such as relatively poor electrical conductivity, poor cyclic stability, and low energy density, the combination of graphene and CPs demonstrates how these problems may be solved. This paper has reviewed graphene and CPs including PANI, PPy and PEDOT, as composite electrodes for supercapacitors. It has been shown that graphene/CP composites may be constructed to possess desirable 3D porous structures. In summary of the synergistic effects, graphene is able to reduce the volume change during charge/discharge cycles, improving the cyclic stability of CPs via reducing the mechanical stresses on them, while at the same time the graphene improves the electrical conductivity and resultant power handling. Meanwhile, CPs can be grown in situ on the surface of graphene which typically reduces the aggregation of the GO precursor during reduction processes, maintaining higher interfacial area within the electrode for improved double layer capacitance. Furthermore, under certain synthesis conditions the excellent mechanical properties of some graphene/CPs composite films demonstrate that the materials can be drawn into yarns suitable for flexible and wearable energy storage devices. In recent years, the functionalization of graphene with organic molecules has been investigated for energy storage. There are a number of reports where functionalized graphene based electrodes show outstanding specific capacitances due to the high pseudocapacitance contribution of the organic molecules. Although the number of reports discussing organic molecules for energy storage devices is growing, up to now, only a few amino or hydroxyl group-containing molecules have been investigated, leaving many other molecules remaining to be studied. In addition, details of the energy storage mechanisms of these molecules is still to be clearly understood. In most cases discussed, the reported high energy densities and favourable specific capacitances were obtained using small amounts of active materials as electrodes in lab-scale test cells and/or devices. However, for practical applications, supercapacitor devices are typically much larger and require more active materials constructed into larger electrodes. Scaling-up the production of many of the active material combinations discussed in this review is a serious challenge for researchers and engineers. In addition to this broad limitation, the composites in most of the cited literature can only be used in aqueous electrolyte systems, whose potential window is limited to around 1.2 V. This may be compared to organic-solvent or ionic liquid electrolyte systems which may reach around 3 V. Although the relationship has not been discussed in detail, lower maximum voltage severely limits the energy density of devices. While aqueous systems may find some genuine niches, such as for biological implants, the relatively lower energy density generally limits their application. Thus, to improve the energy able to be stored in supercapacitors, electrolyte systems that are able to support a wider potential window are always being sought, especially for graphene/CP composites where the present choices are more restricted by the chemistry of the active materials. In conclusion, graphene/CP composites are promising materials for supercapacitor applications. However, currently the available CPs for supercapacitors are limited to PANI, PPy, and PEDOT. Hence their drawbacks in terms of poor cycling stability and narrow voltage window in aqueous electrolyte are anticipated to be solved by the synthesis of new-type CPs. Building asymmetric supercapacitors by using two dissimilar CPs, one as the negative electrode and the other one as the positive electrode, can be an efficient way towards high performance supercapacitors with high specific capacitance and broad voltage window beyond the crytical voltage of water splitting (~1.23 V). With continued effort made to overcome the identified obstacles, and continued research into new-type CPs with better cycling stability, higher specific capacitance, and broader voltage window in aqueous electrolyte, completely novel energy storage devices based on graphene/CPs composites could become commercially available in the not-too-distant future.


Glioma, especially glioblastoma, is pathologically characterized by high aggressiveness, which largely contributed to the ineffectiveness of current therapies. It has been recently reported that intrinsic PD-L1 can regulate tumor malignancy, whereas underlying mechanisms remain mostly unclear. Here, we report a novel mechanism by which PD-L1 promotes glioma cell infiltration. In orthotopic glioma models, PD-L1 expression was up-regulated predominantly in glioma cells in the infiltrating front. For PD-L1-overexpressed glioma cells, PI3K/Akt and actin regulations were among the top six most altered signaling pathways as detected by RNA-sequencing. PD-L1 significantly activated Akt/F-actin signaling while suppressed autophagic signaling upon cell starvation. Mechanistically, PD-L1 preferentially bound to Akt among various PI3K/Akt signaling proteins. Serial truncation identified the interaction between the 128-237aa fragment of PD-L1 and the 112-480aa fragment of Akt, which facilitates the membrane translocation/activation of Akt, and was unaffected by Perifosin (specific p-Akt inhibitor targeting Akt PH-domain). Taken together, our data indicate that in glioma cells, PD-L1 is induced to prevent autophagic cytoskeleton collapse via Akt binding/activation, facilitating glioma cell invasion upon starvation stress. Glioma, the most common primary intracranial tumor, originates from various types of cells. The most malignant glioma is glioblastoma, or glioblastoma multiforme (GBM), which accounts for around 21% of glioma and can be primary or secondary from grade II-III glioma. Recently, glioma is also classified into three types based on its molecular markers: 1p/19q deletion, IDH mutation and TERT promoter mutation. Among them, grade IV glioma mainly has the TERT promoter mutation accompanied by EGFR/PTEN mutation. Current treatments for glioma remain conventional surgery, radiotherapy and chemotherapy. However, glioma is extremely easy to relapse after treatment, with an average survival time of only 14 months for GBM patients after treatment  is a key negative regulator for immune inhibitory axis signaling controlling T-lymphocyte infiltration inside solid tumors. Previous studies reported that PD-L1 is widely expressed in glioma cell lines , 13 and PD-L1 is recently considered to be oncogenic. PD-L1 knockdown significantly decreases tumor volume in murine ovarian cancer, melanoma , murine in vivo. Our data revealed that PD-L1 promoted glioma cell infiltration via starvation-induced Akt/autophagy/F-actin signaling. Particularly, we dissected PD-L1-Akt binding fragments and elucidated how PD-L1/Akt interactions activated its downstream cascades.In the present study, we first reported that PD-L1 was mostly prominent in those highly aggressive infiltrating glioma cells +/K+ ATPase α1 , β-tubulin  were commercially purchased.Cloroquine  was used at a final concentration of 25 μM. LY294002  was used at a final concentration of 0–50 μM. Perifosine  was used at a final concentration of 0–25 μM. Primary antibodies against phospho-Akt , total Akt (#9272), phospho-mTOR, total mTOR,pP62, phospho-P70S6K, LC3B were purchased from Cell Signaling Technology . Antibodies against PD-L1/CD274 , PD-L1 , GST , Beclin1 , N-terminal GFP (Sigma), β-actin , NaGFP-LC3 plasmid was a gift from Dr. He Li (Huazhong University of Science and Technology). GST-kRas/PTEN/Akt1/Akt2/Akt3/EGFR VIII/PKA plasmids were gifts from Dr. Haian Fu (Emory University). PD-L1-EGFP expressing-plasmid was purchased from GeneChem . Full-length and truncated human PD-L1 coding cDNA was PCR amplified from PD-L1-EGFP plasmids and cloned into NV  vector including NV-PD-L1 FL (full length 1-290 aa), NV-PD-L1-T1-259 (truncate 1-259aa), T128-259, T128-239, T1-178. PD-L1 cloned into pDEST-26 or p-FU-Venus vector was used to express GST-PD-L1 and Venus-PD-L1 FL1-290 or T19-290. Full-length and truncated human Akt1 cDNA was PCR amplified from GST-Akt1 plasmid and cloned into p-FU-Venus vector for Venus-Akt1, Akt1-T1-111, and Akt1-T122-480. Akt1-Y176A and Akt1-K14R mutants were constructed by using overlapping-PCR from p-FU-Venus-Akt1 plasmid. Transfection of plasmids was performed by using Lipofectamine 2000  according to the manufacturer's instructions.P-values were adjusted using the Benjamini and Hochberg's approach for controlling the false discovery rate. Differently expressed genes (DEGs) with P < 0.005, adjusted P < 0.05 (Padj) and absolute changing fold ≥1.2 were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) enrichment analysis. KEGG pathways and GO terms with Padj < 0.05 were considered significantly enriched by DEGs. We have submitted our data to NCBI (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE107581).Three 100-mm dishes of U251/PD-L1 and U251/Vec stable cell lines were subjected to total mRNA isolation, cDNA libraries construction and sequencing at Illumina HiSeq sequence platform (PE150) with 6G clean data by Novogene Bioinformatics Institute . Differential gene expression analysis between U251/PD-L1 and U251/Vec groups was performed using the DESeq2 R package (1.10.1). The resulting ad libitum access to food and water. Briefly, adult Kunming male mice (18–20 g) were anesthetized with chloral hydrate (350 mg/kg) and a burr hole was drilled in the skull 0.5 mm posterior to the bregma and 2.0 mm lateral to the midline. A 10-μl Hamilton syringe  containing 20,000 G422 cells (mouse GBM cell line) in 1 μl of PBS was advanced to a depth of 3.5 mm from the skull surface and then withdrawal 0.3 mm. Cell suspension was delivered at the rate of 1 μl/min. After cell implantation, the needle was left in place for 6 min before withdrawal. After 6–14 d of cell inoculation, the mice were perfused with 4% paraformaldehyde (PFA) and the brains were paraffin-embedded.All animal handling and experiments were performed in accordance with NIH guidelines and approved by the Ethics Committees of Huazhong University of Science and Technology. The mice were group housed in the Animal Core Facility of Tongji Medical College under a 12 h light/dark cycle with IHC was performed as previously reported . ParaffiHuman glioblastoma cell lines U251, LN229, and human embryonic kidney 293T cell line were purchased from American Tissue Culture Collection  or China Center for Type Culture Collection . U251, LN228, U87MG with stable PD-L1 overexpression  or knockout (U251/sgGFP or sgPD-L1) were generated as previously described  were considered autophagy-positive. An average percentage of puncta-positive cells from nine fields/culture  was calculated and used for statistical analysis.GST-pull down assay was performed as previously described . 293 cel+/K+ ATPase α1 and β-tubulin as internal control of membrane and cytosol proteins respectively.U251 cells were transfected with p-FU-Venus or p-FU-Venus-PD-L1 plasmid for 24–36 h. Membrane and cytosol proteins were extracted using Membrane and Cytosol Protein Extraction Kit  according to manufature's instructions. Membrane and cytosol proteins were then subjected to Western blotting with Nain vitro experiments. Paired Student's test was used to compare between two groups of animal experiments. Comparisons among multiple groups were performed through one-way ANOVA with Student–Newman–Keuls post-test. P < 0.05 was considered statistically significant.All experiments were repeated independently for at least three times. The values were expressed as means ± SEM. Unpaired Student's test was used to compare between two groups of To ascertain the relationships between intrinsic PD-L1 and glioma cell invasive behavior during GBM development, we examined PD-L1 expression in the whole brain at various stages of GBM development in a highly aggressive orthotopic mouse G422 model . PD-L1 a+-GBM cells in the infiltration frontier, mRNA sequencing was conducted in PD-L1-overexpressed U251 glioma cells. Differential expressed genes (DEGs) between U251/PD-L1 and U251/Vec cells  were selected by three criteria  and the selected DEGs were subjected to various systemic analysis. DO (Disease Ontology) enrichment showed that PD-L1-altered gene expression was significantly associated with malignant glioma (http://gepia.cancer-pku.cn) revealed that PD-L1 (CD274) was negatively associated with the prognosis of glioma patients  of Akt based on its structure  , it is iFinally, we examined the effects of PD-L1 on F-actin structure in starved glioma cells. Fluorescent images of Palloidin staining clearly showed that F-actin formation was evidently increased in PD-L1-overexpressed U251 cells under normal culture conditions as well as EBBS-12 h incubation . Taken tIn our present study, we discovered that PD-L1 elevation occurred predominantly in highly aggressive glioma cells. RNA sequencing revealed that high levels of PD-L1 in glioma cells mainly mediated cell migration and PI3K/Akt/actin signaling. PD-L1 reduced starvation-induced Akt inhibition, autophagic influx and F-actin collapse in glioma cells. Mechanistically, PD-L1 directly interacted with Akt, and this process involved PD-L1 128-237aa and Akt 112-480aa. The binding of PD-L1 to Akt facilitated membrane-translocation of Akt and thus elicited downstream biological effects in frontier glioma cells. These findings reveal a novel invasive mechanism in GBM.+-glioma cells were highly aggressive .The datasets generated for this study can be found in the NCBI (The animal study was reviewed and approved by Experimental Animal Ethics Committee, Tongji Medical College, Huazhong University of Science and Technology.RC, XX, XQ, FL, CL, YL, XL, and GJ performed the experiment. RC, FP, XQ, and XC data analysis, wrote the manuscript, and contributed to discussion. FH and DL contributed to discussion. RC, FP, XQ, and XC designed and wrote 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.


The stretchability and the volumetric capacitance are increased to 150% and 86 mF/cm3 (0.181 mF/cm2), respectively. This additive nanofabrication method is highly desirable for the development of self-sustainable stretchable energy storage integrated with wearable technologies. The flexible and stretchable energy storage with a high energy density opens the new opportunity for on-chip sensing, imaging, and monitoring.Direct laser writing with an ultrashort laser beam pulses has emerged as a cost-effective single step technology for realizing high spatial resolution features of three-dimensional structures in confined footprints with potential for large area fabrication. Here we present the two-photon direct laser writing technology to develop high-performance stretchable biomimetic three-dimensional micro-supercapacitors with the fractal electrode distance down to 1 µm. With multilayered graphene oxide films, we show the charge transfer capability enhanced by order of 10 The development of on-chip in-plane micro-supercapacitors (MSCs) using laser scribed graphene (LSG) has attracted tremendous attention due to the integration possibilities with the small-scale devices3. However, the deliverable aerial capacitance from that MSCs4 is minimal for powering micro-devices which limit the potential of autonomous systems.Energy storage demands for the technological advancements in micro-stretchable, wearable, and portable applications5. However, the current research in this area has been mainly focused on the use of pseudocapacitive materials6 and gold nanoparticles for LSG7, which makes those MSCs lacking the stretchability8 essential for wearable applications9 due to the fragile nature of gold. Although direct laser writing (DLW) with ultrashot femtosecond (fs) laser beams has been adopted to generate 3D MSCs8, two-photon excitation induced in the focus of a high-numerical objective has not been explored. Due to the 3D confinement in the tight focus10, this technology has opened the enormous potential for the industrial scale production11. As a result, this single-step fabrication technique has generated various complex 3D and free-standing structures at micron and sub-micron scales in telecommunications12, biomedical applications13, free-form optics14 and displays15.To address this challenge, three-dimensional (3D) energy storages have emerged as a new concept which can potentially enable the miniaturization and integrability in a single platform without losing the performance of energy storages by the effective utilization of the volumetric capacitance performance4. On the other hand, the MSCs fabricated using electron beam lithography (EBL) limits the possibilities of extension to three-dimensional which inturn affects the capacitance16 in limited aerial footprints.In this paper, we demonstrate the fabrication of a bioinspired 3D two-photon-induced (2PI)-graphene MSC directly on an elastomeric polydimethylsiloxane (PDMS) substrate, using the two-photon DLW approach to generate the electrode inter-distance down to 1 mm Fig.  in 40 mi2 and the energy storage density exceeding the current lithium-ion batteries. More importantly, the stretchability and the volumetric capacitance reach 150% and 86 mF/cm3 (0.181 mF/cm2), respectively, which are enhanced by more than three times and 100 times compared with the state of the art results17.The adoption of PDMS substrate, in addition, allows us to achieve the stretchable feature. Our result shows the charge transfer capability enhanced by order of 104 19 which is the measure of the local size of a set of numbers and can be calculated by using the Box-counting method20. The relationship between HD, the linear scaling (L) and the resulting increase in size (S) which is the number of self-similar units generated after scaling for a layer by layer plane construction of 3D object can be written as below1 for a total 2PI-graphene film thickness of 23 µm, as shown in Fig. The 2PI multilayered electrode structure is achieved with the 3D extension of the two-dimensional structure based on the fractal pattern derived from fern tree leaves4 Fig.  with int2. Beyond the miniaturization aspect, the high spatial resolution of the focused fs-laser beams can lead to the theoretically predicted ideal inter-electrode distance below 25 µm in the MSC for the optimum electrolyte ionic path21.We use a fs-laser beam to achieve the multilayer graphene oxide (MLGO) reduction for the fabrication of a 3D 2PI-graphene MSC in a footprint of 1 mm22, which involve the multiphoton absorption within the time scales of less than picosecond resulting in the suppression of heat diffusion and the elimination of the heat affected zone. As a result, this fabrication method can lead to structures with high-resolution. However, the graphene oxides photoreduction at a wavelength of 800 nm may be contributed by a combination of photothermal and photochemical23 assisted with shock waves as the pulse repetition rate becomes higher. In order to understand the phenomenon in detail, we conducted a detailed analysis of the photoreduction of graphene oxide film under different repetition rates of 80 MHz, and 10 kHz which can be found in Supplementary Notes and the best conditions obtained under 80 MHz is considered for the fabrication of 2PI-graphene MSCs.It has been demonstrated that the microstructures fabricated by fs pulses are mainly a non-thermal fabrication process24. The PL measurements in single-layer graphene oxide film as a function of the laser fluence under a repetition rate of 80 MHz confirms the two-photon absorption  measurementsion Fig.  during tion Fig. . The temThe 2PI phenomena lead to polycrystalline nature for the obtained 2PI-graphene film as observed from the transmission electron microscopy (TEM) image Fig. . In addiD/IG ratios obtained were 1.39 and 1.08 for positive and negative graphene oxides respectively which indicates the increase of defects in the positive graphene oxide layers. The Raman spectra of MLGO were given in Fig. D/IG ratio was calculated to be 1.26.For the improvement of the MSC energy storage density, the thickness of the graphene oxide film is a significant factor. In our studies, we observe that the focused laser beam with an objective of NA 1.4 generates the stronger thickness reduction in the 2PI-graphene film compared with an objective of NA 0.6 Fig. . The optNanoscribe with a pulse width of 100 fs. The temperature involved in the reduction process of graphene oxides using 80 MHz is calculated to be 800 °C  spectroscopies  shows an almost quasi-rectangular behavior upto a 150% stretchability as given in Fig. −1 with a step size of 0.1 mV and the obtained volumetric capacitance is around 86 mF cm−3  substrate by using a commercial direct writing setup Nanoscribe which has a wavelength of 800 nm, pulse width 120 fs and repetition rate of 80 MHz The PDMS substrate  was prepared in a mixing ratio of 10:1. The air was evacuated from the PDMS mixture using the vacuum. The polymer was crosslinked at 80 °C for one hour in a convection oven. The laser fluence used for writing the graphene oxide film was 0.18 J/cm2 with a 20x objective of 0.6 NA. The fabrication of 2PI-graphene MSCs was followed by filling the MSCs with electrolyte ionic gel and allowed to dry overnight. 41. 1.3 mg/ml of each graphene oxides were dispersed in water and drop cast on a glass substrate. Each layer of graphene oxides was dried at 60 °C, and the process was repeated until the desired number of alternate layers of graphene oxides (MLGO) was achieved.Graphene oxide films were prepared through the layer by layer assembly using the electrostatic attraction between the positive and negative graphene oxide layers. Hummer’s method was used to synthesize the negatively charged graphene oxides (Sigma-Aldrich)2] (Sigma-Aldrich) in a ratio of 0.03 g/1.0 g and the mixture was stirred under a nitrogen atmosphere for about 5 hours. The electrochemical window of the electrolyte is 2.5 V.We used an ionic gel electrolyte. The ionogel was prepared by mixing fumed silica nanopowder  with the ionic liquid (1-butyl-3-methylimidazolium bis (trifluoromethyl sulfonyl) imide [BMIM] [NTfRaman spectra were obtained for different layers of graphene oxides using a Renishaw Raman spectrometer equipped with an Nd-YAG laser beam of wavelength 514 nm. Scans were performed between 1,000 and 2,700 wavenumbers with a laser spot size of 1 µm. The background corrected spectrum was normalized by dividing the data by the maximum intensity. The XPS measurements were conducted using the Thermo K-alpha X-ray Photoelectron Spectrometer (XPS) using monochromatic, Micro-focused Al K-α source.−9 Torr and TEM measurements were conducted using the JEOL 2010 with samples fixed on a copper grid. Porosity measurements of the obtained film is determined using three methods: the diameter analysis of the SEM images using Image J software, BJH method using Surface Area and Porosity Analyser  and Small-angle-X-ray scattering (SAXS) measurements. SAXS was conducted on a microcalix SAXS system (Bruker) using Cu Ka radiation . The sample was placed into a 1.5 mm outer diameter quartz capillary. Background measurement was on an empty capillary. Measurements were carried out under vacuum at 20 °C with an exposure time of 2 hours. Primary masking, data reduction and background subtraction and were carried out in BioXTAS Raw42. The pores were modeled as a collection of polydisperse spheres of air within the 2PI graphene matrix28, and fitting was carried out in SasView43 using a standard model44 with a Gaussian distribution. The best fit yielded an average radius of 18.7 nm with a polydispersity of 14.6%.SEM images were conducted using Philips XL30 SEM with a vacuum of 10Two-point conductivity measurements on 2PI-graphene films reduced under different repetition rates were conducted using the Keithley 2400 series source meter wired through two micro-positioners using tungsten probes of tip diameter 5 µm. Measurements were taken by varying the applied voltage between 0.1 V to 4.5 V. The rectangle patterns of area 300 µm × 300 µm were connected to gold electrical pads on each side. The electrical conductivity study was performed by keeping the probes on each side of gold electrical pads. With the thickness information, the bulk conductivity was evaluated using the following equation:2 as the current collectors. Open-circuit measurements were performed initially for about one hour before the electrochemical tests to ensure the stable conditions. We used an ionic gel electrolyte to study the performance of obtained 3D 2PI-MSCs under stretched and non-stretched conditions. The CV measurements with a step size of 0.1 mV from 0 to 2.5 V at a scan rate of 1–3,000 mV s−1 with an average of 10 cycles.The electrochemical measurements were undertaken using the Keithley ECE 2400 source meter with a tungsten probe of diameter 5 µm and an exposed area 2.25 mm21,i is the applied current (A), and dV/dt is the slope of the discharge curve .The capacitance was calculated from the CC curves at different current densities using the formula25. The volume specific capacitance was calculated using the formula,total is the total capacitance obtained from the 3D 2PI-graphene electrode connected with the gold probe electrodes, C (Gold+MLGO) is the capacitance contributed by the gold and MLGO film separator, V3D 2PI is the volume of the 3D 2PI-graphene MSCs.The energy and power densities of the 3D 2PI-graphene MSCs were calculated by taking away the effect of the capacitance from the gold pad, and MLGO filmden, and the power density, Pden (rate of charge transfer) were calculated as the following:The energy density, Etotal, and Ptotal of the device can be calculated by the formula:total is the energy density in Wh/cm3, Cv is the volumetric capacitance, ΔE is the operating voltage window in Volts, RESR is internal voltage drop at the beginning of the discharge, Vdrop, at a constant current density (i) calculated from the CC measurements and given asTotal energy, ESupplementary Information


To enhance the wettability between Ag atoms and graphene of graphene-reinforced silver-based composite filler, the adsorption behavior of Ag atoms on graphene was studied by first-principles calculation. This was based on band structure analysis, both p-type doping and n-type doping form, of the vacancy-defected and Ce-doped graphene. It was verified by the subsequent investigation on the density of states. According to the charge transfer calculation, p-type doping can promote the electron transport ability between Ag atoms and graphene. The adsorption energy and population analysis show that both defect and Ce doping can improve the wettability and stability of the Ag-graphene system. Seen from these theoretical calculations, this study provides useful guidance for the preparation of Ag-graphene composite fillers. Silver-based filler and graphene are the two main materials that will be discussed in this study. Silver-based filler is a very important kind of brazing material, which has the advantages of a high strength, good mechanical properties, a high electrical conductivity, corrosion resistance, and so on ,2,3. It In this study, the adsorption behavior of Ag atoms on vacancy-deficient graphene (VG) and Ce-doped graphene was calculated by first-principles theory. By analyzing the adsorption energy, band structure, and population, the effects of vacancy defects and Ce doping on the adsorption behavior of Ag on graphene were investigated. The interaction between Ag and graphene and electron transfer were discussed. This research will help to improve the wettability between graphene and silver-based composite fillers, thus providing theoretical guidance and technical support for the application of graphene-reinforced composite fillers.−5 eV/atom.The calculations were performed using the CASTEP code , which iC) site directly above a C atom, and the top (TCe) site above the Ce atom. The models of VG and Ce-doped graphene are shown in A 4 × 4 supercell with a periodic boundary condition along the x-y plane was employed for the infinite graphene sheet model. The vacuum was set with 20 Å in the z direction, which could avoid the interaction between periodic graphene layers . By remoadE) can be computed byAg-grapheneE, AgE, and grapheneE represent the total energy of the graphene-Ag system, the energy of the Ag atom, and the energy of graphene, respectively.The adsorption energy of an Ag atom on graphene  is energetically favored for Ag atom adsorption in comparison with the adsorption on either hollow (H) or bridge (B) sites in intrinsic graphene and Ce-doped graphene . This re−3 in −3, which proves that the wettability of the system has been improved.The system change of VG adsorption of Ag atoms is the largest, as shown in To further investigate the mechanism and effect of doping, different band structures were computed, including the intrinsic graphene, VG, and Ce-doped graphene before and after Ag adsorption. The Dirac point ,45 formsThe partial density of states (DOS) in −1, indicating that the defect causes chemical adsorption between Ag atoms and graphene, and there is a stable chemical bond, which is consistent with the previous adsorption energy, adsorption height, and DOS analysis. Compared with intrinsic graphene, the population of the Ce-doped graphene-Ag system between the Ce and Ag atoms is 0.28, and its number of population per unit bond length is 0.933 charge·nm−1, implying the formation of a stable chemical bond between them.To further study the interaction between graphene and the adsorbed Ag atom, population analysis was completed for intrinsic graphene, VG, and Ce-doped graphene after adsorption of the Ag atom. The results are summarized in Vacancy defects in graphene trigger electron deficiency and p-type doping, which could significantly improve the adsorption energy, charge transfer, and wettability between graphene and the adsorbed Ag atom. Conversely, for Ce-doped graphene, n-type doping formed and thus increased the Fermi level of graphene, which might hinder the electron transfer between Ag and graphene. However, the adsorption energy and population analysis shows that Ce doping could enhance the stability of the Ag–graphene system and wettability.The adopted research methods, mechanism cognitions, and obtained conclusion in this study might contribute to the future development of efficient and stable brazing materials, and provide useful guidance for the preparation of graphene-reinforced Ag filler with fine properties.


Mechanical exfoliation of graphite via sonication produced graphene sheets, which were stabilized by the surface adsorption of a cationic surfactant (CTAB). In a subsequent step, varying amount of a cobalt complex [sodium hexanitrocobaltate(III)] was added which selectively bound with the positively charged head of surfactant. In the last step, cobalt complex was reduced with sodium borohydride to obtain Co/G NSs catalyst. The catalyst showed lower overpotential (280 mV) as compared to benchmark catalysts and decent stability and turnover frequency (TOF: 0.089 s One of the renewable and alternative energy sources is water, which could undergo photochemical and electrochemical splitting to produce oxygen and hydrogen green fuel5. In comparison to two-electron hydrogen evolution reaction (HER)7, four-electron oxygen evolution reaction (OER) possesses sluggish kinetics requiring higher overpotential (η) for the achievement of benchmark 10 mA/cm2 current density8. In the past decade various molecular9 and heterogeneous catalysts12 have been developed to achieve oxidation of water at lower overpotential. Focus has been on the earth-abundant metal catalysts15 that could possibly replace the benchmark OER catalysts based on scarcely available Pt, Ir and Ru16. Among other abundantly available transition metals, much attention has been gained by Co catalyst since the pioneer work of Nocera17 and several catalysts based on oxides, chalcogenides, phosphides and phosphates of cobalt have been developed for water splitting22 and energy storage27. However these catalysts based on earth-abundant metals need to be improved to achieve low overpotentials, low Tafel slopes, high Faraday efficiency, high turnover frequency (TOF) and stability28. Among others, carbon29 and graphene-supported cobalt heterostructures have shown high current density, low overpotential and significant stability for OER35. Graphene, a hexagonally arranged two dimensional material made up of sp2-carbon atoms possesses extraordinary properties like room temperature electron mobility, high thermal conductivity and ability to withstand high current densities36. Very high mechanical strength, surface area (>2600 m2/g) and density of surface active sites make graphene particularly suitable for catalysis39. Electrochemical properties of graphene greatly depend upon the synthetic method and can potentially be enhanced by the incorporation of heteroatoms in the graphene lattice which modulates the spin density and charge distribution of carbon atoms thereby creating the catalytically active sites for OER42. However conventional synthesis of graphene by Hummer’s method43 and its modifications45 involves the use of hazardous chemicals or their products whose removal, reuse and disposal is an issue. Chemical vapor deposition (CVD)50 and liquid-phase exfoliation (LE)53 are the only alternative (green) methods55 for the fabrication of highest-quality graphene known for its superior properties. The former method however, is still relatively expensive as it demands large energy and the removal of the substrate36. Recently materials based on cobalt(II) hydroxide has shown potential in electrochemical water splitting58. However poor electrical conductivity of the material needs to be improved in order to obtain a significant OER activity63. Inspired by the pioneer work of Coleman et al.68 we report a one-pot procedure for the solution-phase synthesis69 of graphene-Co(OH)2 nanosheets (Co/G NSs) which catalyze water oxidation at remarkably low overpotential of 280 mV providing high TOF of 0.089 s−1.The present energy resources are insufficient to fulfill global energy in near future due to their rapid depletion, therefore new technological advances to quest clean, sustainable and viable energy resource are highly demanded65. The surfactant gets adsorbed on the surface of so produced graphene via Van der Wall’s interactions and stabilizes the 2-dimensional material 6]: CoL) was added whose anion ([Co(NO2)6]3-) electrostatically binds with the the positive head of the surfactant , to produce graphene flakes in water by mechanical exfoliation of graphite via ultrasonication Fig. 65. The sl)71. For the calculation of extinction coefficient (α), a graphene dispersion (500 ml) was prepared using graphite (3 mg/mL) and CTAB (0.1 mg/mL). After centrifugation and decantation, absorption spectrum was taken at 660 nm. 60 mL of this dispersion was filtered through pre-weighted filter units, dried and weighed to find the graphene concentration. The value of absorptivity constant was found to be 1256 mL/mg/l which is close to the reported value of 1390 mL/mg/l65.UV-vis absorption spectrum of graphene dispersion was found featureless and flat Fig.  at and aG) and CTAB (CCTAB) were also optimized and optimum yield of graphene was found with CG = 3 mg/mL and CCTAB = 0.1 mg/mL  was purchased from Sigma Aldrich. All the solutions were prepared in deionized water (DI) and methanol.Graphite obtained from used zinc-carbon batteries was soaked and washed with hydrochloric acid to leach metal impurities. Cetyl trimethylammonium bromide (CTAB), Sodium borohydride  and surface morphology were examined by using FEI NOVA nano SEM 450 equipped with Oxford EDX detector. Raman spectra were taken using Raman Microscope Renishaw, UK. Surface analysis of the catalysts was conducted using X-ray photoelectron spectrometer . Charging of the spectra was corrected against the graphitic-like C 1 s peak (from the instrument itself) taken at 284 eV and used as a reference. CASAXPS software (version 2.3.16) was used for peak fitting.Transmission electron microscopy (TEM) was carried out by using a Varian LEO 9220 (200 kV) and a JOEL JEM-2200FS instrument. The sample was suspended in chloroform and sonicated for 5 min. Subsequently a drop of the suspended sample was placed on a grid (Plano S 166-3) and allowed to dry.Electrochemical analysis was executed by using Gamray Reference 600 Potentiostat/galavanostat/ZRA with three-electrode system.4 was added and dispersion was stirred at room temperature for 2 hours.Graphene was prepared by a simple mechanical exfoliation method. Typically, graphite and CTAB were mixed in 3.3:1 mass ratio in DI water and the mixture was sonicated for 10 hr in ultrasonic bath (Elma X-tra 70 H) whose temperature was kept below 50 °C. CTAB-stabilized graphene suspension was centrifuged (Centrifuge -5810R) for one hour to remove undissolved graphite and unstable graphene. In the next step, Cobalt complex was added to graphene dispersion (Co to graphene mass ratio = 1:100) and sonicated for 30 min. Afterwards, freshly prepared solution of NaBHFor characterization, graphene dispersion was filtered through syringe filter (0.22 μm) and vacuum dried. For TEM and Raman analysis, dispersions were precipitated at 14000 rpm for 30 minutes.2) electrode (GCE) was scrubbed with alumina powder (0.05 µm) before using as the working electrode. A silver/ silver chloride electrode (Ag/AgCl) was used as reference electrode and platinum wire as counter electrode. All the potentials were converted into RHE by using following Nernst equation.All the electrochemical experiments were performed at room temperature (25 °C) using standard three-electrode system. A glassy carbon  and current density (j) by using Tafel equation.2) and ‘m’ is the loading mass of electrocatalyst (0.017 mg/cm2).Theoretically, mass activity was calculated by using the following equation.−1) and n is the number moles of electrocatalyst.The turnover frequency (TOF) can also be theoretically calculated by considering that every metal atom is involved in catalysis.96.Electrochemically surface area (ECSA) for each system can be measured from double layer charging capacitance of electrocatalytic surface determined from non-faradic region of cyclic voltammetry at multiple scan rates from 10 mV/sec to 200 mV/sec of cyclic voltamogramdl) which yield a straight line equation with a slope which is equal to Cdl according to following equation97 (Fig. c is the double layer charging current, v is the scan rate and Cdl is the double layer charging capacitance. The value of Cdl for electrodeposited Co/G NSs catalyst is 1.17 mF obtained from CV of different scan rates (Fig It is assumed that non- faradic current is due to double layer charging capacitance Figs  and S5. n97 Fig. .\documenates Fig . Then, Es is the specific capacitance due to double layer of smooth surface of deposited sample on electrode. Values of Cs for different metal electrodes have been reported in literature for acidic and alkaline solutions lies in the range of 0.022−0.130 mF cm−2 for KOH and NaOH. The reported value of specific capacitance for cobalt surface is 0.043 mF cm−2 in 0.1 M KOH and consider it as a ‘typical’ value for such materials. The calculated electrochemically active surface area of Co/G NSs is 16 cm2 according to our calculations by using Cdl value.Here CSupplementary Information