Previous studies investigated the prognostic role of programmed death-ligand 1 (PD-L1) expression in patients with biliary tract cancer (BTC); however, the results remained controversial. Therefore, we conducted the current meta-analysis with the aim of clarifying the association between PD-L1 expression and prognosis as well as with several important clinicopathological features of BTC. We searched PubMed, Embase, and Web of Science for relevant studies. Studies that detected PD-L1 expression in tumor cells by using immunohistochemistry (IHC) were selected. Pooled hazard ratios (HRs) and pooled odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to estimate the correlations. In total, 15 independent studies with 1,776 patients were included in this meta-analysis. The pooled data demonstrated that high PD-L1 expression was associated with poor overall survival . The correlation between PD-L1 expression and disease-free survival was not significant . In addition, no significant correlation was observed between PD-L1 expression and clinical features in patients with BTC. Our study results showed that PD-L1 expression could play a pivotal role as an effective factor of poor prognosis in patients with BTC. Biliary tract cancer (BTC) is the second most common cancer among hepatobiliary cancers, and it is a heterogeneous group of gastrointestinal tumors . BTC incFurther understanding of the tumor immune microenvironment has led to the development of immunotherapy, which has garnered much attention in recent years , 9. ProgThe process of literature selection is presented in The characteristics of the included studies are shown in I2=25.9%, p=0.167); therefore, a fixed-effects model was adopted. As shown in The data between PD-L1 expression and OS were extracted from all the 15 included studies with 1,776 patients –25. The I2=65%, p=0.016), a random-effects model was applied. The pooled HR indicated that the correlation between PD-L1 expression and DFS was not significant . Therefore, we examined each included study separately. A total of 11 included studies , 18–25 rAlthough we strictly performed this meta-analysis according to the PRISMA guidelines and selected eligible studies with uniform criteria, the study has several limitations. First, although all included studies used IHC to detect PD-L1 expression, the cut-off values defining low/high PD-L1 levels were different, which may result in heterogeneity. Second, in studies that enrolled patients with iCCA+eCCA , 22, 23,In summary, our meta-analysis demonstrated that PD-L1 overexpression was associated with worse OS but not DFS of patients with BTC. The prognostic value of PD-L1 expression was significant for OS of patients with iCCA and eCCA. However, no significant correlation was observed between PD-L1 expression and clinical features of patients with BTC. These results indicate that PD-L1 could play a pivotal role as an effective factor of poor prognosis in patients with BTC. Nevertheless, as the study had several limitations, further large-scale, well-designed studies are needed to confirm our results.This meta-analysis was performed on the basis of the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines . The datThe inclusion criteria for eligible studies were as follows: (1) BTC was diagnosed on histopathological examination; (2) immunohistochemistry (IHC) analysis of PD-L1 expression was conducted; (3) association between PD-L1 expression and OS and/or disease-free survival (DFS) was presented, or sufficient information was provided to compute the hazard ratio (HR) and 95% confidence interval (CI) ; (4) theData from candidate studies were evaluated and extracted by two independent investigators (C.L. and X.P.). Any disagreements were resolved through discussion. The following information was extracted from eligible studies: the first author’s name, publication year, number of cases, country, ethnicity, study period, patient age, tumor type, detection method, cut-off value for high expression of PD-L1, treatment method, HR and the corresponding 95% CI for OS and DFS, and clinicopathological characteristics. The quality of the included studies was assessed by using the Newcastle-Ottawa Scale (NOS) . The NOSI2 values > 50% and p-values <0.10 were considered to indicate significant heterogeneity, and the random effects model was applied. Otherwise, a fixed-effect model was implemented. Subgroup analysis—stratified by tumor type, ethnicity, and sample size—was performed. Sensitivity analysis was conducted to assess the reliability of the results. Meta-aggression analysis was conducted to identify the source of heterogeneity. Publication bias was measured by using both the Begg’s test and Egger’s test. P-values <0.05 were considered statistically significant.All statistical analyses were performed using Stata version 12.0 . The HRs and 95% CIs were used to assess the prognostic role of PD-L1 expression on the survival of patients with BTC. An HR >1 with a p-value <0.05 indicated a poor prognosis in patients with PD-L1 overexpression. The odd ratios (ORs) with 95% CIs were calculated to determine the correlations between PD-L1 expression and clinicopathological parameters. Statistical heterogeneity among studies was determined using the Cochran Q-test and I-squared test , 41. I2 Supplementary Figure 1


Due to its complex nature, it is necessary to demonstrate batch consistency in its biological activity. Potency is the quantitative measure of biological activity and is also a quality attribute of drugs. Here we developed and validated a proliferation assay using Jurkat cells exposed to azathioprine, which is intended to determine the potency of Transferon® according to international guidelines for pharmaceuticals. The assay showed a linear response (2.5 to 40 µg/mL), coefficients of variation from 0.7 to 13.6% demonstrated that the method is precise, while r2 = 0.97 between the nominal and measured values obtained from dilutional linearity showed that the method is accurate. We also demonstrated that the cell proliferation response was specific for Transferon® and was not induced by its vehicle nor by other peptide complex mixtures (glatiramer acetate or hydrolyzed collagen). The bioassay validated here was used to assess the relative potency of eight released batches of Transferon® with respect to a reference standard, showing consistent results. The collective information from the validation and the assessment of several batches indicate that the bioassay is suitable for the release of Transferon®.Transferon Dialyzable leukocyte extracts (DLEs) are blood products obtained from healthy human donors. These extracts are composed of complex mixtures of peptides that induce immunomodulatory activity ,2,3. Several DLEs have been used worldwide as food supplements but only a few are considered drug products after establishing several controls in the process and in the quality of the finished product. Among the controls, the consistency of batches is essential to ensure the efficacy and safety of a drug product. Consistency is demonstrated through the compliance of the criteria established in quality specifications using suitable analytical methods to assess identity, purity, heterogeneity, and potency ,4. The development of analytical methods to be included in the specifications is key to guaranteeing the robustness of the results. Therefore, these methodologies must be validated to demonstrate that the system is suitable for its intended use under a quality control environment ,6. Howev®, such as polydispersity of low-molecular-weight peptides, confirm that it is a complex drug [®, a drug product made of DLEs, has been tested in several models. Whereas the in vitro assays allow for obtaining basic knowledge [The properties of Transferonlex drug . In mostnowledge ,10, the nowledge . In humanowledge , hypersenowledge , and somnowledge ,15.® include an in vivo assay, which evaluates the effect of Transferon® on the survival of mice infected with herpes simplex virus 1 [® to induce interferon-γ expression in Jurkat cells [®, both also present some disadvantages. For instance, the in vivo assay is affected by multiple variables, and implies complex logistics, high costs, and bioethical issues. Despite the issues involved with the in vitro assay, it is a more feasible alternative since it does not exhibit a dose–response curve because it is a limit test. Thus, new bioassays for the evaluation of DLEs potency are still required.So far, the batch release tests of Transferon virus 1 , and an at cells . Althoug®, as rendered in the demonstration of the consistency of this attribute.Recently, the capacity of DLEs to induce Jurkat cell proliferation exposed to azathioprine has been reported without describing the mechanism of action involved for this biological effect, additionally the assay lacks potency estimation with respect to a reference standard and system suitability for a linear response , which a® and azathioprine avoided this effect in a concentration range from 1 to 180 µg/mL. The obtained dose–response curve exhibited a sigmoidal behavior with a half-maximal effective concentration (EC50) of 13.07 µg/mL, which fitted into the four-parameters logistic model (r2 = 0.95) (® from 2.5 to 40 µg/mL to obtain a linear response (r2 = 0.94) . These results are evidence of a favorable effect of Transferon® on cell proliferation  A. Based  = 0.94) B. Transfferation . Due to c model r = 0.95  values lower than 25% at each concentration level of the dose–response curve. The intermediate precision (IP) values  remained within the established acceptance criterion (≤25%) . All theanalysis . r2 ≥ 0.85 during the linear regression analysis and r2 = 0.97 between the nominal and the measured potency  = 3.086, p = 0.2210), Copaxone®  = 0.023, p = 0.8934), and the vehicle control  = 2.836, p = 0.2342) did not show significant differences with respect to zero during the F-test. The response obtained with Transferon®  = 69.06, p = 0.0142) remained within the acceptance criterion for this attribute  with curves constructed with five data points. The %CV was established between 0.5 to 22. All the evaluated batches showed a response ratio ≥ 2.5 compared to the control containing cells treated only with azathioprine. This means that Transferon® doubled the proliferation response  of Jurkat cells despite the presence of a cell proliferation inhibitor (azathioprine).A linear response was observed in a concentration range (from 5 to 30 µg/mL) for all the evaluated batches  = 0.7519, p = 0.5721) showed equality of slopes in all the evaluated batches, demonstrating the parallelism among curves. The consistency of relative potency among batches was observed between 87 and 113%, while the C.I. 95% range was from 79 to 121% for the eight evaluated batches, complying with the acceptance criteria for this attribute , Roswell Park Memorial Institute (RPMI) medium , fetal bovine serum (FBS) , cell proliferation assay kit -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) , and azathioprine .®) acquired from Teva Pharmaceutical Industries  and hydrolyzed collagen (Colagenart®) acquired from LEMAR S.A.P.I. de C.V. .Two products containing a complex mixture of peptides were used as controls, glatiramer acetate (Copaxone®)  provided by Pharma-FT . Batch 18E13 was used as a reference standard.The development and validation of the bioassay were performed using nine commercial batches of human DLEs  and different concentrations of Transferon® (2.5–40 µg/mL) in a final volume of 200 µL/well with RPMI medium supplemented with 10% FBS. Cultures were incubated at 37 °C, 5% CO2 for 72 h. Azathioprine-untreated cells and vehicle (water for injection) plus azathioprine were used as negative controls. After the incubation, MTS was added to every well of the plate and incubated for 4 h at 37 °C in 5% CO2. The O.D. was measured at 490 nm using a microplate reader .Jurkat E6.1 cells were grown in RPMI-1640 medium with 10% FBS and placed at 37 °C in 5% COAll the work in this paper was executed following the recommendations of Chapters 1032 , 1033 1, and 103® was capable of inducing the proliferation of Jurkat cells exposed to azathioprine, and second, to establish the experimental conditions and parameters to minimize variability in the assay. In this stage, we found the concentration range that allowed for evaluating non-linear and linear responses. Additionally, we evaluated the effect of only Transferon® on the proliferation of Jurkat cells as a control for the assay. The proliferation percentage (%) was calculated by dividing the O.D. of the cells treated with Transferon® by the O.D. of the untreated cells with and without exposure to azathioprine.The aims of the development was, first, to evaluate whether Transferon≤ 25% for each dilution level.Precision was evaluated at the level of repeatability and IP. Repeatability was estimated through the %CV between three independent replicates during an analytic run. The IP was measured through the %CV between two analysts, two instruments, and two independent analytical runs. Five concentration levels with independent triplicates were included in all the assays. The established acceptance criterion was a CV r2 ≥ 0.80 between the nominal and the measured values. Accuracy was evaluated as the dilutional linearity at dilution levels of 60, 70, 100, 130, and 140% for the dose–response curve. Relative potency was calculated with PLA analysis using the Softmax Pro 7.0.3 GxP software , and the potency was relative at the 100% linearity level. The acceptance criterion for linearity was ® with respect to other products composed of a complex mixture of peptides, including glatiramer acetate (Copaxone®) and hydrolyzed collagen (Colagenart®). Additionally, the obtained response was compared to the vehicle control (water for injection). The established acceptance criterion was the expected value for the characteristic biological response with Transferon® while complex mixtures of peptides or the vehicle control did not show biological effect.Specificity was evaluated by comparing the biological response of Transferonr2 ≥ 0.8); ratio (Δ) between the maximum response and control response ≥ 2, using at least four points in the construction of the straight line; and CV ≤ 25%.System suitability was established according to the linear model and precision. The acceptance criteria were: linear response  between 74–136%.Relative potency was determined in eight TransferonF distribution was used to test for equality of slopes. The analysis was performed using the Graph Pad Prism 6.0 software . The fitting of raw data to the linear model and relative potency analysis were performed using the Sofmax Pro 7.0.3 GxP software. An analysis of covariance (ANCOVA) with the ®, which confirmed the consistency of the quality attributes. The collective information from this study will allow for the establishment of relative potency as a critical attribute of Transferon®, along with the rapid implementation of this assay in a quality control laboratory as batch release analysis.The validation of the bioassay according to international guidelines for pharmaceutical products showed that it was suitable to evaluate the biological activity of DLEs and to determine the relative potency. In addition, we confirmed that this bioassay was also suitable to determine the relative potency among different batches of Transferon


Cystic fibrosis (CF) is the commonest life-limiting inherited disorder in the UK. It affects many parts of the body including the lungs and gut leading to increased infection and problems digesting food. People with CF need to undergo many treatments each day throughout their whole lives. These include tablets, inhalers and breathing exercises, which are a huge burden, taking up several hours every dayIt is therefore, really important that the treatments we give are supported by good evidence, usually gathered from clinical trials. Unfortunately, we do not have good evidence for many of the CF treatments. We recently ran an exercise known as a James Lind Alliance Priority Setting Partnership (JLA PSP) to find out which the CF community feel are the top priority research questions. People with CF and those who look after them suggested questions to be answered by clinical trials. Through a series of online surveys and workshops these were then shortlisted to give a final top ten.Due to infection risk people with CF are advised not to mix, this meant we had to do things differently to the usual way JLA PSPs are carried out. We used videoconferencing to enable multiple people with CF to participate. Surveys were accessible online and promoted through social media.BackgroundThe James Lind Alliance (JLA) method is well recognised for setting research priorities. The JLA approach involves a combination of surveys and workshop interactions between patients, carers and health care professionals to identify and agree on a “top ten” list of research questions. Respiratory infection is one of the hallmarks of cystic fibrosis (CF). To avoid cross infection, patients are advised not to meet face to face, preventing us following standard JLA methodology. Here we describe adaptations made during our recent JLA Priority Setting Partnership (PSP) in CF.MethodsWe elicited and prioritised research questions, using sequential online surveys, promoted through social media. People with CF participated in steering committee meetings and the final workshop, using videoconferencing. Alterations to workshop methodology enabled participants attending in person and those joining remotely, to contribute equally. We also altered the JLA methodology to include “lone” questions, asked by only one survey respondent. We are now working with the CF community to co-produce research projects that answer these top ten.ResultsThere were 482 respondents, from 23 countries, who submitted 1080 questions. Increases in the number of responses occurred just after promotion on social media. Use of videoconferencing enabled participation of multiple people with CF and ensured participation from anywhere in the world, including hospital inpatients. Inclusion of lone questions resulted in one being included in our top ten.ConclusionsThere is no “one-size-fits-all” for patient involvement methodologies. Through altering the JLA methods to fit our patient group we achieved wide participation. We believe that methods used in our project may also be applied to future partnerships to increase participation, especially where people may be hospitalised or be unable to travel. The methodology we are developing through the JLA PSP CF2 project may be useful for other PSPs to follow. Cystic Fibrosis (CF) is a life limiting inherited multi-system disorder with a high treatment burden but relatively little good quality evidence to guide treatment decisions . In the The James Lind Alliance (JLA) method is well respected for setting research priorities. Their established methodology  involvesRecently we undertook a JLA PSP in CF . RespiraA James Lind Alliance Priority Setting Partnership (JLA PSP) in CF was carried out at the University of Nottingham and commenced in March 2016. The top ten research priorities for clinical research in CF were agreed at a final workshop meeting in Jan 2017 [The James Lind Alliance methodology is well defined and usually follows a pathway which includes; development of a steering group with face to face meetings; an online/paper survey to gather uncertainties; organization of responses and checking against current evidence; interim priority setting to shorten the list of questions; a final workshop with a mixture of small and whole group discussions where all participants are present in person. The workshop brings together representatives from both the professional and lay communities who jointly refine the final top ten priorities.Below we outline where our methodology deviated from the traditional methods and this is also represented in Fig. An elicitation survey to collect research questions from the patient and clinical communityA prioritisation survey, where the patient and clinical communities ranked the final questions in order of importance.We used two online surveys:These surveys were designed to be completed easily on a mobile device (e.g. smart phone). They collected a minimum of demographic information to ensure they could be completed in a short time. The elicitation survey allowed up to five free text research questions to be submitted, with a suggested outcome measure for each Fig. . The priA bespoke Twitter account was set up @questionCF with the associated hashtag #questionCF. This was managed by members of the steering group and aimed to promote the online surveys and increase participation.To prevent risk of spread of infection, people with CF are advised not to meet face to face with others who have the condition . This prPrior to the workshop, test calls were carried out with all those participants planning on joining remotely. This allowed them to gain familiarity with the software and any necessary trouble shooting to be undertaken. Basic etiquette rules to facilitate the workshop such as muting of microphones when not speaking to avoid background noise and not having light source behind the participant were also covered.To ensure that those joining the final workshop remotely had their voice heard, a non-participating “room-buddy” was present in person at the workshop. The “room buddy” was a steering group member with the specific task of advocacy for one remote participant.Usually in JLA PSP workshops question cards are moved around on tables to help the group decide on rankings , 9. The At the end of the process a short survey was sent out to remote participants to find out about their experience of participating via videoconferencing.Through the elicitation survey, we asked participants to tell us what they thought were the most important questions that needed answering by clinical research. We set aside any questions which did not relate to treatment uncertainties and those which had already been answered. Prior to commencing this analysis we undertook a systematic review of gaps in the evidence for treatment decisions in CF which helped with this process . We sortThe final top ten questions were shared with the patient and clinical community immediately after the final workshop, via our dedicated Twitter account (@questionCF). We felt it was important to release the top ten immediately so that those who had contributed would find out straight away. We produced a press release and key stakeholders, such as the CF trust and NIHR, were updated. Postcards with the Top ten were produced and shared by steering group members with the patient community and with their clinical colleagues. Several discussions were held with the National Institute for Health Research (NIHR) to discuss taking ideas from the top 20 questions forward and funding opportunities for them.Through the use of online survey accessible to all, we reached 2.5% of the UK’s CF population and 0.45% of the world’s CF population .Over the course of the PSP our @questionCF twitter account gained 732 followers with 151,000 impressions . Our hashtag #questionCF was used 320 times with 1806 engagements (the number of interactions people have with our content) Fig. . Over haThe use of videoconferencing at steering group meetings allowed two people with CF to participate in each meeting. Had we not used this method, only one person with CF could have attended the meeting in person. Our steering group members were drawn from all over the UK and videoconferencing allowed colleagues to take part who could not travel, or who had conflicting clinical commitments. Six people attended the final workshop remotely, including one who was a hospital inpatient and an observer from the Cystic Fibrosis Foundation in the USA.We evaluated participants’ experience of joining the workshop remotely and five of the six remote participants completed feedback; three said overall they were “very satisfied” with the experience and two “satisfied”. Quotes from feedback from the remote participants are shown in Fig. “Which therapies are effective in delaying or preventing progression of lung disease in early life in people with Cystic Fibrosis?”. Figure From a total of 39 questions classified as “lone questions”, six were carried forward to the public ranking survey. One of these was in the final top ten at position number four Our immediate dissemination of the Top 10 via Twitter™ proved very popular with the tweet of the Top 10 questions gaining a potential reach of 59,061 people from the many retweets and commenting on the post. From the press release several online news outlets picked up the story and the CF trust produced a short video on the top ten. The postcards we produced have been very useful in providing interested parties with a “take home” visual guide to the Top 10.We are pleased that following on from our discussions with the NIHR, a Health Technology Assessment (HTA) call was released in April 2018, with seven of the eight areas with calls for funding related to our JLA top ten. A further call to apply for NIHR funding to support clinical research in CF, with a particular focus on the JLA top 10, is planned.The importance of cross infection in CF has meant that our PSP had to adapt the standard approach, described in the JLA Handbook . The useWhilst some of the issues we encountered were unique to CF, there are many other instances where our adapted methodology may enhance inclusivity. The Digital Technology for Mental Health PSP used an online Twitter™ chat to gather uncertainties amongst a population where an online survey would be difficult for some participants . Other PIn the planning and delivery of our workshop, we aimed to ensure equality amongst the contributors to allow everyone to have an equal say, regardless of whether they were in the room or joining remotely. Future PSPs may be able to take this process further and run virtual workshops where all participants join remotely which may achieve greater equality. In addition to widening access to participation, there could be other benefits such as cost savings and reduced need for travel. However it is also likely that PSP engagement through online surveys and social media may not be comfortable for all potential participants (e.g. the elderly). This may warrant a well-designed study to compare the standard with an adapted methodology to compare the two.The demographic distribution of the CF population means that most are familiar with social media and it is a safe and popular way to interact with other people with CF without fear of cross infection , 17. OurMany PSPs in other areas have discarded questions where they are suggested by one respondent alone. When we summarised our submitted questions, we kept lone questions in a separate list. We decided to allow these “lone” questions to progress by adding them to the list of summarised questions. One of these remained in the process all the way to the final top ten. This would have been lost from the top ten if we had followed the practice of earlier PSPs. It highlights the importance of the steering group in guiding the refining of questions and ensuring the process remains true to what the CF community were telling us.What are the most burdensome aspects of treatment for people with CF?Does this vary with age, sex, and employment or education status?Would it be acceptable to the person with CF to have a reduction in treatment burden at the expense of a more rapid decline in lung function?The NIHR plans to invite funding applications for CF research based on our JLA top ten. We feel that co-production of CF research should continue beyond setting research priorities. Some of the top ten questions are still very broad, for example: “What are the effective ways of simplifying the treatment burden of people with Cystic Fibrosis?” To develop this question into a testable hypothesis we need to understand:We are currently undertaking a project to explore a number of the top ten questions in greater depth in partnership with the CF community, using a combination of online surveys and focus groups (known as JLA PSP CF2). Our goal is to formulate testable hypotheses for clinical research studies. Our protocol for this is published on the University of Nottingham Research Repository .Although the focus of the JLA is to further the evidence base through clinical trials of treatment interventions, other research methodologies, such as registry studies, may also help resolve clinical uncertainties in CF. In the US, this approach has been used by the US CF Foundation, through the “Insight CF” survey . This enThere is no “one-size-fits-all” for patient involvement methodologies. Through altering the JLA methods to fit our patient group we increased participation. We believe that methods used in our project may also be applied to future partnerships to increase participation, especially in conditions where people may be hospitalized, may be unable or too busy to travel. The methodology we are developing through JLA PSP CF2 / QuestionCF2 project will be useful for other PSPs to follow to help turn their top ten questions into fundable research projects.


Electrode materials are crucial for the electrochemical performance of supercapacitors. In view of the high specific surface area, high conductivity of graphene nanosheets and the high pseudocapacitance of polyaniline (PANI), the combination of graphene with PANI has become a research hotspot. In this work, we summarize the recent advance on the synthesis of PANI and graphene/PANI composites, and their application in supercapacitors. The synthesis of PANI is the basis of preparing graphene/PANI composites, so we first introduce the synthesis methods of PANI. Then, the advances of two dimensional (2D) and three dimensional (3D) graphene/PANI composites are summarized according to the inherent feature of graphene. The 2D composites of pristine graphene and functionalized graphene with PANI are introduced separately; furthermore, the 3D composites are classified into three sections, including flexible graphene/PANI composites, graphene framework based composites, and printable graphene/PANI composites. At last, aiming at solving the current challenges of graphene/PANI composites, we put forward some strategies for preparing high performance graphene/PANI composite electrodes. Moreover, these materials often show a stable long-term cycling performance. The other electrode materials have pseudocapacitive feature, which achieve the electron transfer and charge storage through redox reaction . T. THard tn et al.  synthesi network a. The cor et al.  preparedSoft templates. In the synthesis of PANI, surfactants are also named as soft templates. Using different surfactants, Ma et al.  triazine (TCTA) and PDA . T. T68]. Tg et al.  grew PANn et al.  preparedn et al.  depositeCompared to other 3D carbon frameworks, graphene frameworks have an abundant interconnected porous structure, high specific surface area and excellent conductivity, which can be used to grow a great many of PANI nanoparticles. In this section, we introduce the advance of 3D graphene/PANI composites according to the preparation methods of 3D graphene framework, including chemical vapor deposition (CVD), porous template method, hydrothermal treatment, freeze-drying method and self-assembly of graphene. −1 in 1 M HClO4, with 93.2% capacitance retention after 1000 cycles. Furthermore, 3D graphene network derived from CVD method was served as porous conducting network for growing PANI nanofibers in H2SO4 solution (−1 at 1 mA cm−2. In another work . I. I−1 in her work , PANI naramework . After e−1 or 205.4 F cm−3. Furthermore, the capacitance of this device retained 80% after 5000 cycles. Except for dipping and drying method, Wu et al. [2, hierarchical porous 3D frGO-F skeleton was prepared and in situ polymerized PANI nanowires. The flexible frGO-F/PANI composites were obtained with macro/mesoporous structure. Attributed to the synergistic effect of 3D bicontinuous hierarchical porous framework and PANI nanowires, as-assembled symmetric supercapacitor delivered a specific capacitance of 939 F g−1, and high capacitance retention of 88.7% for 5000 cycles. Besides porous NF, carbon foam was also used as 3D freestanding porous template. In Hong’s work [−1 at 1 A g−1, and the PANI capacitance reached 1003 F g−1.Besides CVD method, porous NF can be served as a porous template to prepare 3D porous rGO foam (rGO-F) by repeatedly dipping and drying. With this way, Yu et al.  preparedu et al.  preparedu et al. , a “temp−1 at 2 A g−1, and an excellent rate capability of 73% capacitance retention at 100 A g−1. Zhao et al. [−1 at 10 mV s−1. Moreover, based on the highly concentrated GO/PANI gels casted on the PTFE tape, 3D porous rGO/PANI hybrid film was prepared through steamed water regulation after hydrothermally treating at 200 °C for 5 h [−1 at 1 A g−1. Meanwhile, the film electrodes also had a high capacitance of 808 F g−1 and an energy density of 28.06 Wh kg−1 in a symmetric supercapacitor.Hydrothermal method is a simple way to prepare 3D porous graphene hydrogels. Under the high pressure water vapor, the self-assembly of graphene nanosheets occurred with the removal of oxygen-containing groups of GO, and the π-π interaction of hydrophobic rGO sheets induced the formation of macroporous structure. In recent years, the hydrothermal method has been widely adopted to fabricate 3D graphene hydrogels (GH) with highly conductive, mechanical robust and macroporous structure . Based oo et al.  also pre for 5 h . When us−1 at 0.5 A g−1 in a three-electrode cell. In another work, the PANI was grown on rGO firstly by interfacial polymerization. Then, through a reassembly of as-prepared GO@PANI and GO sheets in a hydrothermal reaction, 3D hierarchical porous rGO-PANI aerogels were fabricated [2 g−1), high conductivity of graphene framework and the sandwiched PANI/graphene/PANI layered structure, the hybrid aerogel electrodes delivered a high areal capacitance of 453 mF cm−2 in symmetric supercapacitors. Hoa et al. [2. In the second step, a hydrothermal treatment was conducted to prepare rGO-g-PANI aerogels. After coated on the glassy carbon electrode, the composite exhibited a high specific capacitance of 1600 F g−1 at 12 A g−1 and capacitance retention of 91.3% for 3000 cycles.Commonly speaking, a two-step procedure is required to prepare 3D graphene/PANI composites through hydrothermal method. The first step is the preparation of graphene hydrogels, and the second step is the synthesis of PANI on the hydrogel. However, in order to effectively avoid the restacking of rGO sheets, PANI can be grown on the graphene sheets firstly. For example, Wang et al.  preparedbricated . Due to a et al.  prepared−1 at 2 mV s−1. Except for hydrothermal temperature, the polymerization temperature of aniline monomers also affected the electrochemical performance of graphene/PANI composites. Ates et al. [−2 (or 323.9 F g−1) in symmetric supercapacitors, which was much higher than the composite synthesized at 0 °C. In order to increase the active sites on graphene nanosheets to grow more PANI, GO sheets were chemically treated in HNO3 and H2O2, and then hydrothermally treated to prepare 3D multi-growth site graphene (MSG) foam [−1 from 432 F g−1, and the capacitance retention rate was 89.5% after 10,000 cycles at 10 A g−1.In order to explore the influence of hydrothermal conditions on the PANI morphologies and electrochemical performance, Wang et al.  synthesis et al.  preparedSG) foam . Compare3·H2O solution. Zhu et al. [−1 (0.1 A g−1), and a high capacitance retention rate of 95.9% for 5000 cycles. Similar to NH3·H2O, the urea was also employed as both doping nitrogen source and reducing agent. After one-step hydrothermal treatment, 3D nitrogen-doped graphene/PANI hydrogels (RGNP) were obtained. The RGNP electrode exhibited a specific capacitance of 589.3 F g−1 at 3 mA cm−2 in a three-electrode cell. In addition, by using glucose as the reducing agent and CaCO3 particles as templates, 3D-rGO porous framework was obtained by a hydrothermal reaction [3 particles were removed in HCl solution, and PANI nanowires were grown on 3D-rGO framework by in situ polymerization. The prepared 3D-rGO/PANI composite electrode delivered a capacitance of 243 F g−1 at 1 A g−1.Nitrogen doped graphene framework can also be synthesized by hydrothermal reaction in NHu et al.  preparedreaction , then Ca2 as the oxidants. As-prepared N-3D-rGO/PANI-B had a capacitance of 282 F g−1 at 1 A g−1, this performance was much better than that of the composite prepared by using only APS oxidant. During the freezing process, the control of the temperature gradient also affected the performance of graphene aerogel. Wu et al. [−1 (1 A g−1), much higher than that of conventional graphene aerogel/PANI composite (345 F g−1).Different from hydrothermal method, freeze-drying method is much easier, with no hydrothermal process. Liu et al.  adopted u et al.  prepared−1 at 1 A g−1. However, in stretchable solid-state asymmetric supercapacitors consisted of 3D-G/PANI and 3D-G, the energy density reached 77.8 Wh Kg−1, and the capacitance retention was 95.6% for 10,000 cycles, moreover, the capacitance retained 91.2% after stretching for 100 cycles. In another work, Li et al. [−1 (847 F cm−3), with a stable cycling for 10,000 cycles.Through heating the mixture of GO and in-situ polymerized GO/PANI in sodium ascorbate solution at 95 °C, Li et al.  prepared−1 at 1 A g−1, and the capacitance retained 96.14% after 10,000 cycles at 10 A g−1. In addition, Wu et al. [N-methyl-2-pyrrolidone and formed PANI@rGO nanosheets. In the second assembly, the reduction process of GO at 90 °C induced the 3D assembly of PANI@GO nanosheets, and PANI coated rGO framework was obtained. The composite electrode exhibited a high specific capacitance of 808 F g−1 (53.33 A g−1) or 5.717 F cm−2 at 377.4 mA cm−2.By using a self-assembly strategy, a kind of self-suspended polyaniline (S-PANI) containing PEG segment  was usedu et al.  prepared−1 at 0.5 A g−1, and the capacitance of PANI nanoparticles was calculated as 763 F g−1.By using a electrostatic layer-by-layer (LbL) assembly method and electropolymerization, Gupta et al.  preparedWith the rapid development of printing techniques , some el−1 (1 A g−1) and a capacitance retention of 85% after 10,000 cycles. Chi et al. [−1 (1 A g−1) or 190.6 mF cm−2 (0.5 mA cm−2), and the energy density was 24.02 Wh kg−1 at 400.33 W kg−1. In addition, Wang et al. [−2. By using a reproducible printing method and bubbling delamination, a flexible freestanding rGO paper a was prei et al.  preparedg et al.  develope−2 (16.55 F cm−3). Moreover, this micro-device also showed a stable capacitance after bending and twisting tests, which was suitable for wearable and portable electronic devices.Different from traditional 3D printing techniques, Song et al.  developeWhen compared to 2D graphene/PANI, 3D graphene/PANI composites can be directly served as electrodes for their freestanding feature, especially in the two-electrode supercapacitors. Moreover, 3D graphene/PANI composites exhibit a superior specific capacitance and cycling performance, as given in The hybrid electrodes of graphene and PANI have been widely reported in high performance supercapacitors. In this review, we summarized the progress of synthesis methods of PANI and graphene/PANI composites, and their application in supercapacitors. −1, and only 6.78% works (four papers) achieved the higher capacitance (≥800 F g−1) in two-electrode systems. From the long-term cycling performance, 16.95% works (10 papers) listed the capacitance retention over 10,000 cycles, and only 10.17% works (six papers) had a high capacitance retention (≥85%). Therefore, there is still a long way to further enhance the electrochemical performance of graphene/PANI composite electrodes.As shown in A great progress has been achieved in the field of PANI and graphene/PANI composites, and the specific capacitance and long-term cycling performance of graphene/PANI electrodes have enhanced greatly. However, there are still some challenges with graphene/PANI composite electrodes, such as, the controllable synthesis of PANI nanostructures, the agglomeration of PANI particles and graphene nanosheets, the optimized ratio of graphene and PANI, high cost of graphene and the design of high performance graphene frameworks. These challenges are just retarding the rapid development and the commercialization of graphene/PANI electrodes in supercapacitors. In order to solve these problems, we put forward some strategies to develop graphene/PANI composites for high performance supercapacitors.(1) Selecting suitable synthesis method of PANI and considering the combination of chemical synthesis and surface treatment. According to the features of graphene, a suitable synthesis method should be adopted firstly. For example, chemical oxidative method and interfacial polymerization are fit for growing PANI nanostructures on the well dispersed graphene suspension or porous graphene frameworks; while electrochemical deposition method are suitable for growing PANI on the flexible graphene film. In addition, the surface treatment of PANI in chloroform could greatly enhance the specific surface area and pore volume of PANI, which improve the rate capability of PANI. Therefore, the positive effect of surface treatment on PANI should not be ignored. (2) Improving the cost performance of graphene/PANI composites based on functionalized graphene. Compared to the simple hybridization, covalently-grafted graphene can initiate the polymerization of aniline monomers. The functionalized graphene/PANI composites exhibit a superior electrochemical performance than that of ordinary graphene/PANI composites. In addition, the controllable synthesis of PANI is really crucial for improving the cost performance, for example, synthesizing PANI by adjusting polymerization temperature, adopting compound oxidants, or selecting suitable dopants. (3) Designing hierarchical porous graphene framework and hybridizing with an optimized loading of PANI nanostructures. Freestanding porous graphene framework overcomes the blocking effect of polymer binders and ensures an excellent conductivity; moreover, it also facilitates the rapid transfer of ions, which greatly improve the rate capability of supercapacitors. In addition, based on the graphene framework, the PANI nanostructures and their loading amount should be controlled to achieve the high performance supercapacitors.(4) Developing suitable graphene/PANI composite inks and corresponding printing techniques. In recent years, printing technique has been developed to fabricate complicated electrodes repeatedly and quickly. However, the inherent feature of inks will decide the performance of printed supercapacitors. Therefore, high performance graphene/PANI inks can be prepared by adjusting their mass ratio, conductivity, flowability and mechanical strength. Printable inks and suitable printing techniques will promote the fast development of intelligent energy storage microdevices and flexible wearable supercapacitors.


The influence of humidity on the Brillouin backscattering power and linewidth are presented for the first time to our knowledge. The Brillouin backscattering power at a pump wavelength of 1319 nm is affected by temperature and humidity. The Brillouin linewidth is observed to be a function of temperature but not of humidity. The strain coefficient of the BFS is determined to be In this paper perfluorinated graded-index polymer optical fibers are characterized with respect to the influence of relative humidity changes on spectral transmission absorption and Rayleigh backscattering. The hygroscopic and thermal expansion coefficient of the fiber are determined to be  The influence of humidity on numerous polymers has been investigated in the last decades ,2,3,4. TPerfluorinated graded-index POFs (PFGI-POFs) use cyclic transparent optical polymer (CYTOP) as core base material and polycarbonate as over-cladding material. CYTOP fibers are characterized by lower water absorption, lower attenuation and higher bandwidth in the near infrared region in comparison to PMMA ,13. ThesIn a previous paper  we preseThe optical transmission spectra of the PFGI-POF were measured at 4 temperature and 5 humidity values in a climate cabinet. A broadband light source (Yokogawa AQ4305) launched light into a 200 m GigaPOF-50SR by Chromis Fiberoptics , which  1319 nm . An overThe absorption peaks correlate to vibrational absorption bands of the C–F bond (multiple peaks around 1130 nm ) and theThe wavelengths of es at λ1 ,30 mightt λ1 [λ2 . The wavent with . Increasent with . FurtherThe measurement results are consistent to . In addiSwept-wavelength interferometry (Luna OBR 4413) was used to performed the following measurements. The Luna OBR4413 has a center wavelength of 1306 nm and the sweep range of 0.8 nm was set to achieve a spatial resolution of 5 cm. In contrast to the OSA measurements, a 1 m single mode silica fiber was used to connect the PFGI-POF to the Luna device. Consequently, the measured signals are dominated by the Rayleigh scattering of the fundamental mode, due to mode selective coupling. Nevertheless, the Rayleigh influence of higher optical modes on the measurement signal can not be excluded due to the strong mode coupling of the PFGI-POF . Two exaIncreasing humidity values were observed to form a sharper declining slope in the backscatter traces. A linear regression function was computed for each measured backscatter trace. The determined slope values are shown as a function of relative humidity for specified temperatures in −1 and −1 within the given measurement ranges. The given uncertainty values are the threefold standard deviation from the linear regression applied on all measured data points, respectively. On the basis of these coefficients, the measured fiber length l can be described as a function of temperature and humidity as presented in Equation  and the 1550 nm  and humi 1550 nm . Unfortu−1) is significant compared to polyimide and acrylate coated silica single mode fibers at standard layer thickness of 15 −1 and 0.2 −1, respectively [−1 for 876 The humidity-induced measured fiber length change may originate from the humidity-induced change of the fiber core properties and from the humidity-depending behavior of polycarbonate ,4, used ectively ). Thoughhickness ).In  we reportemperature-induced reduction of the SBS power level can be considered to be dominantly caused by the thermoplastic characteristic of CYTOP and the vibrational absorption dependence .The BGS relating to the first 90 m fiber segment is affected by temperature fluctuations and strain changes, whereas the BGS of the non-strained segment is dominantly influenced by temperature fluctuations. The BGSs of the two fiber segments are separating from each other, since the applied strain causes a greater BFS change compared to the BFS change caused by temperature fluctuations. Thus, two Lorentzian regression functions were computed for each measured electrical spectrum to determine the BFS corresponding to each fiber segment (see BFSHowever, the spectral separation of the two BGSs was not feasible for strain values below 0.6%, as the two BGSs are superposed. Therefore, a Lorentzian regression calculation was applied to the complete measured spectrum. The determination of strain-related BFS using the complete spectrum was possible since the SBS power of the strained section was dominant compared to the unstrained section. Unfortunately, the obtained BFS contains strain and temperature information which increases the uncertainty of the measurements compared to the BGS separation approach. The strain-induced BFS change was determined by subtracting the BFS of the strained fiber from the BFS of the unstrained fiber. All strain measurements were performed at room temperature. The maximum temperature drift during a single measurement was observed to be ±0.5 K.The BFS change caused by applied strain is plotted in CS= −1.5 ± 0.9  1550 nm  in the s 1550 nm . Figure In a previous publication  we demon−1 and At all temperatures a BFS decrease with rising humidity values was observed. As previously shown in The isothermal BFS graphs demonstrate divergence from each other with rising humidity values. However, a detailed analysis of the temperature behavior of the humidity-induced core property changes apart of fiber strain can not be given based on this data set. The coefficients uced BFS .When taking into account the fiber strain and the change of the refractive index and/or density, the lack of a humidity-induced BFS hysteresis presented in  can be e−1 and In this paper the influence of humidity on SBS in PFGI-POF is described. Fiber characterization measurements demonstrated the fiber attenuation to be a function of wavelength, temperature and humidity due to vibrational absorptions. Furthermore, the fiber expansion coefficients for humidity and temperature are determined to be


Furthermore, CLSM shows excellent correlation with conventional optical microscopy, atomic force microscopy, Kelvin probe force microscopy, conductive atomic force microscopy, scanning electron microscopy and Raman mapping.Two-dimensional (2D) materials such as graphene have become the focus of extensive research efforts in condensed matter physics. They provide opportunities for both fundamental research and applications across a wide range of industries. Ideally, characterization of graphene requires non-invasive techniques with single-atomic-layer thickness resolution and nanometer lateral resolution. Moreover, commercial application of graphene requires fast and large-area scanning capability. We demonstrate the optimized balance of image resolution and acquisition time of non-invasive confocal laser scanning microscopy (CLSM), rendering it an indispensable tool for rapid analysis of mass-produced graphene. It is powerful for analysis of 1-5 layers of exfoliated graphene on Si/SiO Single-domain epitaxial graphene (EG) grown on the silicon face of SiC(0001)18 has several advantages, such as removing the need to transfer the graphene onto an insulating substrate for device processing, as is the case for chemical vapor deposition (CVD) growth. Recent progress in CVD and EG growth demonstrates the potential for mass production of homogeneous graphene at the wafer-scale22, and increases the demand for a characterization method that is fast, accurate, and accessible. Previously, optical microscopy was demonstrated to be a useful tool for rapid identification of layer inhomogeneities in EG over hundreds of micrometers23. However, low contrast and poor spatial resolution are significant limiting factors. Currently, Raman spectroscopy and scanning probe microscopy (SPM), including Kelvin probe force microscopy (KPFM), are the most widely used methods of characterizing the material quality. Raman spectroscopy is a nondestructive tool for structural analysis of graphene, and is furthermore sensitive to the doping level and strain in graphene29. For one-layer graphene (1LG) the fingerprint in the Raman spectrum is a symmetric 2D peak at ∼2700 cm−1 that can be fitted by a single Lorentzian30. However, a careful analysis of the shape of the 2D peak is required to identify increasing number of GLs. The topography imaged by atomic force microscopy (AFM) is the another method for determining layer thickness of CVD or exfoliated graphene on various substrates. However, for graphene on SiC, identification of EG layers from the topography is far from straight-forward using AFM alone31, due to terrace structure of the SiC substrate, which develops concurrently with the EG and thus has a strong influence on the layer growth and uniformity32. Recently, KPFM was shown to be a more reliable method for distinguishing the number of EG layers33. Nonetheless, Raman and SPM methods are time consuming and typically limit the scan size to a few tens of micrometers. Scaling up the production process requires fast and accurate characterization of the material quality at the wafer scale, while at the same time retaining submicrometer spatial resolution. We believe that confocal laser scanning microscopy (CLSM) is the tool that meets all of the aforementioned requirements with applications in real-time observation of graphene growth34 and nanomaterials in biological systems36.Wafer-scale graphene material is of interest for quantum Hall resistance standards2 and SiC, compared to conventional optical microscopy (OM), Raman spectroscopy, AFM, conductive AFM (C-AFM), KPFM, and scanning electron microscope (SEM) methods. CLSM can simultaneously produce intensity and topography images as well as has a lateral resolution that can be pushed beyond the optical diffraction limit. The depth-of-field is enhanced by digitally selecting in-focus regions from multiple images at different focal planes, enabling high resolution over larger areas. First, we discuss CLSM results on various thicknesses of exfoliated graphene transferred to Si/SiO2 substrate. By comparing the results to AFM and Raman measurements, we present a method for assessing the CLSM intensity and height for graphene of different thicknesses. Next, we apply CLSM and Raman spectroscopy to CVD-grown graphene transferred to Si/SiO2 substrate to demonstrate fast and accurate, largescale analysis. Finally, we apply CLSM to EG on SiC demonstrating the speed, accuracy, and versatility of CLSM compared to OM, SEM, AFM, KPFM, C-AFM, and Raman microscopy.In this report, we demonstrate that reflection mode CLSM is a superior tool for rapid characterization of large-area graphene and graphene nanostructures on Si/SiO2 substrates. The OM images of the sample was carried out on a Nikon L200N optical microscope [see 2. Both the imaging techniques are performed in reflection mode and compared to the corresponding AFM image in 37. The same region imaged with the CLSM shows a signal-to-noise ratio three times higher than OM as well as provides in situ map of the height. Raman spectra were recorded at the nine different regions of the sample as indicated by red dots in −1, respectively) and the height ratios of G/2D peaks are ∼0.7  also increases approximately linearly with the layer number , The thickness of the first graphene layer (P1b) as measured by AFM is 1.35 ±0.1 nm , with su2 substrate. The CLSM image reveals finer structures that are not clearly visible in the OM image, such as the wrinkle at point labeled as P2 in 40. Furthermore, the graphene layers in the scroll are not tightly packed, which broadens the 2D-peak. The G/2D peak ratio is also larger due to the presence of several layers of graphene within the scroll.39.We first present the EG sample with partial coverage of graphene, as shown in 25 that may accompany the decomposition of single, SiC atomic layers.The darker regions in OM and CLSM intensity images are the electrically insulating interfacial layer or bare SiC, as verified with a several SPM and Raman techniques . ImagingThe CLSM intensity image of We obtained additional measurements with AFM, KPFM, SEM, C-AFM, and Raman microscopy, on EG nanoribbons for accurate identification of their structure. The CLSM intensity image in 41. The SEM is able to clearly distinguish graphene regions from IFL with high spatial resolution .2 substrate, CLSM images show excellent correlation to OM, Raman spectroscopy, and AFM height mapping, where the latter two can be used to calibrate the CLSM intensity and height to directly determine graphene layer thickness. For graphene on SiC substrate, the measured reflected intensity from 1LG is ≈3% higher than that from adjacent IFL regions and the reflected intensity from 2LG is ≈2% higher than the 1LG  and a feedback loop tracks the surface of the sample by adjusting the Z-piezo height to maintain a constant amplitude of the cantilever oscillation. The phase of the cantilever oscillation is also compared to the sine wave driving the cantilever oscillation, and thus, AFM achieves simultaneous mapping of the topography and tapping phase, which is a measure of the energy dissipation between the probe and sample, thus encompassing variations in adhesion, composition, friction, viscoelasticity, and other mechanical properties of the sample43.AFM was performed in tapping-mode in air using a Bruker Dimension FastScan SPM. In this mode, the probe oscillates at its fundamental resonance , AC voltage (VAC) is applied to the electrically conductive probe, which shifts the cantilever resonance due to the electrostatic attraction/repulsion and thus produces side lobes at f0±fmod. When the FM-KPFM feedback loop applies an additional DC voltage to the probe (VDC), the amplitude of the side lobes is proportional to the difference between VDC and the surface potential of the sample . The surface potential is determined by the VDC minimizing the side lobes, i.e., when potential of the probe is equal to the potential of the sample. The surface potential map is obtained by recording VDC pixel by pixel. The surface potential values of the sample can be converted to a work function using, ΦSample = Φprobe − eΔVCPD, provided the work function of the probe (Φprobe) is known. For further details see ref. 44KPFM was performed by means of frequency modulation (FM) using a Bruker Dimension Icon SPM2 were acquired under ambient conditions with 514.5 nm excitation (Renishaw InVia), which is focused to an approximately 1 μm spot on the sample through a ×50 objective (0.75 NA). The Raman spectra and mapping of EG on SiC were acquired under ambient conditions with 532 nm excitation (Renishaw InVia), which is focused to an approximately 0.8μm spot on the samples through a ×100 objective (0.85 NA). Raman maps were performed by raster scanning the laser with a step size of 100 nm and collecting the spectra with an exposure time of 1 s for each point, 1800l/mm grating and high confocality (20μm slit opening). Raman maps of the G-and 2D-peaks area, intensity, width and shift were generated from fitting the data.Raman spectra of graphene on Si/SiOThe datasets generated and analyzed during the current study are available from the corresponding author on request.Supp1


Graphene has been widely used in novel optoelectronic devices in decades. Nowadays, fabrication of large size monolayer graphene with spectral selectivity is highly demanded. Here, we report a simple method for synthesizing large size monolayer graphene with chemical functionalized groups in solution. The few layer nano-graphene can be exfoliated into monolayer nano-graphene under short time UV irradiation in protic solution. The exfoliated monolayer nano-graphene could experience deoxygenation during long time UV exposure. At the same time, the edge of nano-graphene could be activated under deep UV exposure and small size nano-graphene sheets further aggregate horizontally in solution. The size of aggregated rGO increase from 40 nm to a maximum of 1 μm. This approach could be one promising cheap method for synthesizing large size monolayer reduced graphene oxide in the future. Herein, we report a new strategy to fabricate large size chemical functionalized monolayer graphene by deep UV irradiation. The layered nano-graphene can be exfoliated to monolayer under short time UV exposure. The new sp2 domain can be restored during long time UV exposure. Furthermore, the edge carbon atom can be activated during UV irradiation, leading several monolayer nano-graphene sheets to aggregate horizontally to form large size monolayer graphene.Graphene is a potential material for ultrathin optoelectronic and photodetection devices because of its high carrier mobility and high optical transparency , 2. The 2 domain , which rGraphene oxide (GO) was synthesized from natural graphite by modifying the Hummer’s method as reported in our previous work . The resN,N-dimethylformamide (DMF)) was added. The mixture was heated to 176 °C for 5 h. The supernatant was filtered through a 0.22-μm microporous membrane. Finally, the colloidal solution was the few layer nano-graphene solution.4.4 mg GO solid was transferred to Teflon-lined autoclave and 12 mL ethanol  were obtained by deep UV light  irradiating, as illustrated in Scheme The photoluminescence (PL) and Fourier-transformed infrared (FTIR) were measured on steady-state fluorescence spectrometer  and FTIR spectrometer , respectively. The morphology and height were characterized by atomic force microscopy (AFM) operating in tapping mode at room temperature on Si substrate (NT-MDT Prima). The crystallinity of the sample was performed by high resolution transmission electron microscope (HRTEM) .AFM was used to characterize the morphology and size of nano-graphene. The results of nano-graphene under various UV irradiation time are shown in Fig. Figure D and G (ID/IG) model of Raman scattering can reflect how hydrogen atom is attached to the graphene. To investigate the exfoliation mechanism, the Raman spectra of few layer nano-graphene under various UV exposure times were measured. The results are shown in Fig. ID/IG increases from 1.21 to 1.43, indicating the presence of binding hydrogen on the few layer nano-graphene surface after UV exposure. C atom moves toward the out-plane direction and lead to breaking of the vdW force between graphene layers [As reported by Lee et al. , hydroge−1). The absorption peak at 1740 cm−1 and 3129 cm−1 correspond to the stretching mode of C atom bonded carboxyl (-COOH). The absorption at 2850 cm-1 and 2920 cm−1 reveal the existence of C-H. The broad band absorption (3100–3700 cm−1) peaked at 3450 cm−1 is attributed to the hydroxyl groups (-C-OH). The C=C absorption of intrinsic sp2 domain at 1641 cm−1 is also presented. After a short time (240 s) UV irradiation, the peaks of epoxy group, hydroxyl group, carboxyl group, and C=C did not change significantly. However, the absorption peak of C-H becomes more pronounced. This is consistent with the result we obtained from Raman, indicating that the H atom binds to the C atom and enhances the absorption of C-H. It also shows that short-time UV irradiation does not lead to the reduction of nano-graphene and oxygen functional groups in nano-graphene do not change during the exfoliating process.The surface of the graphene oxide is generally rich in oxygen-containing functional groups such as -COOH, C-OH, C-O-C, and so on. These oxygen-containing functional groups introduce defect states, which are important sources of graphene oxide luminescence. Therefore, it is very important to study the changes of the functional group of nano-graphene during UV irradiation. Infrared absorption is a very effective method for studying chemical functional group changes. The exfoliation mechanism can be more deeply understood by measuring the changes in the infrared absorption spectra of the nano-graphene irradiated by ultraviolet light. Figure −1 is significantly reduced. At the same time, new C-O-C absorption occurs and it overlaps with previous C-O-C absorption, resulting in extensive C-O-C absorption. Secondly, absorption of C=O moves from 1740 cm−1 to low wavenumber direction (1720 cm−1). This is due to the increase of the conjugate system. The third major change is the appearance of a new C=C in-plane absorption peak at 1562 cm−1. This is because that photo-reduction process of GO can induce deoxygenation and restore the sp2 domain [On the other hand, the FTIR spectra of nano-graphene changes significantly Fig. b after a2 domain , 23, 24.In order to study the effect of layer changes on the optical proprieties, we measure the steady-state fluorescence of nano-graphene under different UV exposure time. Figure 2 domain (302–380 nm) and sp3 defect luminescence caused by the oxygen-containing functional group [2 domain due to the stacking induced band bending. The few layer nano-graphene emit pure UV light. We assign luminescence with peak at 307 nm to the fluorescence of the eigenstate of the sp2 domain. Meanwhile, the visible portion (peak at 500 nm) is derived from the luminescence of the defect states as literature reports [2 domain gradually disappears as the nano-graphene is exfoliated into monolayer and we believe that thickness change of nano-graphene is the main reason for the fluorescence change.Fluorescence of graphene has been systematically studied. Origin of luminescence is mainly assigned to eigenstate-induced fluorescence caused by the spal group , 25–27.  reports , 25. It −1 (Fig. For a clearer, intuitive representation of the change in graphene oxide functional groups during the reduction process, we measured steady-state fluorescence during the reduction process Fig. . The flu−1 Fig. . Thus, t2 domain [Accompanied with the visible emission change, a surprising phenomenon, UV emission centered at 326 nm with a shoulder at 360 nm is shown and increase with reduction time. The reduction of graphene oxide is usually accompanied by the formation of a new sp2 domain . We specTo further examine the changes of physical structure for nano-graphene after long time UV irradiation, we measured the morphology of rGO using AFM. As illustrated in Fig. N and the Langmuir adsorption isotherm can be written asBecause the intensity is directly related to the aggregation, we investigate the aggregation dynamics of nano-graphene by analyzing the PL in the UV range. The UV exposure time was normalized for the concentration of nano-graphene bonded to each other. Figure N0 is the initial UV intensity. The associated equilibrium constant k can be obtained by fitting the experimental data. For a system at chemical equilibrium, the Gibbs free energy is givenHere, k to the Eq. ∆G ≈ − 4.43 kJ/mol. The ∆G < 0 indicates that this reaction is thermodynamics favored at room temperature [2 structure but hardly contains any functional groups for spectral selectivity. Our UV induced nano-graphene aggregation could contain various functional groups which have unique optical property and can be used as identification sites for selective detection.When we applied perature . Thus, s2O. Figure 2O. The PL of nano-graphene in DMF performs deep UV emission, which origin from the stacking of nano-graphene layer. There is only slight change even after 115-min UV irradiation. It indicates that after a long period of exposure to ultraviolet light, no exfoliation or aggregation happens. The height distribution of nano-graphene in DMF is shown in Fig. 2O is centered at ~ 475 nm (Fig. 2O is < 1.0 nm (Fig. 2O. The results above show that stacking of nano-graphene can be controlled by using different solvents.In order to study the effect of solvents on the structure of nano-graphene, we prepared nano-graphene in aprotic solvent DMF and protic solvent H nm Fig. c. There  nm Fig. d and it In summary, we discovered that few layer nano-graphene can be exfoliated to monolayer nano-graphene due to the bonded H atom on the surface under short time deep UV irradiation in protic solution. The monolayer nano-graphene could aggregate into large size monolayer rGO under excess time UV irradiation. The AFM results clearly show that large size monolayer rGO is formed by aggregation of several small nano-graphene sheets. The aggregation of small nano-graphene agrees with the Langmuir adsorption isotherm model, which indicates that the edge of nano-graphene can be activated and can bond with other nano-graphene. This UV-induced growth method may promote the low-cost, large-scale fabrication for monolayer graphene in the future.


PYCR1) gene in humans caused cutis laxa (ARCL) disease, with progeroid appearance, lax and wrinkled skin, joint laxity, osteopenia, and mental retardation phenotypes. In this study, we aimed to generate pycr1 knockout (KO) zebrafish and carried out biochemical characterizations and behavior analyses. Marked apoptosis and senescence were detected in pycr1 KO zebrafish, which started from embryos/larvae stage. Biochemical assays showed that adult pycr1 KO fish have significantly reduced proline and extracellular matrix contents, lowered energy, and diminished superoxide dismutase (SOD) and telomerase activity when compared to the wild type fish, which suggested the pycr1 KO fish may have dysfunction in mitochondria. The pycr1 KO fish were viable; however, displayed progeria-like phenotype from the 4 months old and reach 50% mortality around six months old. In adult stage, we found that pycr1 KO fish showed reduced locomotion activity, aggression, predator avoidance, social interaction interest, as well as dysregulated color preference and circadian rhythm. In summary, we have identified multiple behavioral alterations in a novel fish model for aging with pycr1 gene loss-of-function by behavioral tests. This animal model may not only provide a unique vertebrate model to screen potential anti-aging drugs in the future, but also be an excellent in vivo model towards a better understanding of the corresponding behavioral alterations that accompany aging.Aging is a natural process that internal gene control and external stimuli mediate. Clinical data pointed out that homozygotic or heterozygotic mutation in the pyrroline-5-carboxylate reductase 1 ( PYCR1 gene displayed typical premature aging symptoms of lax wrinkled skin, osteopenia, joint hyper laxity, and postnatal growth delay [PYCS (Pyrroline-5-Carboxylate Synthetase) and PYCR (Pyrroline-5-Carboxylate Reductase) are two key enzymes that control the formation of L-proline from L-glutamate ,2. The eth delay ,7,8,9,10Several animal models, such as worm ,12, fly The fish model was suitable for studying aging-associated phenotypes and the underlying mechanisms for number of reasons. First, several biomarkers, such as lipofuscin, SA-β-gal, and permeability on Smurf dye, have been validated to be associated with senescence or aging ,23,24,25pycr1 KO zebrafish model by Transcription activator-like effector nuclease (TALEN)-mediated gene knockout approach to study how pycr1 affects aging to facilitate to screen potential chemicals that restore premature aging phenotypes. We observed that the pycr1 KO fish exhibit premature aging phenotypes, such as dwarfism, slow swimming ability, loss of fertility at the age of six months, and increased mortality when compared to the wild type fish at same age. In addition, we also identified several behavioral alterations in pycr1 KO zebrafish. Therefore, our pycr1 knockout zebrafish model may have potential to serve as a platform to screen the potential anti-aging therapeutics in the future.We established a pycr1 gene, and showed that the decreased expression of pycr1 induced massive apoptosis in both zebrafish and Xenopus embryos [pycr1 on aging-related processes. In humans, the PYCR1 gene contained eight exons on chromosome 17, while the pcyr1 gene in zebrafish contained eight exons on chromosome 3. One TALEN pair targeting zebrafish pycr1 exon 4 was assembled  when compared with the un-injected group (green color)  B. The futerminus C,D. F1 pition 54 C,D. On tition 54 B.pycr1 TALEN specifically targeted to pycr1 exon 4. This question was addressed by searching the potential off-target sites within zebrafish genome and examining whether any mutation occurred on the potential off-target sites by HRMA. We identified four potential off-target sites , which carried high similarity on sequence to the on-target site with 4 to 5 bp mismatch in either the left or the right arm of TALEN pair  fish. The results showed that the degrees of cell death  assay and the senescence level by SA-β-Gal assay at the embryonic stage to address how early the aging phenotype can be detected in ll death A and senll death  were sigonth-old C and sixonth-old D pycr1 Kstaining D. The styear-old D. The Smnogaster ,41 and lnogaster , showingnogaster . This reno assay . Therefopycr1 homozygotic mutation was not lethal, which makes it possible to investigate the adult phenotype. The fecundity in pycr1 KO fish was greatly reduced when compared with the WT siblings. Due to premature aging, the reproductive performance was greatly reduced in the pcyr1 KO fish from four months old onwards. The onset of gonadal degeneration was considered to be a reproductive aging process [pycr1-deficient fish can only be successfully bred to produce progeny within the age from three to six months. Mutant fish that were older than six months displayed aging phenotype, including infertile. The gonadal development in both genders of the pycr1 KO fish was greatly impaired in tissue sectioning, leading to complete infertility at the age older than six months. Morphometrically, the pycr1 the KO fish was significantly different from those of their WT and heterozygotic siblings in morphometric analysis, where the pycr1 KO fish showed slim and internally reduced abdomen  with proline-specific antibody, a significantly reduced level of proline in the whole-fish body-extract of the pycr1 KO fish at adult stage was observed  effectors are essential in embryonic development and survival ,51. TotaPYCR1 in human patients caused abnormalities in the central nervous system, such as mental retardation, microcephaly, corpus callosum agenesis, and epilepsy [pycr1 KO fish was conducted. Similar to the rodent open field test, the novel tank assay is a method of evaluating zebrafish anxiety level by exploiting the innate behavior of zebrafish to seek protection in an unfamiliar environment [pycr1 KO fish, was demonstrated in a novel tank. When introduced into a novel environment, zebrafish have a natural tendency to spend the majority of time at the bottom and then expand their position of swimming to include the higher portions of the test tank gradually over a period of minutes [pycr1 KO fish. The latency to enter the top portion of the tank , mutated epilepsy . Furtherironment . The ada minutes . The deg minutes . Using tthe tank E and thethe tank F of the me point . The locArchocentrus nigrofasciatus) between the pycr1 KO and the WT fish. It was intriguing to find that the pycr1 KO fish displayed less fear response as triggered by the predator fish, which was represented by significantly more predator approaching time   N–P as coovement) . Figure pycr1 mutant fish during the light and dark cycles. We discovered that the pycr1 KO fish displayed a dysregulated circadian rhythm phenotype. In the light cycle, less average speed       C, green/ 0.0001) D, red/ye 0.0001) E, green/ 0.0433) B, and bl 0.0001) F.Since PYCR1 deficiency in human caused mild mental retradation , it is ipycr1 KO animal has not yet been reported in other model animals. We measured different neurotransmitter levels in the fish body and brain to understand the mechanism. By ELISA assays with target-specific antibodies for neurotransmitters, including GABA, dopamine, serotonin, melatonin, epinephrine, norepinephrine, acetylcholine, etc., we found that all of the neurotransmitter levels displayed no significant difference in brain tissues between WT and pycr1 KO fish  in the pycr1 KO when compared to the WT fish  aberration. Generally, mitochondrial dysfunction has been classified as one hallmark of aging. The PYCR1 protein has been reported to be essential in cell proliferation and maintaining the structural integrity of mitochondria [pycr1 KO fish could be caused by the degenerated mitochondria due to a loss of integrity. This energy deficiency might be insufficient to maintain normal cell function, such as transcription and translation control. This might be the reason that explains why most of the biomarkers we measured were downregulated in pycr1 KO fish.In addition, according to our results, the aging phenotypes in the chondria . Therefopycr1 KO zebrafish model displayed multiple behavioral changes that are associated with aging. However, no significant change of neurotransmitter levels was detected in the brain tissues of mutant. Instead, we observed the lower level of neurotransmitters in fish body extract of mutants when compared with wild type fish . The zebrafish were fed twice a day with lab-grown brine shrimp. For behavioral tests, we used adult zebrafish aged around 6–10 months.The Committee for Animal Experimentation of the Chung Yuan Christian University approved all of the experimental protocols and procedures involving zebrafish . All of the experiments were performed in accordance with the guidelines for laboratory animals. Wild-type AB strain and pycr1 +/− and pycr −/− zebrafish were separately raised in 10L tanks until they reached three months old (n=60 for each group). From three months onwards, WT (pycr1 +/+), pycr1 +/− and pycr1 −/− zebrafish were transferred and subsequently raised in 100L tanks. All of the fish were fed with artemia twice per day and kept at constant temperature of 28°C. The standard body length, body weight, and survival rate were measured every month when fish were aged from three months onwards until seven months.Mixed gender of WT, For histology, the tissues were dissected and embedded in Technovit 7100 resin (Heraeus Kulzer) or 4% Paraformaldehyde (PFA). The samples were sectioned at 5 μm interval and counter-stained with H&E (Merck) to visualize the nuclear position according to previously described protocol . For thehttp://life.bio.sunysb.edu/morph/soft-utility.html). We digitized the image landmark using TpsDig2 tool. Later, we perofrmed procrustes analysis for zebrafish using MorphoJ software (http://www.flywings.org.uk/morphoj_page.htm). This MorphoJ software can generate a covariance matrix and then Principal Component Analysis (PCA) can be performed to compare the morphometric difference between the wild type and pycr1 mutant. The dtail protocol for morphometric analysis can be found in supplemetary protocol.The image files were first converted to .tps format using TpsUtil for morphometric analysis . The TALEN vectors were linearized with Not1 restriction enzyme and transcribed using mMACHINE SP6 kit  to synthesize 5′capping TALEN mRNAs. Following the completion of transcription, poly(A) tailing reaction and DNase I treatment were performed according to the manufacturer’s instructions for the TALEN mRNAs. The in vitro transcribed TALEN mRNAs were filtered and enriched by YM30 column to increase the purity of the TALEN mRNAs.The TALEN mRNAs were co-injected with Transposase mRNA into one-cell stage zebrafish embryos. Each embryo was injected with approximately 2 nl of solution containing 50 pg/nl of TALEN mRNA. On the next day, the injected embryos were inspected under a stereoscope. Only embryos that developed normally were used for further analyses. Genomic DNA was extracted from embryos one day-after injection of TALEN mRNAs.E. coli bacteria DH5α strain. Thus, each colony represented one PCR amplified sequence. To determine the somatic mutation rates, the National Taiwan University Hospital DNA Sequencing Core sequenced the plasmids DNA isolated from single colonies. The indel mutation rates were determined by the number of colonies containing mutant sequences divided by the total number of colonies sequenced.Genomic DNA was extracted from the pools of 10 controls or injected embryos. Targeted genomic loci were amplified using primers that were designed to anneal approximately 150 to 200 base pairs upstream and downstream, respectively, from the expected FokI cut site and KOD FX high-fidelity DNA polymerase , according to the manufacturer’s instructions. All of the primers used in this study have been described previously . HRMA waThe potential founders were crossed with the wild-type zebrafish. Three to four days post-fertilization, 20 progenies were pooled and lysed in 25 microliters of the alkaline lysis buffer  and heated at 95 °C for 30 min. Subsequently, the DNA solution was neutralized using 25 microliters of the neutralization buffer . The samples were spun at 13,000 g for five minutes to remove the debris, and the supernatant contained extracted genomic DNA. In general, 20 embryos from each potential founder were screened for the presence of indel mutations by HRMA amplifying the region that surrounds the relevant TALEN FokI cleavage site using a forward primer and a reverse primer. For sequence confirmation, genomic DNA from single embryos was amplified using targeted loci-specific primers. The PCR products were then submitted for Sanger sequencing.For the TUNEL assay, embryos that were aged 24-h post-fertilization (hpf) were pre-treated with PTU and then processed to 4% PFA fixation for at least 6 h at 48 hpf. Following washing in PBST for 10 min, embryos were stored in 100% methanol at −20 °C for 2 h or longer. Embryos were subsequently incubated with 0.1 M sodium citrate for 30 min. at 70 °C to enhance embryo permeability. Thereafter, the embryos were rinsed twice with PBS and incubated with 5 μL of enzyme solution plus 45 μL of label solution at 37 °C for 1hr following the manufacturer’s instructions . Finally, the embryos were washed three times in PBS and ready for detecting of red fluorescent signals for cell death.Zebrafish adults and embryos were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) at 4 °C (for three days for adults and overnight for embryos), and then washed three times for 1 h in PBS (pH 7.4) and once for 1 h in PBS (pH 6.0) at 4 °C. Staining was performed overnight at 37 °C in the staining solution . All of the animals were photographed under the same conditions using transmitted light under a dissecting microscope. SA-β-gal activity in each animal was quantitated using a selection tool in Adobe Photoshop, following the method described previously. For analyses of embryos, the SA-β-gal-positive areas in the tail fin were counted and normalized to the total tail fin size.Adult zebrafish were immersed in working concentration of Smurf dye  for 30 min. and then rinsed in fish water for another 30 min. for de-staining, according to previous published protocol [The novel tank, mirror biting, predator avoidance, social interaction, and shoaling tests were conducted by a special designed zebrafish tower according to our previous published protocol . A specig or 15 min. and the crude homogenates were stored in 100 µL aliquots at −80 °C until required.After the behavioral analysis, the fish were treated in ice water to death. Subsequently, the brain and the fish body were separated and used for homogenate preparation. The tissues were minced and homogenized at medium speed with a Bullet blender  with 10 volumes of (v/w) ice cold phosphate saline buffer adjusted to pH 7.2. The samples were incubated on ice for 30 min. and further centrifuged at 13,000× Pierce BCA (Bicinchoninic acid) Protein Assay Kit determined the total protein concentration . The color formation was analyzed at 562 nm using a microplate reader .2O2 level) was measured by commercial target-specific ELISA kit . The tissue anti-oxidative capacity markers of catalase and SOD were measured by commercial target-specific ELISA kits . A commercial colorimetric kit measured the total anti-oxidative capacity (T-AOC) . Commercial target-specific ELISA kits measured the stress hormone of cortisol . Some neurotransmitters, such as dopamine, GABA, serotonin, melatonin, norepinephrine, epinephrine, acetylcholine, acetylcholine esterase, glutamate, glycine, and histamine were estimated by commercial target-specific ELISA kits . The tissue energy markers of creatine kinase, ATP and CoQ10, were measured by commercial target-specific ELISA kits . The telomerase activity was measured by commercial target-specific ELISA kit . The target-specific ELISA kits used in this study were based on Sandwich ELISA principle. First, the target-specific antibodies were immobilized onto 96-well microplates. Later, the tissue homogenates and HRP (horseradish peroxidase)-conjugated target-specific antibodies were applied onto microplate and then incubated at 37 °C for 1 h. After washing with washing buffer, chromogen A and B were applied onto microplate and incubated at 37 °C for 15 min. Finally, stop solution was applied to stop color development and the absorbance was analyzed at 450 nm while using a microplate reader . The relative concentration of target protein was then quantified by comparing to the standard curve that is generated from the standard provided by commercial kits.The proline content was measured by using commercial colorimetric kit  or target-specific ELISA kits . The five extracellular matrix markers of hydroxyproline, chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate were measured by commercial target-specific ELISA kits . ROS (by measuring H®RT (Invitrogen) and quantified with microplate reader  at OD260 nm. RevertAid first cDNA synthesis kit  was used to synthesize first-strand cDNA from total zebrafish RNA, according to the manufacturer’s instructions. RT-qPCR was performed using iQ SYBR Green Supermix  on a MyGO Pro  using β-actin as control, and the data were analyzed using the ∆∆Ct method [pycr1 gene are forward: 5′-GCCTCATATCATTCCCTTTGTCC-3′ and reversed: 5′-CTGCAGCAGTTTCTTCTCGA-3′.Total RNA from different tissues of adult zebrafish that were aged at around six-month-old were extracted by the RNAZolt method . The pri


The development of chemically stable and high conductive membranes is one of the most important issues to improve the performance of vanadium flow batteries (VFBs). Herein, poly(vinylidene fluoride) (PVDF)/graphene composite nanoporous membranes were easily fabricated by manipulating crystallization processes. The graphene was used to enhance membrane selectivity and conductivity. In the nanoscale channels of the membranes, the graphene nanosheets reduced the apertures among the crystal grains, thus restraining vanadium ions crossover due to the size exclusion effect. Moreover, the oxygen groups on the graphene improved the surface hydrophilicity and formed hydrogen bonds with the PVDF polymer chains, which facilitated the proton transport. The composite membranes, with a 0.15 wt % graphene loading, showed a selectivity of 38.2 and conductivity of 37.1 mS/cm. The single cell exhibited a coulomb efficiency of 94.7%, a voltage efficiency of 88.5%, and an energy efficiency of 83.8%, which was 13% higher than that of the pristine PVDF membranes. The composite membranes showed excellent stability during 100 charge-discharge cycles. All these results indicate that the PVDF/graphene composite membrane is a promising candidate for VFB applications.  OwinProton conductivity reflects the migration of a proton through the membranes in an electric field. Using membranes with high conductivity can reduce the internal resistance and improve the efficiency of batteries. With an appropriate graphene loading content, the PVDF/G-0.15 composite membranes had a higher proton conductivity of 37.1 mS/cm, compared with pristine PVDF membranes of 23.5 mS/cm. This indicated that the incorporation of highly active graphene improved the proton migration through membranes. After the crystallization, the graphene nanosheets were located in the nanoscale pores of the membranes. The oxygen functional groups on the graphene nanosheets promoted the formation of hydrogen bonds between the PVDF polymer chains and graphene . Moreove2 are shown in 2  of the cell was recorded at the beginning of a 50% state of charge. The electrolyte was cycled by pumps during the test. The test was finished when the OCV was less than 0.8 V. As shown in 2, the CE increased from 95.4% to 97.7%, while the VE decreased from 88.3% to 74.7%, owing to the shorter charging time and higher polarization, respectively [2. At 80 mA/cm2, PVDF/G-0.15 showed a 79.6% EE, while a 65.5% EE was obtained for the PVDF membranes. This significant improvement originated from the high ion selectivity and proton conductivity of the composite membranes.Furthermore, the performance of cells assembled with PVDF and PVDF/G-0.15 membranes under different current densities was tested and compared. For PVDF/G-0.15, as shown in ectively . Therefo2. As shown in Considering the strongly acidic and oxidizing conditions in electrolytes, the stability of membranes and steady operation are essential for commercial VFB application. The battery cycle performance of PVDF/G-0.15 was investigated at 50 mA/cmHighly active graphene nanosheets were incorporated in nanoporous membranes for VFB application. The physicochemical and electrochemical properties of the composite membranes were investigated. The addition of graphene enhanced the proton conductivity and ion selectivity of nanoporous PVDF membranes. During crystallization in the fabrication process, graphene was located in the nanoscale channels among PVDF spherulites, which reduced the pore size and limited the vanadium ions crossover. Moreover, oxygen groups on the edge of the graphene nanosheets improved the surface hydrophilicity of the channels and formed hydrogen bonds with the PVDF polymer chains, which benefited proton conduction through the membranes. Correspondingly, the battery assembled with PVDF/G-0.15 exhibited an EE of 83.8%, which was 13% higher than that of the pristine PVDF membranes. The composite membranes showed excellent stability in 100 charge-discharge cycles and low cost. In brief, the incorporation of graphene nanosheets into PVDF nanoporous membranes is a facile and effective modification method and is a promising candidate for VFB application.