C/EBPβ expression was reduced by α-MSH and Foxc2. FFA level was elevated by α-MSH and pc-Foxc2 treatment along with increased FAO in white and brown adipocytes. The expression of FAO key enzymes, medium-chain acyl-CoA dehydrogenase (MCAD) and long-chain acyl-CoA dehydrogenase (LCAD) were increased in α-MSH and pc-Foxc2 group. Combination of α-MSH and Foxc2 treatment synergistically promoted FAO through increasing the activity of CPT-1 and phosphorylation of ACC. We found C/EBPβ bind to MC5R and Foxc2 promoter regions and inhibited FAO. cAMP level was increased by α-MSH and Foxc2 individually treated or combined treatment. Furthermore, cAMP/PKA pathway-specific inhibitor (H89) blocked the FAO, despite in α-MSH and Foxc2 both added group. While forskolin, the cAMP agonist, promoted FAO and enhanced the effect of α-MSH and Foxc2. Collectively, α-MSH and Foxc2 mutual promote FAO in WAT and BAT via cAMP/PKA signal pathway. And C/EBPβ as a transcription suppressor inhibits α-MSH and Foxc2 expression and FAO.Alpha melanocyte stimulating hormone (α-MSH) and Forkhead box C2 protein (Foxc2) enhance lipolysis in multiple tissues. However, their relationship in adipose fatty acid oxidation (FAO) remains unclear. Here, we demonstrated that α-MSH and Foxc2 increased palmitate oxidation to CO Obesity, type 2 diabetes, cardiovascular and cerebrovascular diseases are increased risks for human health. Promotion of cellular lipid metabolism comes to be a valid therapeutic strategy to treat these diseases2357et al.2021Melanocortins also affects energy homeostasis by acting on peripheral tissues. Alpha melanocyte stimulating hormone (α-MSH), produced by proopiomelanocortin (POMC) neurons, act via the interaction with its receptors in reducing food intake and increasing energy consumption of the peripheral tissues and organs910161718Forkhead box C2 protein (Foxc2) is a member of the forkhead/winged helix transcription factor family in various organs and tissues, such as cardiac system, mammary gland, liver and adipose tissue. Our previous studies found Foxc2 enhanced mice pre-adipocytes proliferation and inhibited apoptosis via activating Akt/mTORC1 signaling pathway2426Here, we provide evidences for the profound effects of C/EBPβ in α-MSH-mediated and Foxc2-mediated fatty acid oxidation in both white and brown adipose tissues. Furthermore, we show that cAMP/PKA signal pathway which activated by α-MSH and Foxc2 is essential in stimulating FAO in adipose tissues.14C]-palmitate into different adipose tissues  showed α-MSH injection remarkably produced a 0.3-fold to 0.5-fold decrease in the palmitate incorporation into iWAT, eWAT and iBAT . α-MSH also elevated the activity of CPT-1, and increased fatty acid oxidation most effectively during lipolysis in white and brown adipose tissues. Meanwhile, α-MSH elevated the mRNA levels of MC5R and Foxc2. These data suggested α-MSH exhibits positive regulation on fatty acid oxidation in adipose tissue and Foxc2 effects during this process.α-MSH also control energy expenditure and lipid metabolism in peripheral tissues and organs, especially in adipose tissue by binding to specific receptors3435202014C]-palmitate incorporation into white and brown adipose tissues. CPT-1 activity and phosphorylation of ACC were increased in iWAT and iBAT. This effect was more obvious in iBAT which has more energy expenditure and thermogenesis. Our results indicated α-MSH and Foxc2 had the same effects on fatty acid oxidation in white and brown adipose tissues.Foxc2 is originally found in mice embryo trunk, head, and limbs in the mesoderm26402 production. By treating white and brown adipocytes with α-MSH and Foxc2, we observed that these two factors combined together to promote FFA release and increase palmitate oxidation to CO2. The mitochondrial UCP, a distinctive and specific marker for FAO, has been a major factor on the transcriptional control of MCAD, LCAD and other-oxidation enzymes in vivo44Mitochondrial β-oxidation is one of the major biochemical factors to maintain adipocytes function in both white and brown adipocytes42C/EBPβ plays an important role in pre-adipocyte differentiation and adipogenesis461949In summary, our study provided new insights into the mechanisms required for the regulation of α-MSH and Foxc2 on fatty acid oxidation in white and brown adipose tissues. Our data indicated α-MSH and Foxc2 were mediated by cAMP/PKA activation, and C/EBPβ was a novel transcriptional suppressor of MC5R and Foxc2 . Our finad libitum water and standard laboratory chow diet purchased from Animal Center of the Fourth Military Medical University. Animal room was maintained at 25 ± 1 °C, humidity at 55 ± 5%, and a 12 h light/12-dark cycle. Four-week old mice were injected either PBS or α-MSH (500 nM) intraperitoneally once a day for two days before dark cycle. Inguinal adipose tissue (iWAT) was removed at 0.5 h, 1 h, 2 h, 4 h and 6 h after the last injection from the treatment group. Vehicle, si-Foxc2 and pc-Foxc2 were administered as a daily intraperitoneal injection for 6 days. Tissue samples of epididymal white adipose tissue (eWAT), inguinal WAT (iWAT), and interscapular brown fat (iBAT) pads were removed for future studies.Three-week old C57BL/6J male mice were purchased from the Laboratory Animal Center of the Fourth Military Medical University . All mice handling methods and all experimental protocols were conducted following guidelines and regulations approved by the Animal Ethics Committee of Northwest A&F University. Mice were provided 4 cells/dish, and incubated at 37 °C under a humidified atmosphere of 5% CO2 and 95% air until confluence. Differentiation of white pre-adipocytes was performed as following. Cells grown to 100% confluence (Day 0) were exposed to the induction DMEM/F12 containing dexamethasone , insulin , IBMX  and 10% FBS. Four days after the induction (from Day 2), cells were maintained in the induction medium containing insulin  and 10% FBS until the day for cell harvest. And brown adipocytes were induced to differentiate using DEME/F12 with 15% FBS, 0.5 mM IBMX; 0.25 mM indomethacin; 2 μg/mL dexamethasone; 1 nM T3, 20 nM insulin for 2 days and subsequently maintained on differentiation media .For collected iWAT samples, the connective fiber and blood vessels were removed and washed three times with PBS buffer containing 200 U/mL penicillin  and 200 U/mL streptomycin . The adipocyte culture was carried out according to our previous publication. Briefly, pre-adipocytes were seeded onto 35-mm culture dishes at a density of 8 × 109 IFU/mL for Foxc2. The in vivo experiments were performed after 6–8 days of infection. H89 and Forsklin were bought from Selleck.cn . Thirty minutes before α-MSH stimuli, H89 (10 μM) or Forskolin (10 μM) were used to treat adipocytes. cAMP was measured using the FlashPlateTM method according to the FlashPlate  protocol.α-MSH was purchased from Sigma , and the working solution (500 nM) was prepared to treat adipocytes for 1 h. The recombinant adenovirus overexpression vector of Foxc2 (pc-Foxc2), recombinant lentiviral interference vector of Foxc2 (si-Foxc2), and lentiviral interference vector of MC5R (si-MC5R) were constructed in our lab. The overexpression plasmid vector of C/EBPβ (pc-C/EBPβ) and interference vector of C/EBPβ (si-C/EBPβ) were constructed by the Shanghai GenePharma Co., Ltd . The adenovirus and lentiviral recombinant vectors were infection in a titer of 1 × 102 by tissue homogenate and the incorporation of palmitate into lipids were measured according to previous published method51525314C] palmitic acid and measured for trapped CO2. For in vivo study of palmitate oxidation to CO2, we used the modified method from Sebastián and our pervious study552 measurement, a parafilm-sealed system was used. The reaction was stopped by the addition of 40% perchloric acid through a syringe that pierced the parafilm. Serum and adipocytes triglycerides (TG) and free fatty acids (FFA) contents were determined by using commercial ELISA kits  according to the instructions.Palmitate oxidation to CO3H]carnitine) with or without different levels of malonyl-CoA. Samples were incubated at 37 °C for 3 min. And then stopped by the addition of 0.5 ml of ice-cold perchloric acid. After mitochondrial centrifuged, the mitochondrial pellet was then resuspended in ddH2O and extracted with 600 μL of butanol 300 μL of the butanol-phase was counted by liquid scintillation.The CPT-1 activity assay was performed as described by Pawel and BremerSerum α-MSH, CPT-1, MCAD and LCAD were measured using commercial ELISA kits . Adipose tissues were collected as described previously. Cells were collected and disrupted by ultrasonication .Adipocytes were prepared for chromatin immunoprecipitation (ChIP) assay using a ChIP assay kit  according to the manufacturer’s protocol. Crosslinking was reversed, and purified DNA was subjected to Real-time PCR with SYBR green fluorescent dye .Four fragments containing Foxc2–5′ sequences from −1100 to −264 relative to the transcription initiation site were sub-cloned into pGL3-basic vector . HEK293T cells were cultured in 24-well plates and co-transfected with Foxc2 promoter plasmids and pGL3-basic plasmid (control reporter). Cells were harvested 48 h after transfection, and detected using the Dual-Luciferase Reporter assay system . The MC5R promoter reporter assay and luciferase reporter assay were performed as the same protocol. And the four fragments containing MC5R-5′ sequences were from −1200 to −210 relative to the transcription initiation site.−ΔΔCt.Total RNA from adipose tissues and cells were extracted with TRIpure Reagent kit . 400 ng of total RNA was reverse transcribed using M-MLV reverse transcriptase kit . Primers were synthesized by Shanghai Sangon Ltd . Quantitative PCR was performed in 25 μL reaction system containing specific primers and SYBR Premix EX Taq . The levels of mRNA were normalized using β-actin. The expression of genes was analyzed by method of 2Mice pre-adipocytes and adipose tissues were solubilized in lysing buffer. The solubilization was preceded for 40 min at 4 °C, then the solution was centrifuged at 12,000× g for 15 min at 4 °C, and the supernatants were used for determination of protein concentration. Protein samples (30 μg) were separated by electrophoresis on 12% and 5% SDS-PAGE gels using slab gel apparatus, and transferred to PVDF nitrocellulose membranes , blocked with 5% skim milk powder/Tween 20/TBST at room temperature for 2 h. The membranes were then incubated with primary antibodies against CPT-1, p-ACC, ACC, Cidea, PGC1-α, FABP3, FABP4, UCP1, UCP2 and GAPDH  at 4 °C overnight. Following this step, the appropriate HRP-conjugated secondary antibodies  were added and incubated for 2 h at room temperature. Proteins were visualized using chemiluminescent peroxidase substrate , and then the blots were quantified using ChemiDoc XRS system .p < 0.05 was considered to be significant.Statistical analyses were conducted using SAS v8.0 . Data were analyzed using either one-way ANOVA or two-way ANOVA. Comparisons among individual means were made by Fisher’s least significant difference (LSD). Data were presented as mean ± SD. How to cite this article: Gan, L. et al. α-MSH and Foxc2 promote fatty acid oxidation through C/EBPβ negative transcription in mice adipose tissue. Sci. Rep.6, 36661; doi: 10.1038/srep36661 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Social media is increasingly used by patients to source information for managing chronic disease. The aim of this study was to understand patient information needs about gout by a content analysis of questions posted on the social news website Reddit.We analysed questions posted onto the ‘Gout sufferers unite’ subreddit site. Two reviewers coded questions into categories (inter-reviewer kappa 0.70), with discordant coding resolved by a third reviewer. Data were analysed by calculating the frequency of questions within the categories. Where relevant, categories were further separated into sub-categories to allow organisation and interpretation of the data.We analysed 359 questions in 287 posts by 213 individuals. A wide range of questions arose. The single most common category related to uncertainty of diagnosis (22.3% questions), with questions about disease management common. Information-seeking about medications was generally cautious, with questions about side-effects, risk of flares after starting urate-lowering therapy, and decision to start urate-lowering therapy. Community users experiencing flares posted questions about flare management, including medications, sometimes in real-time. Dietary management questions included the effectiveness of dietary changes as a management strategy, choice of alcoholic beverage, and weight loss strategies. Questions about serum urate levels were rare (2.8% questions).Questions about gout posted on the subreddit site most often related to uncertainty about symptoms and disease management strategies, with infrequent questions about serum urate testing, results or targets. These findings may inform development of strategies to address the information needs of people with gout. Gout is a chronic disease of monosodium urate crystal deposition that typically presents as recurrent flares of painful inflammatory arthritis . It is tGout is poorly controlled worldwide, with low adherence to urate-lowering therapy and infrequent achievement of serum urate target . Strategreddit.com have appeared where registered users can submit content and seek advice. In 2016 Reddit had 542 million monthly visitors and was ranked the 25th most visited website in the world with 8 billion page views per month [Social media is widely and increasingly used by patients to source information about disease . As the er month . Within While it is clear patients are increasingly using social media platforms to inform disease management, very little research has examined the types of questions patients seek answers for on these platforms. The aim of this study was to understand patient information needs about gout by a content analysis of questions posted on Reddit.We analysed questions posted onto the ‘Gout sufferers unite’ subreddit. This open access subreddit has existed for five years, and had 1811 ‘Gout friends’ at the time of writing. We analysed questions in the “Hot” section of the subreddit posted from September 4th 2015 to August 23rd 2016. Posts were extracted from the website with information including title, text, username, and questions in the text. Posts that did not include a question were not extracted for analysis.A sample of questions were initially reviewed by four reviewers , and 13 categories (themes) were agreed upon. Two reviewers (CD and AS) then coded all questions into the categories. Kappa values were analysed using SPPS v23 . Kappa (SE) for inter-reviewer agreement was 0.70 (0.026). Discordant coding was resolved by review by a third author (ND). Data were analysed by calculating the frequency of questions with the categories. After coding and agreement, categories were reviewed and where relevant, further separated into sub-categories to allow organisation and interpretation of the data.According to the University of Auckland Human Participants Ethics Committee policy, this work did not require ethical approval, as it involved analysis of publically available data. Consent was not obtained from individuals or Reddit, as the posts are available in the public domain and were made voluntarily from individuals who could not be identified.We analysed 359 questions in 287 posts by 213 individuals. The majority of individuals  submitted a single post. The mean number of posts per individual was 1.34 (range 1–8). For most posts, it was not possible to determine disease duration of gout, but a new or very recent diagnosis was mentioned by 41/213 (19.2%) users. A wide range of questions arose. The single most common category was about uncertainty of diagnosis (22.3% questions), with management categories  also common  questions about medications. These included questions about specific named drugs, most commonly allopurinol (33 questions) and colchicine (15 questions). Questions about medications were generally cautious or negative, with key sub-categories of medication side effects, risk of flares after starting urate-lowering therapy, and deciding whether to start urate-lowering therapy. Community users experiencing flares posted questions about medications for flare treatment in real-time. Examples of questions are shown in Table % questioThere were 44 (12.3%) questions about dietary management. Sub-categories were questions about general dietary strategies, effectiveness of dietary changes as a management strategy, choice of alcoholic beverage, and the best weight loss strategies for people with gout. Examples of questions are shown in Table There were 37 (10.3%) questions about non-pharmacological management, including questions about the benefits of supplements, the role of activity during flares, choice of footwear, and topical treatments during a flare. Less frequent questions related to exercise strategies, devices and mobility aids during walking, and the value of blood donation or sex to improve gout.There were 13 questions that specifically related to supplements. These supplements included curcumin, theracurmin, multivitamin, black cherry, vitamin C, “enzyme” supplements, herb gel, and alkaline water. Questions about several supplement combinations marketed for urate or gout were also posted including: ‘Gouch’ , celery seed extract, stinging nettle root, ‘Urcinol’ , and ‘Uric Acid Support’ .Really open to suggestions... for anything, really…… I also know so little about gout, so any information is good information, tbh.”). Requests for non-specific advice were also made during the initial flare of disease There were 37 (10.3%) non-specific questions regarding gout management posted. These questions often included a detailed narrative about the course of the person’s experience, with a question requesting general advice from the community , and questions about financial and employment issues such as negotiating health insurance systems, disclosure of the condition to an employer, and the ability to work once a diagnosis of gout has been made. There were also questions about sourcing of expensive medications (“They gave me a script for colchicine and i don't have insurance. ….. Any idea on how to get more in the future for cheaper?”). Questions about reliable information primarily related to electronic sources or apps that can be used to help manage gout.A number of other categories were less frequently observed, with <10% questions per category (Table This analysis of questions posted on the social media website Reddit shows uncertainty about symptoms and management strategies for gout are common. The questions show that medications, particularly urate-lowering therapy, are often viewed negatively, with concern about side effects and the balance between risks and benefits. In this online community sample, there was a strong interest in non-drug strategies, including many different dietary solutions and supplements.The most common single category was uncertainty about symptoms, particularly for attacks that did not conform to typical patterns. A recent detailed symptoms analysis of patients with crystal proven gout has shown that many patients do not present with stereotypical attacks, and that flares may affect more than just ‘classical’ joints such as the 1st metatarsophalangeal joints . It is aManagement categories were also common, emphasizing the information needs for patients for general management advice, medication advice, and non-pharmacological advice. The community users demonstrated concerns about long-term medications, and it appeared that the balance of benefits and potential side effects for some users was uncertain. The concern about flares during initiation of urate-lowering therapy highlights the need for further discussion with patients about the long-term benefits of ULT and efforts to reduce flares during initiation using anti-inflammatory prophylaxis .There was a strong focus on non-drug management, including questions about the role of many dietary factors and supplements, including many with no reported benefits for gout. These findings are consistent with the lay view of gout as being caused by overindulgence in rich food and alcohol . It is aNotable from the analysis of the postings was that there were very few questions concerning serum urate testing, results or targets. Given that serum urate lowering is the central strategy for long-term effective management of gout, these data suggest that information about this fundamental aspect of gout management has not widely penetrated the gout patient community. Together, these findings highlight the importance of changing prevailing social narratives of gout from a self-inflicted condition that can be primarily managed through dietary modification, to contemporary concepts of gout as a chronic disease of urate crystal deposition that can be effectively managed through complementary pharmacological and non-pharmacological urate-lowering strategies .Study limitations include the lack of community user information. Reddit community users are known only by their usernames, and demographic and disease details are not available. Therefore, it is not possible to determine whether the questions are consistent with those of all people with gout, or indeed whether all the community users truly have the disease. Furthermore, disease duration was not available for all community users with gout, and we are unable to systematically determine whether the community user had a new diagnosis of gout, or had longstanding disease. It is possible that the information needs of Reddit community users do not represent all people with gout . This stThis analysis of questions posted on Reddit highlights a number of information needs for people with gout. The key findings are uncertainty about symptoms and questions about disease management, both pharmacological and non-pharmacological. These findings may inform development of strategies to address the information needs of people with gout, with the ultimate goal of improving patient and community understanding about the disease and its management.


The differential diagnosis of immune (ITP) and hereditary macrothrombocytopenia (HM) is key to patient management. The immature platelet fraction (IPF) represents the subset of circulating platelets with higher RNA content, and has been shown to distinguish hypo- from hyperproliferative thrombocytopenias. Here we evaluated the diagnostic accuracy of IPF in the differential diagnosis between HM and other thrombocytopenias in a population of patients with post-chemotherapy thrombocytopenia (n = 56), bone marrow failure (n = 22), ITP (n = 105) and HM (n = 27). TPO levels were also measured in HM and ITP matched for platelet counts. Platelet counts were similar in all patient groups. Higher IPF values were observed in both ITP  and HM  compared to hypoproliferative thrombocytopenias. IPF values were also higher in HM compared to ITP, yielding a diagnostic accuracy of 0.80  to distinguish these two conditions. Intra- and inter-assays reproducibility of IPF in HM patients revealed that this is a stable parameter. In conclusion, IPF is increased in HM compared to both ITP and other thrombocytopenias and contributes to the differentiation between ITP and HM. Further studies are warranted to understand the biological rationale of these findings and to its incorporation in diagnostic algorithms of HM. Recently, the feasibility of using parameters of the complete blood count (CBC) to support this differential diagnosis was illustrated by a series of studies which demonstrated and validated that the mean platelet volume (MPV) can help the segregation of patients with ITP and HM5.The differential diagnosis of thrombocytopenias includes a variety of conditions such as hematologic malignancies, bone marrow failure (BMF), hypersplenism, immune thrombocytopenia (ITP), microangiopathic hemolytic anemias and hereditary macrothrombocytopenia (HM). Among these, the differential diagnosis between ITP and HM can be challenging due to the absence of specific tests, particularly in patients with mild bleeding symptoms6. Initially, the IPF was measured by flow cytometry, and described as reticulated platelets7. Recently, studies have reported the clinical utility of measuring immature platelets in clinical settings using automated hematology analyzers10. The correlation between flow cytometry and hematology analyzers in quantifying this population has been previously demonstrated11.In recent years, new parameters were incorporated to the CBC, including the immature platelet fraction (IPF), which represents a population of newly formed platelets containing a greater amount of residual RNA12, less information is available about its use in the differential diagnosis between ITP and HM. In 2000, Fabris et al. evaluated reticulated platelets by flow cytometry in a population of 29 patients with HM, observing reduced IPF values when compared to ITP13. More recently, Miyazaki et al.14, in a study with 15 patients with HM in which the IPF was measured in an automated hematology analyzer observed significantly higher IPF values in HM compared to ITP. To further elucidate the value of IPF determination in the diagnosis of thrombocytopenias, with focus on the differential diagnosis between ITP and HM, we investigated the diagnostic accuracy and the precision of IPF measurements in a population of patients with different causes of thrombocytopenia, including a well-characterized cohort of patients with HM. In addition, we also evaluated whether thrombopoietin (TPO) levels could further facilitate this diagnosis.Although the utility of the IPF in the differential diagnosis between hypo- and hyperproliferative thrombocytopenias has been already reported15. The study population consisted of patients with thrombocytopenia confirmed in two independent samples and microscopic analysis, in regular clinical follow-up in the Hemostasis outpatient clinic of University of Campinas, or admitted to the hematology ward of the same institution. The inclusion criteria was a confirmed diagnosis of any of the following: (i) ITP (based on previously established guidelines)16; (ii) bone marrow failure (including aplastic anemia and myelodisplatic syndromes with a platelet count below 150 × 109/L)18; (ii) post-chemotherapy (Ctx) thrombocytopenia  with a platelet count below 150 × 109/L; and (iv) HM, with platelet counts below 150 × 109/L. Diagnosis of HM was established by clinical and laboratory criteria as determined by international guidelines21. These included the exclusion of all other causes of thrombocytopenia, objective confirmation of thrombocytopenia in first-degree relatives, platelet aggregation studies, molecular analyses and specific tests such as platelet glycoprotein studies and electron microscopy in selected cases. Exclusion criteria included: a platelet count above 150 × 109/L in ITP patients at the day of enrollment; (ii) the presence of conditions known to influence IPF values such as sepsis and other inflammatory diseases22, or (iii) the use of antiplatelet agents. The study was approved by the Institutional Review Board of University of Campinas (certificate number 411.620/2013) and performed in accordance with the Declaration of Helsinki. All patients provided written informed consent prior to enrollment.This was a cross-sectional diagnostic accuracy study, designed according to STARD (Standards for the Reporting of Diagnostic accuracy studies) guidelinesRecruitment occurred between July 2013 and February 2015. ITP, HM and BMF patients were included consecutively, aiming for an enrollment of 100 patients with ITP, 20 with BMF and all patients with HM. Post-Ctx patients were included using a convenience sampling strategy, which consisted in weekly visits to the hematology ward, with a target of 50 patients.Samples were collected by venipuncture by the same nursing team responsible for the collection of routine clinical samples, and using the same standard operating procedures in both clinical sites. Blood was drawn in Vacutainer® EDTA K2 tubes . IPF measurements were performed within four hours from sample collection, which is the threshold suggested in the equipment operational procedures. After IPF analysis, tubes were centrifuged , and plasma aliquots were frozen at −80 °C until analysis.10. Finally, we also evaluated the intra- and inter-assay reproducibility of IPF measurements in a subgroup of patients with HM. Platelet counts and mean platelet volume (MPV) were obtained from the same samples, by the same hematological analyzer, which measures the MPV by impedance. In the laboratory where IPF measurements were performed, commercial internal quality control samples  are run before the beginning of each 8 h-routine, as well as after any corrective or preventive maintenance of the hematological analyzer. In addition, the laboratory participates in a regular national external quality control program and is within the scope of the University laboratory quality management system.Whole blood samples were analyzed in a Sysmex XE 5000 hematology analyzer . Briefly, the IPF fraction was identified in the reticulocyte channel, employing a fluorescent dyes containing polymethine and oxazine. These dyes penetrate the cell membrane, staining residual RNA of red blood cells (reticulocytes) and platelets. In addition, these two populations are separated by cell size. A computer algorithm then discriminates mature platelets and the IPF, which was expressed as a percentage of total platelets (IPF%). In addition, we also calculated the absolute count of the IPF (A-IPF), which has been previously reported as superior to the IPF% in some clinical settings9/L were used as a control group for the measurement of TPO levels. All of these patients were off-ITP treatment for at least one year and had platelet counts above 250 × 109/L at the time of testing.Circulating TPO levels were measured in plasma samples using a commercial immunoassay , based on a sandwich ELISA technique, according to the manufacturer’s protocol, in all HM patients and in seventy-four ITP patients. Of note, none of these patients were using TPO receptor agonists. A group of eight patients with a history of ITP diagnosis that were not excluded from the study due to platelet counts above 150 × 10Results are expressed as median and range, or means ± SD, unless mentioned otherwise. Differences between quantitative variables were evaluated by Mann-Whitney or Kruskal-Wallis with Dunn’s post-test for comparison with 2 or more than 2 variables respectively. Associations involving categorical clinical variables were analyzed using the Fisher exact test. Correlation analyses were performed using the Pearson or Spearman correlation (rho) test, according to data distribution. The diagnostic accuracy of IPF for the segregation of patients by each diagnosis was estimated by the ROC (receiver operating characteristic) procedure, which allows the simultaneous analysis of sensitivity and specificity of each test in relation to selected clinical outcomes. Results are reported with confidence intervals and level of significance. A P value < 0.05 was considered statistically significant.9/L . In total, 210 patients were analyzed, divided in the following subgroups: Post-Ctx (n = 56), BMF (n = 22), ITP (n = 105) and HM (n = 27). Demographic and clinical characteristics are shown in Table 5. In all other samples, IPF was normally reported. Of note, no differences were found in platelet counts between ITP and HM patients.Two-hundred forty-eight patients were enrolled in this study, of which 38 were excluded due to platelet counts above 150 × 10Similar IPF values were observed between patients with Post-Ctx thrombocytopenia and BMF. All other comparisons yielded significant differences in IPF between different patient groups Fig. , with boAn inverse correlation between IPF and platelet counts was observed in BMF, HM, and ITP, but not in Pos-Ctx patients Fig. . IPF valThe diagnostic accuracy of IPF% and A-IPF measurements for the differential diagnosis of thrombocytopenia was estimated using ROC analysis. The AUC for the differential diagnosis between ITP and HM was 0.80 . Using an arbitrary IPF% cut-off value of 17.4%, which presented the best combination of sensitivity and specificity, ITP could be distinguished from HM with a sensitivity of 70% and a specificity of 90%. The positive and negative predictive values were 81.48% and 71.43 respectively. A similar result was obtained using the A-IPF, which yielded and AUC of 0.77 . Therefore, all additional analyses were performed using the IPF%.23 despite the former group having much lower platelet counts than the latter. Even when compared to HM patients with similar levels of thrombocytopenia, ITP patients presented significantly lower TPO levels  or Bernard-Soulier syndrome , lower IPF values were observed in the remaining patients classified as non-specified hereditary macrothrombocytopenia .Despite the low number of patients with HM, we performed a subgroup analysis to investigate whether IPF values differed between patients with different types of HM Table . While n4, a finding that was later validated in a multicenter study using different instruments to measure this parameter5. Using a population of 210 patients with different causes of thrombocytopenia, the main result of our study was that another platelet parameter derived from automated an hematology analyzer reliably differentiates HM from ITP, with an estimated diagnostic accuracy similar to that reported in the literature for MPV5.The differential diagnosis between ITP and HM represents a significant clinical problem since tests used to distinguish these two conditions are not available in most clinical laboratories, and even when available, cannot discern among all clinical etiologies. The development of hematological analyzers in the last decades has allowed the incorporation of new parameters in the diagnosis of hematological disorders; and the feasibility of this strategy for the diagnosis of HM has been shown by a series of studies evaluating platelet size in these patients. These studies demonstrated that MPV is significantly higher in patients with HM compared to ITP9/L for both groups). It should be noted that in contrast to other clinical settings, no relevant difference was observed in when IPF was reported as a percentage or in absolute values.In our population, IPF% values were significantly higher  in the HM group compared to hypoproliferative thrombocytopenias such as BMF and Post-Ctx. More importantly, IPF% values were higher in HM than ITP patients with similar platelet counts. An inverse correlation between platelet counts and IPF% was observed not only in ITP patients but also in patients with BMF and HM. The absence of such correlation in patients with Post-Ctx thrombocytopenia is most likely due to the blunted thrombopoietic response secondary to chemotherapy. The ROC analysis estimated significant diagnostic accuracies of IPF% and A-IPF in the differential diagnosis of ITP and HM even in a group of patients with similar levels of thrombocytopenia  compared to TPO. In regard to the fact that no correlation was found between TPO levels with either platelet counts or IPF in HM, this suggests that increased IPF is not associated with increased thrombopoietic activity in HM.25 is probably not sufficient to explain the 200–300% increase of IPF in these patients. In addition, formation of platelet aggregates was ruled out by the microscopic evaluation of blood smears, and by a review of the scatter plots of IPF measurements from all patients with HM. The strong correlation between IPF measurements performed one year apart suggests that the higher RNA content of these platelets could be a stable characteristic associated with the cellular mechanisms of thrombocytopenia in these patients. Cellular mechanisms responsible for thrombocytopenia in HM are complex and dependent on the molecular etiology of each disease entity. Increased counts of immature platelets were described in an animal model of HM due to RASA3 mutations in which platelet turnover is increased26. However, most of our patients presented with more classical forms of thrombocytopenia such as BSS and MYH9-related disorders in which thrombocytopenia is attributed to defects in proplatelet formation29. Interestingly, in MYH9 megakaryocytes, the physiological suppression of proplatelet extension exerted by the interaction with type I collagen is lost leading to premature or abnormal platelet release from the bone marrow29. We therefore speculate that the extremely high IPF values observed in most of our patients with HM are an expression of the premature release of platelets to the circulation. Interestingly, patients in whom the diagnosis of MYH9-related platelet disorders or BSS were confirmed by molecular or flow cytometry assays presented significantly higher IPF values than patients with a diagnosis of non-specified HMT. However, due to the limited number of patients in this subgroup analysis, these data should be considered preliminary. The confirmation of this hypothesis in future studies would provide a biological rationale to the use of IPF as a diagnostic tool in HM.The mechanisms underlying the increase of IPF in HM remain to be elucidated. The previously reported increase in platelet volume of around 50 to 100%23, our analysis demonstrated significantly lower TPO levels in ITP patients compared to control subjects with normal platelet counts. Nevertheless, suboptimal TPO increase did not preclude the demonstration of a marked negative correlation between TPO levels and platelet counts in ITP (rho = −0.50), which was even stronger when only patients using steroid were evaluated (data not shown). This suggests that in ITP, increase in TPO levels is blunted by yet unknown mechanisms which are apparently improved by steroid therapy.In accordance with the concept that ITP is a condition associated with suboptimal increases in TPO levelset al.13 or using automated hematological analyzers, as in the study by Myiazaki14 and ours. Few studies have formally evaluated the correlation of IPF measurements by these two methods but in two of the most recent ones, this correlation was limited to ITP patients30. Considering the challenges of standardizing platelet flow cytometry analyses compared to a CBC, we believe that it is fair to state that the former method should not be regarded as a gold-standard for IPF measurement. In fact, the vast majority of studies addressing other uses of IPF do not perform parallel flow cytometry analysis. Moreover, the concordance of our results with those from Myiazaki et al. in two independent populations supports the conclusion that IPF is increased in HM, possibly showing the premature release of platelets into the circulation. In addition, the analyzer used in our study measures MPV by impedance, which is a method that in patients with HM is known to report MPV results in less than half of the cases due to abnormalities in the platelet distribution curve5. This technical limitation was also present in our study, and precluded some interesting analyses such as the comparison of the estimated diagnostic accuracy of IPF and MPV for the differential diagnosis between HM and ITP, and the evaluation of the combined performance of IPF and MPV in this context. On the other hand, it illustrates the challenges of using the MPV as a diagnostic test for HM, highlighting the importance of additional assays such as the IPF. Interestingly, when MPV values from our patients obtained from the same sample in a different hematological analyzer (Advia 2120) were used, the estimated diagnostic accuracy for the differential diagnosis of HM and ITP  was lower than that of the IPF.Our study presents limitations that need to be acknowledged, with lack of flow cytometry data being possibly the most relevant. As mentioned earlier divergent results were obtained when the IPF was measured in HM patients by flow cytometry in the study by Fabris In conclusion, our results validate previous demonstrations that IPF is increased in HM in an independent and larger population of patients. We also demonstrate the reproducibility of this method, thus supporting the feasibility of its incorporation in the diagnostic approach of HM. Given the easy accessibility to this parameter for routine clinical laboratories, and the importance of differentiating HM and ITP, further studies are warranted to the incorporation of the IPF in diagnostic algorithms of HM, and to further investigate new questions raised by our study such as the mechanisms of IPF elevation in HM.


The understanding of interactions between electrons and phonons in atomically thin heterostructures is crucial for the engineering of novel two-dimensional devices. Electron–phonon (el–ph) interactions in layered materials can occur involving electrons in the same layer or in different layers. Here we report on the possibility of distinguishing intralayer and interlayer el–ph interactions in samples of twisted bilayer graphene and of probing the intralayer process in graphene/h-BN by using Raman spectroscopy. In the intralayer process, the el–ph scattering occurs in a single graphene layer and the other layer (graphene or h-BN) imposes a periodic potential that backscatters the excited electron, whereas for the interlayer process the el–ph scattering occurs between states in the Dirac cones of adjacent graphene layers. Our methodology of using Raman spectroscopy to probe different types of el–ph interactions can be extended to study any kind of graphene-based heterostructure. Electron–phonon interactions in van der Waals layered materials can occur either within the same layer  or in different layers (interlayer). Here, the authors use multi-wavelength Raman spectroscopy to probe intra- and inter-layer electron–phonon interactions in twisted graphene heterostructures. For twisted bilayer graphene (TBG), the interaction between electrons of different layers generates van Hove singularities (vHs) in the density of electronic states (DOS), whose energies are θ dependent3. Electron–phonon coupling is also a fundamental interaction that affects a broad range of phenomena in condensed matter physics, such as electron mobility and thermal conductivity. In atomically thin heterostructures, the interaction can involve electrons in the same layer  or in adjacent layers (interlayer el–ph interaction). The interlayer el–ph interaction has been recently observed in WSe2/h-BN heterostructures4.Interlayer electron–electron and electron–phonon (el–ph) scattering processes emerge from the coupling of atomic layers in two-dimensional (2D) heterostructures, and are essential for describing their physical properties and technological applications. The additional possibility of controlling the twisting angle θ. Prominent new peaks are observed in the Raman spectra of TBG samples and come from phonons within the interior of the Brillouin zone (BZ) of graphene that are folded to the centre of the reduced Moiré pattern BZ. The frequencies of these phonons depend on the twisting angle θ11. We show here that they can be activated either by the intralayer or the interlayer processes. The intralayer process was also observed in the Raman spectra of gr/h-BN samples and allows the experimental determination of their mismatch angle between the crystallographic axes of graphene and h-BN. In this case, the el–ph process occurs in a graphene monolayer and the h-BN surface imposes a periodic potential needed for the electron backscattering in the resonant Raman process. This effect is expected to be sensitive to the strength of the interaction between monolayer graphene and any other single layer or crystalline surface.Here, we report the ability of Raman spectroscopy to probe and distinguish interlayer and intralayer el–ph interactions in graphene heterostructures. This is experimentally attained by tuning the energy of the excitation photon and observing the resonances of the Raman modes in different samples of TBG and graphene on the top of h-BN (gr/h-BN), with previously determined mismatch twisting angle θ can be determined from the optical images using the procedure reported in ref. 12 . The ratio of ITBG/ISLG = 1.8 eV for the G-band intensity (around 1580 cm−1) of the θ = 6° sample shown in Fig. 12. However, for the θ = 13° sample shown in Fig. ITBG/ISLG is more than 50. This huge increase of the G-band intensity has already been observed in previous Raman studies of TBGs16 and explained by the resonance of the incident photons with the transition between vHs in density of electronics states of a TBG.Experiments were first conducted in TBG flakes, that appear in an optical microscope as an external hexagon (first layer) and an internal one (with two layers), where the twisting angle −1. Extra peaks have been observed in many previous Raman studies of TBGs11. They arise from phonons of graphene with momentum ħqM, where qM is a vector of the Moiré pattern reciprocal lattice, that are folded to the centre of the reduced BZ and become Raman active11. In 2010, Gupta et al.5 proposed that the extra peaks were activated by double-resonance Raman (DRR) process where momentum conservation is provided by the potential of the Moiré reciprocal lattice. Carozo et al.7 attributed the new peaks below and above the G-band position to the intervalley and intravalley DRR processes, and estimated the resonance energies as a function of θ for these two processes. The valleys considered in this work belong to the same graphene layer and, therefore, both cases correspond to an intralayer el–ph scattering process.In addition to the G band, we can observe in Fig. 10 and Wang et al.11 reported measurements of TBG samples with intermediate twisting angles (13°–16°) using different laser lines. In both works, the appearance of new peaks in the range 1380–1450 cm−1 was observed to occur in the same spectra where the G band was enhanced. This resonance behaviour could not be explained by the predictions of the DRR process involving the periodic potential of the Moiré7, and revealed that a different el–ph process involving phonons with momentum ħqM may exist. We will show below that the new phonons observed in these works11 are activated by the interlayer el–ph scattering process.In 2013, Carozo et al.R and R′ peaks10. Since they can be activated either by the intralayer or the interlayer el–ph scattering process, we will adopt for these peak notation Aα, where A = T or L refers to the branch  of the unfolded phonon and α refers to the el–ph scattering mechanism . It will be clear below that the extra peaks above and below the G band shown in Fig. a and Te, respectively.The extra peaks below and above the G-band position have been called in the literature as the θ = 6 and 13°, respectively. The vertical scale corresponds to the energy of the incident photon, and the peak intensities are represented by the colour bar on the right side. We can observe in Fig. ITBG/ISLG of the θ = 6° sample does not depend on the photon energy and is always around 1.8, an expected value far from resonance conditions12. On the other hand, the La peak clearly exhibits a resonance behaviour and reaches the maximum intensity for photon energies around 2.2 eV, where it becomes as intense as the G band in SLG. Figure θ = 13° sample exhibit a different behaviour. Now, both the G band and the Te peak exhibit a resonance behaviour and are enhanced in the same excitation energy range.In order to explain the different resonance behaviour of the Raman peaks in TBGs, we first made multiple-excitation Raman measurements using many different laser lines in the visible range (see Methods section), which allowed us to obtain the accurate Raman excitation profile (REP) of Raman peaks in samples with small and intermediate twisting angles. Figure a and Te peaks, that is, the intensity of each peak as a function of the photon energy, for the θ = 6 and 13° samples, respectively. Figure a peak exhibits maximum enhancement at approximately 2.2 eV and has a width γ around 0.7 eV. This value of γ agrees with the result in a previous ultraviolet (UV) Raman study of TBGs8. In the case of the θ = 13° sample shown in Fig. e peak exhibit very similar REPs, except for the fact that the intensity of the extra peak was multiplied by ≈ 100 for comparison. The data in Fig. 10, where the fitting parameters are the energies of the optical transitions among vHs, EvHs, and the width γ of the REP. The values of the parameters that fit the experimental data in Fig. EvHs = 2.37 eV and γ = 0.25 eV. Notice that this value of γ agrees with the width of the peaks in the optical absorption spectra of TBGs17. Several other samples with low (4°–6°) and intermediate (12°–16°) twisting angles were investigated using multiple-excitation Raman measurements, and results similar to those shown in Fig. EvHs and γ for the G band on the twisting angle θ is shown in ref. 18.Figure ħqM to another state in the same layer , whereas in the other case, the excited electron is scattered to a state of the other layer (interlayer process). We will show that these two mechanisms give rise to resonances at different energies.In order to understand the origin of the results presented above, we performed a theoretical simulation considering two different el–ph processes that take part in the Raman scattering. Details of the calculations are given in the Methods section. In one case, the excited electronic state from one layer is scattered by phonon with momentum θ = 13.2° and represented, respectively, by the grey and black hexagons. The vectors q1, q2 and q3 correspond to the unit vectors of the Moiré reciprocal lattice (we will consider in this work that qM = q1 or q2 or q3). The reduced BZ of the Moiré superlattice is shown by the small red hexagon in the centre of Fig. θ = 13.2° TBG and shown in Fig.Ea  creates an electron–hole pair, and a phonon with momentum ħqM  scatters the excited electron to another state in the same Dirac cone (layer B in this case). For simplicity, the change in energy between these two states is not considered in this figure since the phonon energy is much smaller than the energy of visible photons. In the interlayer process also represented in Fig. Ee creates one electron–hole pair, but now the electron from one layer (layer B) is scattered to a state of the other layer (layer A) by a phonon with momentum ħqM. In both cases, the electron is then scattered back elastically to the first excited state by the Moiré potential, with wave vector qM, for electron–hole recombination and emission of the scattered photon.Figure Ee around the Dirac points KB and KA, respectively. An excited electron with momentum ħk from the Dirac cone of one layer (dark green curve) is scattered by a phonon with momentum ħqM (red arrows) to a state with momentum ħk′ in the Dirac cone of the other layer (light green curve). The lowest possible value of Ee occurs when the equi-energies curves of the two layers tangentiate, as shown in Fig. Ee, the difference EvHs between the vHs in the valence and conduction bands of a TBG.Figure A and KB, respectively, correspond to the curves of constant energy Ea. In this case, the excited states with momenta ħk and ħk′ belong to the Dirac cones of the same layer (dark or light blue curves). The intralayer process is similar to the DRR mechanism that gives rise to the disorder-induced D and D′ bands in the Raman spectrum of graphene19. For the disorder-induced bands, the electron is scattered back to the initial excited state by a defect whereas, in the case of TBG, the backscattering is provided by a periodic potential of the Moiré pattern. This DRR process, where momentum conservation is provided by a vector qM of the Moiré lattice, has been called previously as the umklapp DRR process6.The intralayer el–ph scattering process is schematically represented in Fig. qM. In the reduced BZ scheme, they are folded to the centre of the reduced BZ and have zero momentum. The first-order Raman process can be accomplished in this scheme since the two excited states k and k′ in the extended BZ in Fig. k and k′ in the extended BZ in Fig. So far, we discussed the two processes in the extended BZ scheme, where the activated phonons have finite Ea and Ee, we calculated the restricted density of joint electronic states that satisfy the intralayer and interlayer conditions. For the calculation, we divided the graphene BZ in a 2400 × 2400 k-points grid and, for each twisted angle θ, we stored the number of joint electronic states satisfying the restriction α symbolises the valence or the conduction bands and ε = 0.02 eV is an arbitrary tolerance. For the intralayer case, we have k′ = k + qM because the two electronic states, k and k′, are connected by qM while for the interlayer process, k′ = R(θ)k + qM, where R(θ) is the rotation matrix that takes the Dirac cone of one layer into the Dirac cone of the other layer. Considering these two conditions, momentum conservation is always achieved in our model by a given pair of states, k and k′, as shown in Fig. In order to determine the resonance energies θ. The position of the low-energy peaks increases, whereas the position of the high-energy peaks decreases with increasing values of θ. These two maxima are associated with electronic transitions close to K and M points of the graphene, and will be called E− and E+. For the intralayer process, the positions of EvHs.Figure θ represented by the dashed blue, green, pink and red curves, respectively. In Fig. −1 that are observed in samples with small angles (4°–6°). They nicely agree with the calculated Ea versus θ results represented by the dashed blue curve. Details about the experimental values are shown in Supplementary Note −1 for samples with intermediate twisting angles (12–16°) are plotted as open black circles in Fig. θ. The black symbols × in Fig. EvHs measured directly by optical conductivity17. It is interesting to note that, despite the fact that our model does not consider electronic coupling between the layers that opens a minigap which splits the valence and the conduction bands in TBL, the agreement between the minimum possible value 17 is remarkable.Figure ħqM as a function of θ. The frequencies of the extra peaks of samples with small (intermediate) values of θ are plotted as open blue (black) circles in Fig. The red and black curves in Fig. a and Le peaks for samples with intermediate angles. However, Ta and Le were not observed in our multiple-excitation experiments using visible photons. The lack of observation of Le can be due to the huge enhancement of the G band. Since the position of the Le peak is very close to the G-band position in samples with intermediate twisting angles, it is possibly masked by the G-band enhancement. For samples with θ around 10° and measured using the 1.96 eV laser line, Campos-Delgado et al.9 reported the observation of a peak at 1622 cm−1, that might be assigned to the Le. The absence of Ta can be ascribed to the very weak cross-section of TO phonons activated by the intralayer process. Electron–phonon matrix elements calculations would be necessary to better clarify this issue.In principle, the intralayer and interlayer el–ph processes can also activate TO and LO phonons, respectively, and give rise to Tθ in the range of 2–9°, using three laser lines with energies 1.49, 1.70 and 1.82 eV  that was not observed in the results obtained with visible photons and shown in Fig. qM tends to zero. The laser energies where the extra peaks observed in the infrared (IR) Raman spectra in samples with θ in the ranges of 3–5 and 6–9° have the maximum intensity are shown, respectively, by the light blue and red open squares in Fig. The calculated results presented in Fig. θ show that extra peaks are expected to appear in the UV Raman spectra (for excitation energies above 3 eV). Figure −1. In the pioneer Raman study of graphene folded onto itself, Gupta et al.5 observed nondispersive extra peaks in the range of 1370–1395 cm−1, and showed that they were enhanced in the UV Raman spectrum (Elaser = 3.41 eV). Righi et al.6 studied the UV Raman spectra of TBGs and observed several extra peaks in the range of 1370–1420 cm−1, but the twisting angles θ were not determined in this work. In both studies6 the appearance of new peaks was not accompanied by the enhancement of the G band, as expected for an intralayer el–ph process. In contrast, the UV data of Wang et al.11 in TBG samples with large values of θ show the activation of new peaks in spectra where the G band also exhibits an enhancement of 5–20 times, indicating thus an interlayer el–ph scattering. The laser energies where the extra peaks of the samples with large values of θ in the range of 22–28° exhibit the largest intensity and the frequencies of these peaks observed in the UV Raman spectra are represented by black open squares in Fig. 11 are also plotted in Fig. The theoretical predictions in Fig. θ = 23, 25 and 28°, performed with the 3.00 and 3.41 eV excitation energies (details are described in the Methods section). The vertical scale gives the ratio of ITBG/ISLG. We can see in the spectra new peaks with frequencies in range of 1370–1420 cm−1, as predicted in Fig. Figure θ = 23° sample recorded with the 3.00 eV line and shown in Fig. ITBG/ISLG for the G band is ≈ 9, suggesting that the two peaks around 1380 and 1410 cm−1 come from phonons of the TO and LO branches, respectively, and are activated by the interlayer el–ph process (Te and Le peaks). On the other hand, in the spectrum of θ = 28° sample recorded with the 3.41 eV line and shown in Fig. ITBG/ISLG ≈ 2 for the G band reveals the activation by the intralayer el–ph process. We thus assign the extra feature around 1370 cm−1 as a La peak, although we cannot rule out the possibility of being a Ta peak, since, as shown in Fig. θ. In Fig. ITBG/ISLG for the G band is around 4 and only one extra peak is observed. In this case, possibly both processes are occurring simultaneously. The peak position around 1380 cm−1 suggests that it comes from a phonon of the TO branch, and it can be thus assigned as a Ta/Te peak. The laser energies where the extra peaks shown in Fig. In the spectrum of the 20 reported the observation of weak extra peaks both below and above positions of the G band in graphene, and suggested that they arise from the graphene/h-BN interaction.As discussed above, the intralayer el–ph process occurs in one graphene layer, and the second layer only imposes a periodic potential needed for momentum conservation in the DRR process. Therefore, extra peaks are also expected to appear in the Raman spectra of graphene deposited on top of any atomically flat substrate, assuming that the interaction graphene/substrate is strong enough to scatter electrons. In a recent study of graphene on the top of h-BN, Eckmann et al.θ 20. They are much less intense than the extra peaks in bilayer graphene showing that the imposed potential of h-BN on graphene is much weaker than graphene–graphene interaction. Results shown in Fig. In order to check this assumption, we prepared graphene/BN samples by transferring SLG to the top of a h-BN crystal. From the analysis of optical images, where the crystallographic edges of graphene and BN are evidenced, we can estimate the twisting angle qM can be activated by two different resonant el–ph processes: the interlayer process, where the electron scattering occurs between the Dirac cones of different graphene layers, and the intralayer process which occurs in a single graphene layer, and the other layer or substrate only imposes a periodic potential that activates the phonons in the Raman spectrum. The observation of new peaks in the Raman spectrum of SLG on the top of h-BN crystals proves that the intralayer el–ph process occurs not only in TBG, but also whenever graphene is in contact with any periodic layer or substrate. The intensity of these extra peaks is expected to provide the strength of the interaction between monolayer graphene and the substrate. On the other hand, the activation of new phonons by the interlayer el–ph process reported in many previous studies in the literature occurs only in TBGs.We report in this work the observation of intralayer and interlayer el–ph interactions in twisted graphene heterostructures with different mismatch twisting angles by Raman spectroscopy. Measurements performed with many different laser excitation energies allowed us to conclude that phonons of graphene with momenta This work highlights the importance of understanding fundamental el–ph interactions in heterostructures of 2D materials, which affect thermal and transport properties in these systems. The possibility of distinguishing intralayer and interlayer el–ph interactions in bilayer graphene and of studying the interaction of monolayer graphene with a crystalline substrate using light scattering allow new ways to design devices applications of 2D heterostructures and is crucial for the engineering of new devices.θ. Experiments in the visible range were performed in a DILOR XY triple-monochromator spectrometer equipped with a N2-cooled charge-couple device detector, a 1800 g/mm diffraction grating, and using an Ar/Kr with 12 laser lines in the visible range. The NIR Raman measurements were performed on a home-made setup, including an iHR-550 Horiba spectrometer equipped with a liquid-nitrogen-cooled silicon CCD detector. A tunable CW Ti:sapphire laser filtered using tunable laser line filters21 was used for excitation. The scattered light was collected through a ×100 objective (NA = 0.95) using a backscattering configuration. The 3.41 eV Raman spectra were obtained using a Dilor UV Raman spectrometer equipped with a cooled CCD and an argon-ion laser. The laser power on the sample was kept lower than 1 mW with ×40 objective to avoid heating. The Raman spectra at 3.00 eV were obtained using a Jobin-Yvon-Horiba T64000 Raman spectrometer equipped with a cooled CCD with a krypton-ion laser. The laser power on the sample was limited to 0.5 mW with ×100 objective. In the case of G/h-BN heterostructures, the measurements were performed in Witec Alpha 300R system using 633 nm as a pump laser.The Raman measurements of the TBG samples were performed using many different laser lines, in the near IR, visible and UV ranges, for samples with different twisting angle 13. The graphene layers were first covered by a thin layer of polycarbonate, followed by etching in HCl aqueous solution to remove the Cu in the transfer process. The polycarbonate film with attached graphene was then transferred onto different substrates: a 300 nm SiO2/Si, 90 nm SiO2/Si and fused silica. Finally, the polycarbonate film was removed using chloroform. The gr/BN samples were prepared by mechanical exfoliation of graphene and transference to a h-BN substrate.The TBG samples were grown by CVD technique using methane (99.99%) on polycrystalline Cu foils22. The electronic structure calculations are based on a tight-binding approach in which the parameters are fitted to reproduce density functional theory (DFT) calculations with many-body corrections, while the phonon dispersion was obtained from many-body corrected DFT calculations. The many-body corrections change the phonon slope of the highest optical branch near K (with respect to DFT), reproducing the Kohn anomaly and providing a much better agreement with inelastic X-ray scattering measurements for graphite23. These curves describe accurately the D band of graphene when a red shift of 40 cm−1 is applied for phonons close to the K point. However, in order to reproduce the phonons close to the graphene G band, we blue shifted all frequencies by 20 cm−1. Even though the calculations were done only by folding the SLG results, the physics of the intralayer and interlayer processes in TBG is nicely captured.The electronic and phonon structures were obtained by folding the SLG calculation results. For SLG, we follow the calculation procedure given in ref. 17. As far as phonon properties are concerned, Cocemasov et al.24 have shown that the weak van der Waals interactions between layers in TBG do not alter significantly the frequencies of the branches in the range considered here. Although the folding method provides a good description of the phonon frequencies and of the electronic transitions of TBG, the theoretical model used here does not give us the intensities of Raman peaks. For that we also need to consider the values of the matrix elements for the el–ph and electron–photon interactions. The computation of these matrix elements can clarify, through the intensity, if the peak marked as Ta/La in Fig. a or La peak.Concerning the electronic properties, it has been shown that interlayer interactions perturb the bands from each layer producing minigaps in the conduction and valence band of TBG with same energy. However, the allowed transitions do not depend on the size of the minigap, and therefore the folding approach is supposed to give good results concerning the Raman processes investigated hereThe data that support the findings of this study are available from the corresponding author upon request.Supplementary Information(PDF 2759 kb)Peer Review File(PDF 261 kb)


Flexible sensors connected to cell phones are a promising technology that can aid in continuously monitoring signals in our daily lives, such as an individual’s health status and information from buildings, farms, and industry. Among such signals, real-time humidity monitoring is crucial to a comfortable life, as human bodies, plants, and industrial environments require appropriate humidity to be maintained. We propose a hydrophilic polytetrafluoroethylene (H-PTFE)-based flexible humidity sensor integrated with readout circuitry, wireless communication, and a mobile battery. To enhance its sensitivity, linearity, and reliability, treatment with sodium hydroxide implements additional hydroxyl (OH) groups, which further enhance the sensitivity, create a strong linearity with respect to variations in relative humidity, and produce a relatively free hysteresis. Furthermore, to create robust mechanical stability, cyclic upward bending was performed for up to 3000 cycles. The overall electrical and mechanical results demonstrate that the flexible real-time H-PTFE humidity sensor system is suitable for applications such as wearable smart devices. The development of flexible humidity sensors has been gaining interest in several applications, including industrial production processes, environmental monitoring, and healthcare ,2,3. MonAmong several types of humidity sensors, the capacitive humidity sensor has been widely investigated because of its capability to function at high temperatures, fully recover from condensation, and resist chemical vapors ,6. CapacMany materials have been studied as dielectric materials for capacitive humidity sensors, including polymers, ceramics, and composites ,9,10. Ce4, with respect to the variation in relative humidity (RH) over a range of 45~90%. In addition, linear behavior of the sensing performance and a low hysteresis were clearly observed. Moreover, the proposed sensor was not significantly sensitive to temperature fluctuations and showed no decrease in performance, even after a cyclic bending test of 3000 cycles. Finally, we implemented a real-time monitoring system through system-level integration of the fabricated flexible H-PTFE humidity sensor with readout circuits and a Bluetooth module. Finally, the proposed system was attached to a curved surface for real-time monitoring of the moisture of the materials, which demonstrates the potential of our sensor as a humidity-monitoring application.In this report, we present a flexible capacitive humidity sensor using hydrophilic polytetrafluoroethylene (H-PTFE) as a dielectric sensitive film for water uptake. H-PTFE is a polymer-based membrane that has a random porous structure with a 0.45 μm pore size and 80% porosity. Due to its porous structure, H-PTFE has a large volume area and flexibility like a paper. In addition, H-PTFE is known to have thermal stability, chemical stability, and tear resistance, which also provide advantages for monitoring sensors . Further−6 Torr. Titanium was used for an adhesion layer in this process. After deposition, the shadow mask was removed. To enhance the mechanical stability of sensors, poly (PET) film (70 µm) was laminated beneath the thin H-PTFE film.The H-PTFE membrane was obtained from Merck Millipore, Burlington, USA. First, the H-PTFE film was prepared with dimensions of 1.5 × 1.5 cm. In order to form the capacitive electrode on the H-PTFE film, a nickel alloy shadow mask  was used for selective deposition of the electrode source. The pattern of the shadow mask was an interdigitated structure with eight fingers. Each finger width was 250 µm and the finger-to-finger distance was 150 µm. The thickness of the shadow mask is 100 μm, which could be adhered completely to the H-PTFE substrate beforemetal deposition. After fixation of the shadow mask above the H-PTFE film, titanium/gold (20 nm/100 nm) was deposited by e-beam evaporator under a vacuum of 10The fabricated H-PTFE humidity sensor was incubated in NaOH solution  for 2 h at room temperature. This process was performed with stirring. After removing the sensor from the NaOH solution, the sensor was dipped into DI water to rinse it enough, then it was dried by nitrogen gas. Finally, the remaining water in the H-PTFE film was completely removed by annealing in a vacuum oven  with a temperature of 120 °C for 1 h. −1.High-resolution field-emission scanning electron microscopy (FE-SEM) images were acquired using Merlin, Carl Zeiss operating at 10 kV. The optical microscope images were obtained by BX51M, Olympus. Fourier transform infrared (FT-IR) spectra were recorded using a Spectrum One system, Perkin-Elmer, with a wavenumber range of 500–4000 cmThe fabricated sensor was connected with wire using conducting tape for electrical measurements. Then, it was inserted in a humidity and temperature chamber  for testing. The capacitive responses of the sensor were measured as a function of RH with different ranges from 45% to 90% by a CV analyzer  at 40 kHz. The sensor exposure time to each RH environment was 5 min. After measurements, the sensors were stored in a dry environment. Cyclic bending was performed using a multi-modal bending tester  with a controlled radius and number of bending cycles. After bending, the sensing characteristics were measured with re-flat condition.For real-time monitoring, anisotropic conductive film bonder  were utilized to achieve electrical contact between the H-PTFE sensor and readout circuit. We already have mobile application systems used in previous studies. The humidity data of the H-PTFE sensor, transmitted via Bluetooth communication, was displayed in a mobile application.The schematic structure and a photograph of the H-PTFE flexible humidity sensor are shown in + ions have been reported to be very effective in changing the bond structure of PTFE [+ and OH− ions infiltrated the H-PTFE pore structure. Then, the Na+ ions break the strong carbon (C)–fluorine (F) bonds, and produce a small quantity of C–OH bonds [Although H-PTFE plays a significant role in water absorption, the water affinity of the sensor can be enhanced by treatment of NaOH on the H-PTFE humidity sensor. Na of PTFE ,17. The  of PTFE . When thOH bonds . These m2 and CF3 groups at 1201 cm−1, 1146 cm−1, 638 cm−1, and 502 cm−1 [−1 [−1 associated with OH [The FT-IR spectra provided information on the chemical functionalization of the PTFE surface upon treatment with NaOH. 502 cm−1 ,22. The cm−1 [−1 . Also, a with OH . This in4, compared to non-treated H-PTFE sensors under a same RH range. Not only was there the long-range linear behavior, but also the extremely low non-coincidence between absorption and desorption of the H-PTFE sensor with NaOH treatment was represented in In Abundant OH groups in the NaOH-treated H-PTFE film promote the physical and chemical absorption of water molecules, which provides a great advantage in detecting humidity in low-RH regions. Also, in high-RH regions, the water molecules absorbed on the H-PTFE surface make a secondary layer through physical adsorption by adjacent water molecules, which demonstrates a large increase in capacitance responses .To estimate the sensor performance in terms of reliability, we measured the capacitance responses of four H-PTFE sensors with regard to RH variation. There are several requirements to use our H-PTFE humidity sensor for real-time monitoring. High sensitivity, linearity, and reliability have already been mentioned above, and in terms of stability, we conducted additional experiments. First, to verify the sensing stability of our sensor under different temperatures, sensing responses were measured under a temperature range from 10 °C to 40 °C. The RH was fixed at 50% and 90%, respectively. As shown in n) of 100, 1000, and 3000 with a 10 mm bending radius. The back of the H-PTFE film was laminated with a 70 µm thick PET film to downshift the mechanical stress. As shown in Stable operation under mechanical stress also should be ensured for a flexible sensor. Herein, to confirm the performance variation of flexible H-PTFE humidity sensors under mechanical stress, we performed cyclic bending tests with different numbers of bending cycles  are designed, as shown in The flexible H-PTFE sensor was connected with the readout circuit by utilizing anisotropic conductive film bonder which is widely used for contact between electrodes. As shown in inset of In conclusion, we demonstrated the potential of a flexible sensor using H-PTFE as a dielectric sensitive film to perform reliable humidity monitoring. We showed that the sensor response was dependent on the RH. It was confirmed that NaOH treatment of the H-PTFE film further improved the sensitivity, detection dynamic range, and reliability of water uptake. The cycling bending test also proved the high mechanical flexibility of the humidity sensor. Finally, in order to validate the applicability for real-time monitoring, the flexible humidity sensor, integrated with an analog interface and wireless communication, was attached on the curved surface, and then was used to detect humidity changes. Our future efforts will be focused on improving the sensitivity and reproducibility of the sensor, and further extending this platform to humidity detection for industrial, environmental, and medical fields.


Other than the general role of graphene as a high-performance electron acceptor or transporter, the observed enhancement in photocatalytic activity over HTT/graphene can be ascribed to the improved interfacial charge migration from enhanced chemical bonding (Ti–C bonds) during the in-situ compositing process. The formation of Ti–C bonds is confirmed by XPS analysis and the resulting enhanced separation of photoinduced charge carriers is demonstrated by electrochemical impedance spectra and transient photocurrent response.Hydrogen titanate nanotube (HTT)/graphene nanocomposites are synthesized by hydrothermal reduction of graphene oxide (GO) and simultaneous preparation of nanotubular HTT via an alkaline hydrothermal process. By using this facile in-situ compositing strategy, HTT are densely supported upon the surface of graphene sheets with close interface contacts. The as-prepared HTT/graphene nanocomposites possess significantly enhanced visible light catalytic activity for the partial oxidation of benzylic alcohols. The amount of graphene has significant influence on catalytic activity and the optimal content of graphene is 1.0 wt %, giving a normalized rate constant  In the last several decades, the photocatalytic process in semiconductors has been attracting more and more attention as an advanced green technology for contaminant purification, energy production, and selective organic synthesis ,2,3. The2-derived nanosized materials appear to be a potential candidate, owing largely to their excellent performance, long term stability, low toxicity, and cost [2 nanostructures, such as TiO2 nanotubes, nanowires, and nanofibers [2(B) or anatase phase, exhibits higher catalytic performance for pollutant degradation than P25-TiO2 [2 or titanate remains problematic. Numerous strategies have been studied to tailor the photocatalytic process and promote catalytic performance. Particularly, fabrication of carbon-titanate heterojunction composites has been identified as one of the most feasible strategies to enhance the separation of electron-hole pairs [Among various photocatalysts, TiOand cost ,5. In thnofibers ,7,8. Thenofibers ,10. For P25-TiO2 ,12. Howele pairs ,14,15.2WO6 composites with strong covalent bonding between graphene and Bi2WO6 nanoparticles. The improved photocatalytic performance for H2 and O2 production were ascribed to the efficient electron migration and separation of photoinduced charge carriers through the chemically bonded interaction [2 nanoparticles. This chemical bonding effect plays a pivotal role in the interfacial electron migration, which makes TiO2/graphene composites possess an outstanding catalytic activity for the photo-oxidation of formaldehyde, compared to a mechanically mixed sample [Owing to the great surface area of graphene and its excellent electronic transport properties, many studies have been carried out on fabricating composite catalysts of graphene and semiconductor particles in an attempt to promote photocatalytic activity ,17. Undeeraction . Xie et d sample .3N4, HNb3O8 nanosheets, CdS, and TiO2-based composites [2 catalysts using visible light, although the photocatalysts themselves and the reactants can only absorb UV light [The selective oxidation of alcohol to carbonyl compounds is a significant chemical conversion process. Generally, the partial oxidations of alcohols are implemented with hazardous or toxic stoichiometric oxidants under high temperature and pressure conditions . Deteriomposites ,26,27,282 or TiO2 nanohybrids as photocatalysts [2O for hydrogen production [However, previously reported research on photocatalytic selective synthesis principally focused on reaction systems using TiOatalysts ,30,31. Toduction ,33, wherIn this paper, HT nanotube (HTT)/graphene nanohybrids with numerous heterogeneous interfaces are prepared via a facile hydrothermal method under basic conditions. Due to their one-dimensional structural characteristics, HT nanotubes can be uniformly coupled onto graphene to form close interfacial contacts. The presence of chemical Ti–C bonds between nanotubular HT and graphene are confirmed by XPS analysis. The HTT/graphene nanocomposites with chemically bonded interfaces exhibit exceptional photocatalytic reactivity toward the partial oxidation of benzylic alcohols under visible illumination. The influence of graphene content on the catalytic performance is investigated and a possible mechanism based on the experimental results is also proposed.4, H2SO4, H3PO4, H2O2 (30%), NaOH, P25 TiO2, and ethanol were obtained from Aladdin Industrial Inc. . Benzyl alcohol and its derivatives were purchased from J&K Scientific Ltd. . 5,5-Dimethyl-1-pyrroline-N-oxide was obtained from TCI Development Ltd. . All the other chemicals were of analytical reagent grade and used as received without further purification. Deionized water was obtained using a Millipore Milli-Q system and used throughout the experiments.Graphite was purchased from Nanjing XFNANO Materials Tech Co., Ltd. . KMnO2SO4/H3PO4  was added into a mixture of squama graphite and KMnO4 . Then, the obtained mixture was heated for 12 h at 323 K under magnetic stirring. After being cooled down to room temperature, the mixture was poured into an icy solution of 30% H2O2. The solid was recycled by filtration, washed by deionized water, and vacuum-dried at 298 K for 12 h. The obtained graphite oxide (GO) was dispersed in water (0.5 mg/mL) and subjected to sonication for 60 min to form an aqueous dispersion of exfoliated GO.To improve the hydrophilic and oxidation degree of graphite, a modified method was used to prepare graphite oxide . In brie2 was added to 65 mL of concentrated NaOH solution (10.0 M) and was stirred for 30 min to form a uniform suspension. Then, a specific amount of GO dispersion was added to the above-obtained suspension under magnetic stirring. The weight ratios of GO to P25 TiO2 were 0, 0.2, 0.4, 1.0, 2.0, and 4.0%, and the as-prepared samples are labeled as HTT/graphene-x%, where x refers to the mentioned weight ratios. After further stirring for 90 min, the mixture was transferred into a 100 mL stainless steel autoclave and heated at 160 °C for 12 h. The obtained precipitate was separated by centrifugation, washed by deionized water, and re-dispersed for several cycles until a pH value of nearly 8 was obtained. The precipitate was finally washed with absolute ethanol and dried at 90 °C for 10 h to obtain HTT/graphene-x% nanocomposites.In a typical synthesis process for HTT/graphene nanohybrids, 1.3 g of P25 TiO2 and NaOH solution. About 10 mg of graphene was added into 50 mL of deionized water and sonicated for 1 h. Then 1.0 g of as-made HTT was mixed into the graphene suspension under magnetic stirring. After being stirred for 2 h, the homogeneous suspension was treated by centrifugation. The precipitate was washed with deionized water and dried in a vacuum oven at 90 °C for 10 h. The as-obtained product is denoted as HTT/graphene-1.0%-mixed. For the preparation of the P25 TiO2/graphene-1.0% composite, 1.0 g of P25 TiO2 was added to 65 mL of deionized water and stirred for 30 min. 10 mL of GO dispersion (1.0 mg/mL) was added into P25 TiO2 suspension under magnetic stirring. After further stirring for 90 min, the mixture was transferred into a 100 mL Teflon-lined autoclave and heated at 160 °C for 12 h. The obtained precipitate was washed with deionized water and dried at 90 °C overnight. The as-synthesized sample is named as P25 TiO2/graphene-1.0%.For comparison, bare HTT and graphene samples were prepared using a similar process without GO or P25 TiO−1. X-ray photoelectron spectra were recorded using a Thermo Scientific ECALAB 250xi system with a monochromated Al Ka X-ray source. UV-visible diffuse reflectance spectra were collected on a UV-vis spectrophotometer  using BaSO4 as the reflectance standard. The specific surface area of as-synthesized samples were measured by N2 adsorption-desorption with a Micrometrics ASAP 2020 apparatus.X-ray diffraction measurements were conducted using a D8 Advance diffractometer (Bruker) with Cu Kα radiation (λ = 0.15418 nm). Raman spectra were recorded on a Labram HR 800 Raman spectrometer  using 532 nm excitation light at room temperature. The morphological features of HTT/graphene nanohybrids were observed by a transmission electron microscope  operating at 200 kV. FTIR spectra were acquired with a Nicolet 750 FTIR spectrometer in the range 400–4000 cm4 and nitrogen gas was continuously purged through the electrolyte solution. After being immersed in a benzyl alcohol solution for 1.5 h, the working electrode was irradiated using a 300 W Xe lamp with a 400 nm cut-off filter. Electrochemical impedance spectra (EIS) were measured using the above-mentioned three-electrode cell at 0.0 V with 0.1M Na2SO4 aqueous solution. Electron spin resonance (ESR) signals of the radicals trapped by DMPO were collected on a Bruker ER200-SRC spectrometer at ambient temperature.Photoelectrochemical experiments were performed in a standard three-electrode system. Platinum foil and Ag/AgCl were used as the counter and reference electrodes, respectively. The working electrode was prepared by the following method: 40 mg of powder sample was dispersed in 0.2 mL of polyethylene glycol solution and ultrasonicated for 45 min to obtain a uniform slurry. The resulting slurry was then spread on a fluorine doped tin oxide (FTO) conducting glass and was dried at 90 °C to get HTT/graphene/FTO electrodes. The photocurrent was recorded at a bias voltage of 0.6 V on an electrochemical workstation . The electrolyte was 0.1 M LiClO2 for 30 min. This suspension was stirred magnetically for another 60 min to reach the adsorption-desorption equilibrium of the reactant alcohols and catalysts. The mixture was then irradiated using a 300 W Xe lamp with 400 nm cut-off filter under ambient temperature. At certain time intervals, a 0.5 mL mixture was withdrawn and filtered using a Millipore filter (pore size 0.45 μm) to remove the catalyst particles. The concentration changes of reactants in the remaining filtrate were quantitatively analyzed on a gas chromatograph (Agilent 7890) equipped with a standard FID detector and a DM-5 amine capillary column. Oxidation products were identified by comparing the retention times with the standards and further confirmed with GC-MS.The partial conversion of benzyl alcohol was performed in a 20 mL round-bottom quartz glass flask. Benzyl alcohol (0.25 mmol) and the photocatalyst (50 mg) were dispersed into 5 mL of acetonitrile and the obtained mixture was bubbled with Otert-butyl alcohol, triethanolamine, and benzoquinone as the scavenger for the hydroxyl, photogenerated hole, and superoxide radicals, respectively) were performed via an analogous process of benzyl alcohol photo-oxidation, except that the radical scavenger (0.2 mmol) was included. The primary role of the active species were revealed by analyzing the changes of the benzyl alcohol conversion yield.In order to elucidate the photocatalytic mechanism, control experiments using different radical scavengers , suggesting an interfacial interaction between graphene and HTT in the nanocomposites.The presence of graphene in the HTT/graphene nanocomposites could be confirmed using FTIR spectra . Bare GO053 cm−1 . Compare−1 range for GO and HTT/graphene nanocomposites, as shown in 3 defects, and the latter gives information about the in-plane vibrations of sp2 bonded carbon [ID/IG) represents the relative concentration of defects in GO or graphene. Compared with bare GO, the calculated ID/IG of the HTT/graphene composites are lower, demonstrating a lower concentration of defects in graphene. The decreased ID/IG ratios in HTT/graphene nanohybrids imply the employed hydrothermal process facilitates the formation of more graphene without reducing the average size of sp2 domains. Furthermore, it is worth noting that a G band up-shift from 1588 to 1596 cm−1 is observed for HTT/graphene compared with GO and graphene, which gives evidence for chemical bonding of carbon materials [Raman spectroscopy is also widely employed to characterize the electronic structure of carbon products. Two bands are observed in the 850–2000 cmd carbon ,34. The aterials ,38. The The morphologies of HTT/graphene composites can be observed by TEM, as shown in 2 adsorption-desorption experiments were used to investigate the textual properties of the as-synthesized nanocomposites. The specific surface area, SBET was calculated according to the classical Brunauer–Emmett–Teller (BET) theory and the corresponding data are summarized in BET than bare HTT, which is due to the huge surface area of graphene [BET increases from 228.9 to 248.0 m2·g−1 when the content of graphene increases from 0.2% to 4.0% for as-synthesized HTT/graphene composites. There is no doubt that the enhanced SBET can provide more surface active sites and facilitate the adsorption of the reactants, thus promoting heterogeneous catalytic reactions.Ngraphene . Additio2-hybridized carbon of GO and the oxygen-containing carbonaceous bands (C–OH) [The interactions between HTT and graphene are further studied by XPS. The high-resolution C 1s XPS spectrum of bare GO is shown in s (C–OH) ,39. Thes1/2 and Ti 2p3/2 spin-orbital splitting photoelectrons of Ti4+. Two bands at binding energies of 463.3 and 457.7 eV usually correspond to Ti3+ [4+ and Ti3+ in HTT, two other weak peaks located at 465.3 and 459.4 eV are found and are likely due to the forming of Ti–C bonds in the HTT/graphene nanohybrids. This suggests that a chemical interaction between HTT and graphene is present in as-synthesized HTT/graphene composites and absent in the mixed samples.Forming Ti–C bonds is also investigated and identified by analyzing the Ti 2p XPS spectra of the HTT/graphene nanocomposites. As depicted in  to Ti3+ ,36. In F2/graphene -1.0%. As depicted in 2/graphene nanohybrids for contaminant degradation and hydrogen production [To investigate the photocatalytic performance of HTT/graphene nanohybrids, the selective oxidation of benzylic alcohols was chosen as a model reaction. No product of aldehyde was obtained without visible radiation or the photocatalyst, indicating that the selective oxidation of benzyl alcohol is conducted through the photocatalytic reaction. oduction ,20.2/graphene-1.0% and HTT/graphene-1.0%-mixed have also been tested and analyzed under the same experimental conditions. The sample of P25 TiO2/graphene-1.0% shows dramatically lower activity  than the HTT/graphene-1.0% composites, suggesting that more direct contact and the resulting strong interactions between HTT and graphene sheets are critical for the enhancement of photocatalytic activity because of improved electron/hole separation and migration. The physical mixed sample (HTT/graphene-1.0%-mixed) shows a significantly lower conversion yield than that of HTT/graphene-1.0% synthesized via a one-step hydrothermal process. This suggests that simple mechanical-mixing with graphene has a small effect on improving the photocatalytic activity of HTT, and is due to the absence of a chemical-bonding interface between HTT and graphene. This also indicates that the interaction state between HTT and graphene has crucial influence on photocatalytic performance of HTT/graphene nanohybrids.For comparison, the selective conversion of benzyl alcohol in P25 TiO0/Ct)  and the reaction time is displayed, indicating the oxidation is pseudo-first order. The pseudo-first-order rate constant (k) of the HTT/graphene photocatalysts can be obtained from the slope. The rate constant (k) of benzyl alcohol oxidation over the optimal photocatalyst, HTT/graphene-1.0%, is 0.411 h−1, which is 5.3 and 6.4 times higher than those of the P25 TiO2/graphene-1.0% and HTT/graphene-1.0% -mixed samples.Additionally, the reaction kinetics of benzyl alcohol oxidation were investigated and the corresponding results are shown in 3– and CH3O–) facilitate the formation of aldehyde products; for example, 97.6% of 4-methoxybenzyl alcohol can be converted in 4.5 h with 96% selectivity. Whereas, the electron-withdrawing substituents (such as F– and Cl–) retard the conversion of alcohols. This suggests that the existence of an electron-donating group at the p-position of benzyl alcohol benefits the selective conversion over an HTT/graphene photocatalyst.To investigate the general applicability of HTT/graphene composites, the oxidation of various benzylic alcohols over the optimal HTT/graphene-1.0% were examined and the corresponding results are displayed in The stability or recyclability of catalysts is another important practical issue. In order to study the stability and durability of HTT/graphene composites, cycle experiments are performed. After the catalytic reactions, HTT/graphene-1.0% photocatalysts are separated and recovered by washing, and are reused. As depicted in −1) after being adsorbed over HTT/graphene-1.0%. On the other hand, the characteristic peaks belonging to skeletal vibrations of the phenyl ring (at 1495 and 1451 cm−1) and the C–C stretching vibration (at 1207 cm−1) show no changes [2OH groups of the alcohol molecules interact with the unsaturated Ti sites of HTT, forming alcoholate species –CH2–O(H)–Ti.Surface adsorption generally plays a critical role toward photocatalysis-driven selective oxidation of alcohols. The surface adsorption of benzyl alcohol on HTT/graphene composites is first investigated using FTIR spectra A. In com changes ,28. Thes2–O(H)–Ti can produce visible absorption, which provides a prior condition for the selective conversion of benzylic alcohol to product aldehyde. This has been previously shown in research on photocatalytic reactions over TiO2 or Nb2O5 [2/graphene nanohybrids [The formation of surface complexes –CHor Nb2O5 ,41. Baseohybrids . AlthougThe band gap energy of the photocatalyst is an important factor affecting the catalytic performance, especially for a visible light photocatalytic system. The band gap energies of pure HTT, HTT/graphene-1.0%, and HTT/graphene-4.0% are estimated through a Tauc plot of the modified Kubelka–Munk function with linear extrapolations C. The caI-t curves of the above-mentioned three working electrodes in a benzyl alcohol containing electrolyte solution under visible light illumination. The photocurrent is primarily determined by the charge transfer from the adsorbed benzyl alcohols to the HTT conduction band and the subsequent graphene surface. The photocurrent density of the HTT/graphene-1.0% electrode is enhanced ~4.6 times that of pure HTT, indicating the separation efficiency of photoinduced charges can be improved by the interfacial electronic interactions between HTT and graphene. Moreover, the photocurrent density of the in-situ composited HTT/graphene-1.0% is enhanced ~2.3 times that of the mechanical-mixed hybrid (HTT/graphene-1.0%-mixed), which is primarily ascribed to the chemical interactions between HTT and the graphene sheets. Additionally, for the HTT/graphene-1.0% electrode, the photocurrent density in the presence of benzyl alcohol is predominantly higher than that in its absence. This result implies the generation of anodic photocurrent is closely related to the surface adsorption of benzyl alcohol upon the HTT/graphene-1.0% electrode. The photocurrent experiments offer direct evidence for electron migration from the adsorbed benzyl alcohol to the conduction band of HTT and subsequently to graphene surface under visible light.To investigate visible light induced charge transfer during the photo-oxidation of benzylic alcohols over HTT/graphene catalysts, the transient photocurrent responses were recorded over working electrodes made of pure HTT, HTT/graphene-1.0%-mixed and in-situ compositing HTT/graphene-1.0%. SBET normalized rate constants of benzyl alcohol oxidation. This suggests that visible light induced charge transfer plays a pivotal role in the photocatalytic conversion of benzylic alcohol. For graphene-based photocatalytic systems, the enhanced charge separation efficiency is dominated by the high spatial charge transportation properties of graphene [Based on the experimental data in graphene ,44. The graphene ,20,21,22graphene ,20,21,225, 2.2 × 105, and 1.0 × 105 ohm, determined by fitting with the equivalent circuit. This demonstrates that the introduction of graphene facilitates the charge transfer of HTT. Furthermore, the arc radius of the EIS Nyquist curve for HTT/graphene-1.0% is smaller than that of both the physically mixed (HTT/graphene-1.0%-mixed) and pure HTT samples. Generally, the arc radius of an EIS spectrum reflects the resistance of the interface layer at the surface of the electrodes [ectrodes ,45. The ectrodes ,45. Thestert-butyl alcohol (TBA) to the reaction solution [2 by sparging with N2 results in a low conversion of benzyl alcohol (ca. 15%), indicating that O2 plays a critical role in photocatalytic oxidation of benzyl alcohol. When triethanolamine  is put into the reaction system, the oxidation of benzyl alcohol is remarkably inhibited, implying that partial oxidation of benzyl alcohol is primarily driven by photo-holes. Additionally, the introduction of benzoquinone (BQ) results in an obvious decrease in the conversion of benzyl alcohol, suggesting that O2·ˉ is also involved during the photo-oxidation of benzyl alcohol. To confirm the formation of O2·ˉ, active radical species generated over HTT/graphene-1.0% are detected by ESR using DMPO as the spin-trapping agent. As depicted in 2·ˉ.The involved catalytic mechanism of alcohol photo-oxidation on HTT/graphene composites is further studied. Control experiments on radical trapping were carried out and the obtained results are illustrated in solution , the con2 to produce a superoxide anion radical. Meanwhile, the adsorbed benzylic alcohol transforms into an intermediate with a positive charge. Subsequently, the photogenerated hole drives the α-deprotonation of the intermediate and gives rise to a neutral carbon radical. Then the reaction between the substrate radical and superoxide species yields the product aldehyde. Compared to the HTT/graphene-mixed samples, the in-situ composited HTT/graphene catalysts possess significantly enhanced photocatalytic activity for the selective oxidation of benzylic alcohols under visible light illumination, as indicated in According to these results, a possible mechanism for the improved visible light catalytic activity over as-prepared HTT/graphene composites is proposed and depicted in −3 g/m2·h, which exceeds that of pure HTT by more than seven times. Further characterization results indicate that HTT are chemically bonded with graphene surfaces via Ti–C bonds, which is supported by XPS analysis. More importantly, the chemical-bonded nanocomposite exhibits a remarkable enhancement of photocatalytic activity, which is 5.2 times that of a mechanical-mixed sample. Based on the results of transient photocurrent and EIS, this enhancement can be ascribed to the interfacial charge transfer effect, which provides favorable spatial conditions for electrons migration from in-situ generated surface complexes to neighboring HTT and finally to graphene. This effect inhibits the recombination of electrons and holes, and thus yields superior photocatalytic performance. Our study on the efficient interfacial charge transfer via a chemically bonded interface provides new insights for guiding the design of novel photocatalytic reaction systems, particularly for solar-light driven selective organic synthesis.In summary, HTT/graphene nanohybrids with superior photocatalytic activity were fabricated through a facile hydrothermal process. Nanotubular HTT are densely supported upon the surface of graphene with close interfacial contacts. Introducing graphene can markedly improve the catalytic performance for the selective conversion of benzylic alcohols under visible illumination and the contents of graphene have significant effects on the catalytic activity of HTT. The optimal content of graphene is 1.0 wt % and the corresponding normalized rate constant k of benzyl alcohol oxidation is 1.71 × 10