Caenorhabditis elegans model of TDP-43 proteinopathy expressing human mutant TDP-43 pan-neuronally (TDP-43 tg). In TDP-43 tg C. elegans, we conducted a genome-wide RNAi screen covering 16,767 C. elegans genes for loss of function genetic suppressors of TDP-43-driven motor dysfunction. We identified 46 candidate genes that when knocked down partially ameliorate TDP-43 related phenotypes; 24 of these candidate genes have conserved homologs in the human genome. To rigorously validate the RNAi findings, we crossed the TDP-43 transgene into the background of homozygous strong genetic loss of function mutations. We have confirmed 9 of the 24 candidate genes significantly modulate TDP-43 transgenic phenotypes. Among the validated genes we focused on, one of the most consistent genetic modifier genes protecting against pTDP accumulation and motor deficits was the heparan sulfate-modifying enzyme hse-5, the C. elegans homolog of glucuronic acid epimerase (GLCE). We found that knockdown of human GLCE in cultured human cells protects against oxidative stress induced pTDP accumulation. Furthermore, expression of glucuronic acid epimerase is significantly decreased in the brains of FTLD-TDP cases relative to normal controls, demonstrating the potential disease relevance of the candidate genes identified. Taken together these findings nominate glucuronic acid epimerase as a novel candidate therapeutic target for TDP-43 proteinopathies including ALS and FTLD-TDP.Pathological phosphorylated TDP-43 protein (pTDP) deposition drives neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). However, the cellular and genetic mechanisms at work in pathological TDP-43 toxicity are not fully elucidated. To identify genetic modifiers of TDP-43 neurotoxicity, we utilized a  C. elegans neurons, we have modelled aspects of ALS pathobiology. This animal model exhibits severe motor dysfunction, progressive neurodegeneration, and accumulation of abnormally modified TDP-43 protein. To identify genes controlling TDP-43 neurotoxicity in C. elegans, we have conducted a genome-wide reverse genetic screen and found 46 genes that participate in TDP-43 neurotoxicity. We demonstrated that one of them, glucuronic acid epimerase, is decreased in patients with FTLD-TDP suggesting inhibitors of glucuronic acid epimerase could have therapeutic value for ALS and FTLD.The protein TDP-43 forms aggregates in disease-affected neurons in patients with ALS and FTLD-TDP. In addition, mutations in the human gene coding for TDP-43 can cause inherited ALS. By expressing human mutant TDP-43 protein in  TARDBP, have been identified in ~4% of families with heritable ALS directly linking TDP-43 dysfunction with disease  (primary RNAi screen and retesting), CK423 bkIs423[Psnb-1::hTDP-43(M337V)+Pmyo-2::dsRED], and CK426 bkIs426[Psnb-1::hTDP-43(A315T)+Pmyo-2::dsRED] , CK10 bkIs10[Paex-3::hTau(V337M)+Pmyo-2::GFP], and CK144 bkIs144[Paex-3::hTau(WT)+Pmyo-2::GFP] for motility assays. OH1487 hse-5(tm472), DA509 unc-31(e928), and CB1072 unc-29(e1072) were used as controls.Wild-type escribed . Transge::dsRED] , 78. GR1unc-22 (enhancer control) were screened in parallel for all experiments. Follow-up screening of putative positives was carried out on individual 60mm plates and were given a semi-quantitative overall score from 0 to 5, where 0 is unable to differentiate from the empty L4440 vector control, and 5 is complete suppression to N2 wild-type behavior. All RNAi clones from the Ahringer library that passed initial screening were analyzed by Sanger sequencing to validate clone targeting.Initial screening of the feeding RNAi Ahringer library was conducted essentially as described . BacteriC. elegans locomotion were carried out as previously described with minor modifications [Assessments of ications . In briehse-5(tm472) with CK423 and the reporter strain EG1285 oxIs12[Punc-47::GFP + lin-15(+)]. Animals were grown to the indicated stage at 20°C before scoring. Living animals were immobilized on a 2% agarose pad with 0.01% sodium azide, and intact GABA-ergic neurons (L4 or day 1 adult) or axon abnormalities (L4 stage) were scored under fluorescence microscopy on a DeltaVision Elite (GE) imaging system using an Olympus 60x oil objective. Statistical significance was analyzed using Prism statistical software.Strains with GFP marked gamma-aminobutyric acid (GABA)-ergic motor neurons were generated by crossing Images were acquired using a Leica SP5 confocal microscope with a 40x oil immersion lens. Z-plane stacked images were flattened into a maximum intensity projection using Imaris x64 software. Images were minimally processed using Adobe Photoshop to enhance visibility of fluorescent signal.C. elegans were grown to L4 stage at 20°C, and then transferred to NGM plates containing the indicated concentrations of aldicarb (Sigma). Animals were scored for paralysis using a gentle touch on the head and tail with a platinum wire We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.Yours sincerely,Anne C. HartAssociate EditorPLOS GeneticsGregory BarshEditor-in-ChiefPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: This study by Liachko et al perform the first genome-wide RNAi screen for modifiers of TDP-43 M337V toxicity. They find a number of candidates in interesting GO categories such as energy metabolism, protein homeostasis, and ECM/cytoskeleton. They then follow up on a single candidate gene, hse-5. They find that an hse-5 mutant decreases TDP-43 neurodegeneration of GABAergic cells in C. elegans, and that knock down of the ortholog GLCE in a mammaliam model of pTDP-43 is also protective. Finally they see decreased levels of GLCE in human brains with TDP-43 pathology. Overall, this is a well-done, comprehensive screen for modifiers of TDP-43 toxicity. The candidate gene hse-5/GLCE is quite intriguing with solid follow up in cell culture and human neuropath. A few minor additions would strengthen this paper but overall an important, well-done study.1) For the Western blots of TDP-43 and pTDP-43, some quantification of the results would be helpful. The suppressor hits that did not change TDP-43 and pTDP-43 levels would also be interesting to see.2) In regards to TDP-43 levels, the authors are careful to avoid saying that the decreased levels are due to improved clearance, but nonetheless it would be important to see if the candidates change TDP-43 mRNA levels. If they do so, it would not disqualify the candidates, but one would have a better, different idea of mechanism of protection.3) In regards to hse-5, one wonders if adult only (or from L4 onwards) RNAi has an effect and would avoid the potentially protective/potentially harmful axonal abnormalities4) The screen was performed in a mutant TDP-43 line, however the cell culture and FTLD-TDP would presumably express wild-type TDP-43. Therefore, it would be interesting to see if the hse-5 RNAi or mutant is protective against the WT TDP-43.5) Also, Table 2 is referred to in the text, but was not included in the manuscript for review.Reviewer #2: Genome wide analysis reveals heparan sulfate epimerase modulates TDP-43 proteinopathyThe manuscript by Liachko and colleagues is an investigation into finding genetic modifiers of TDP-43 toxicity. These genetic modifiers may inform into the mechanisms underlying TDP-43 linked pathologies, and they may also serve as new targets for therapeutic intervention. The work is done with a well characterized C. elegans model of TDP-43 neuronal toxicity and a genome wide reverse genetics screen using RNA interference to screen for suppression of TDP-43 induced motility phenotypes.The work is well done, the manuscript is clearly written and logical in its presentation. Furthermore, I would like to commend the authors on completing such a labour intensive genetic screen. The results are novel and likely to aid further research in the field. However, there are a few outstanding issues that need to be addressed before considering the work for publication.Major pointsUnbiased genetic screens are powerful ways to advance understanding. However, sometimes it is not evident by which mechanism a modifier achieves its phenotypic effects. Here the authors sequentially limit their candidate genes to a select few before settling on hse-5/GLCE.There is no doubt that this gene has an effect on phosopho TDP-43 phenotypes. But the mechanism for the suppression is not sufficiently accounted for.p. 10 “Preliminary characterization of GLCE suggests it can act to detoxify pTDP and is a therapeutic target worth further examination.”The choice of focusing on one gene, hse-5 is reasonable, but I have several questions.How does a potential ECM protein affect the levels of TDP-43 phosphorylation in worms and tissues? What is the working model? The gap in understanding should be addressed, at least as speculation in the discussion.Has GLCE showed up as a modifier of ALS in GWAS studies?Where is hse-5 expressed in C. elegans and how does this relate to the expression of mutant TDP-43?Were both deletion alleles of hse-5 tested?The fact that hse-5 does not suppress neurodegeneration is unfortunate, perhaps this is the key mechanism, but at the same time maybe it is non-specific. Does hse-5 result in alternative neuronal connections that simply compensate for impaired movement in TDP-43 animals? Do hse-5 animals have generally improved motility? How do they respond to aldicarb and/or levamisole? They say that RNAi candidates working in non-transgenic worms were excluded as part of the selection process, but it is possible that retesting of hse-5 RNAi in N2 worms would miss subtle effects. Thus, do hse-5 mutants have improved motility compared to N2 worms?Furthermore, a control that is missing is tests versus wild type TDP-43 transgenics. The 2010 Liachko et al. manuscript describes the WT control strains, thus these are likely the floor for the amount of suppression possible with this approach and hse-5 should be tested in these WT TDP-43 strains.Suppression of phenotypes associated with human disease proteins is of wide interest. Thus, it would be good to know if suppression of TDP-43 toxicity by hse-5 is specific. Does hse-5 protect against other forms of toxicity, perhaps Tau, given the role of phosphorylation in tau toxicity.Does hse-5 affect the expression of the TDP-43 transgenes? This would be good to know as it could change interpretation of the potential mechanism for reduced pTDP-43. Perhaps for the other 4 candidate genes as well.Finally, does hse-5 affect lifespan? Also good to know in terms of modifiers of age-dependent phenotypes.Minor issuesTable 2 is missing.p. 5 “We found that 5 of the mutants tested had decreased levels of TDP-43 protein accompanied by reduced phosphorylation . Interestingly, these results indicate the remaining 4 suppressor genes that improve TDP-43 motor dysfunction do so without a direct impact on TDP-43 phosphorylation or protein levels.”It looks like there is more of a mix of reduced total TDP-43 versus pTDP-43. It is clear that something is happening, but perhaps a simple relative quantification would aid the figure. Also, what happened to TDP-43 in the gly-8 mutants?Reviewer #3: The authors present data on an unbiased genome-wide RNAi screen for suppressors of tdp-43(M337V) toxicity. After screening 16757 individual gene knockdowns, they identified 46 candidate genes that were required for tdp-43(M337V) toxicity. Based on sequence homology, 24/46 genes appeared to have human homologs. These 24 genes fell into several functional classes. The authors obtained loss-of-function mutants for 17 genes and found that 9 mutants mimicked the RNAi results and suppressed tdp-43(M337V) toxicity. For five of these mutants, the authors showed that overall tdp-43 protein levels were reduced. The authors focus their attention on one gene, hse-5, which consistently suppressed tdp-43(M337V) toxicity. hse-5 appears homologous to the human gene GLCE, which modifies heparin sulfate proteoglycans. An hse-5 mutant, tm472, exhibits improved motor function in the tdp-43(M337V) background. Surprisingly, hse-5(tm472) significantly increases the loss of GABA motor neurons in the tdp-43(M337V) background. The remaining neurons exhibit excessive branching and axonal complexities. This increase in axon abnormalities was additive with tdp-43(M337V). The authors also show that siRNA inhibition of GLCE in human HEK cells substantially reduced endogenous tdp-43 phosphorylation. Finally, the authors examine frontal cortex brain sections from post-mortem FTLD patients and observed significantly reduced GLCE staining in the FTLD patients.The manuscript is concise and clearly written. The introduction clearly frames the significance of the work. However, the discussion section essentially restates the results and does not offer substantial insights into any of the hits or their potential significance to the field. It would be extremely useful to discuss whether any of these genes or their homologs have been identified in the other screens mentioned by the authors. If so, what can be learned? If not, what might be unique about this tdp-43 model that facilitated their identification? Could the over-expression of mutant human tdp-43 in the background of an endogenous WT worm tdp-43 influence the results of the screen? How might this be addressed? I would also like to see more discussion of why a gene involved in (mostly) extracellular protein modification that is predicting to be localized within the secretory pathway is required for toxicity of a nucleo-cytoplasmic protein. This is not clear and is one of the most interesting conclusions from this work. There is also a lot known about the role of hse-5 in neuronal migration and axon regeneration from previous work, which is basically not discussed. Could these functions possibly explain how hse-5 might be protecting against tdp-43? Additional discussion in this area is warranted.I also have several major and minor concerns regarding the data as presented:Major1. In the screen flow chart, the authors indicate that gene knockdowns that are “different for TDP-43 tg versus non-transgenic” are discarded. Given that the phenotype being screened for is improved motility in the tdp-43(M337V) background, how would one see improved motility in a non-tg (ie wild type) background? Were any hits eliminated based on this criteria? Or was the filtering for gene knockdowns that had similar motility in WT and tdp-43(M337V)? This description needs to be clarified. Along these lines, it would be useful to see the motility of WT in Fig 2A and 2B.2. Figure 2 C-G, the authors show that 5 loss of function mutants, including hse-5, exhibit reduced phosphorylated tdp-43(M337V) protein levels, as well as total tdp-43(M337V) levels . The reduction in phospho-tdp could be due to several less interesting effects that have nothing to do with tdp-43, including transgene suppression (ie gly-8 mutants appear to have NO tdp expression) or reduced snb-1 promoter activity and subsequent reductions in tdp mRNA. Were any of these possibilities examined? This is important not only for the authors to interpret their data but also to prevent others from chasing non-existent tdp-43 toxicity mechanisms.3. The authors state that the results from Fig 2C-G “indicate the remaining 4 suppressor genes improve TDP-43 motor dysfunction without a direct impact on TDP-43 phosphorylation or protein levels.” Given that no data were presented for these 4 mutants, there are no data in the manuscript to support this conclusion.4. On pg7, para2, the authors state “Loss of C. elegans hse-5 protects against accumulation of phosphorylated TDP-43. This is not supported by the data, which show a qualitatively similar decrease in both total tdp-43 protein levels and phospho-tdp-43 levels in hse-5 mutants. Therefore, when hse-5 is lost, there is LESS tdp-43 to protect against.Minor1. Why does 2A show motility data in the M337V while 2B shows it in the A315T? Have any suppressors been identified that exhibit allele-specific suppression? Is there something preventing comparisons in the same tdp-43(M337V) background, given that authors went to the trouble to make two unlinked integrated strains?2. There are two Figure 4C panels3. The PDF file that I reviewed lacked Table 2, which is referenced in the paper at least twice. Not sure if this was an author or a journal issue.4. The reduction in GLCE protein in the frontal cortex of FTLD patients is interesting. The hypothesis that reduced GLCE expression in the FTLD patients is protective is quite provocative. If this is true, one might expect GLCE expression levels to be normal in other brain regions not undergoing neurodegeneration. Have other non-degenerating regions been examined? Perhaps this could be more easily addressed in worms. For example, is hse-5 expression down-regulated in tdp-43(M337V) worms?**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: No: Table 2 is referred to in the text, but I cannot find it in the manuscript provided.Reviewer #2: No: Table 2 was missing.Reviewer #3: No: numerical data underlying graphs was not provided in a spreadsheet.**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  and your responses to reviewer comments. If eligible, we will contact you to opt in or out. *Dear Dr Kraemer,Thank you very much for submitting your Research Article entitled 'Genome wide analysis reveals heparan sulfate epimerase modulates TDP-43 proteinopathy' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The manuscript describes important work and the revised manuscript has dealt with all substantive concerns raised by the reviewers. There are still some minor concerns raised by reviewer 3. In my opinion as academic editor, these last small concerns do not preclude accepting the manuscript-- but I want to give you the opportunity to read these comments and consider changing the manuscript text. Adopting these reviewer suggested changes may increase clarity and readability of the manuscript. Therefore, I am returning the manuscript to you for "minor revisions". 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You will be contacted if needed following the screening process.PLOS has incorporated To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.[LINK]Please let us know if you have any questions while making these revisions.Yours sincerely,Anne C. HartAssociate EditorPLOS GeneticsGregory BarshEditor-in-ChiefPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: The authors have addressed my concerns as stated in the earlier review.Reviewer #2: This is a much improved manuscript, and the authors have adequately addressed most of my concerns.Reviewer #3: 1. Were RNAi clones sequenced to confirm their identity? Neither the methods nor the results state that they were sequence verified. Table 2 suggests that many ‘hits’ were not phenocopied using LOF mutants. One possibility for this is that the RNAi clone targeted a different gene. From many RNAi screens, we find that ~10% of Ahringer library RNAi clones do not correspond to the predicted gene when sequenced.2. There are still some nomenclature issuesa. Allele names in Table 2 are not italicizedb. Mutant name in Fig 2A & B, 3A,B,C,G, S1A,B,S2A-E are not italicized. Also, the allele should be listed, not (-). There is no evidence presented that these alleles are null mutants, as implied by the (-) designation3. P7 ‘ To confirm mRNA expression of the TDP-43 transgene in these strains…’. I believe the authors meant to say ‘ To determine if TDP43 mRNA expression levels were altered in these strains…’. To me, statements that aim to confirm imply that the authors are trying to prove their hypothesis is true, rather than attempting to disprove their hypothesis.4. The extended paragraph on hse-5 expression, mutant phenotypes, physiological roles, etc (p7-8) doesn’t seem to belong in the results section and should be moved to the discussion.5. The authors refer to an interaction between hse-5/GLCE and TDP-43 (p9). Since the authors have not demonstrated that there is a physical interaction between these two proteins, they need to be more precise with this statement and indicate that they are referring to a genetic interaction.6. I find the first two pages of the discussion section to be largely uninformative. It simply restates the molecular associations for many of the hits from this screen. Virtually all of that information is found in Table 1 and the discussion does not really expand on anything. On the other hand, the discussion that occurs after these sections is much improved.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********what does this mean?). 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At the onset of cancer, specific biomarkers get elevated or modified in body fluids or tissues. Early diagnosis of these biomarkers can greatly improve the survival rate or facilitate effective treatment with different modalities. Potential nanomaterial-based biosensing and bioimaging are the main techniques in nanodiagnostics because of their ultra-high selectivity and sensitivity. Emerging graphene, including two dimensional (2D) graphene films, three dimensional (3D) graphene architectures and graphene hybrids (GHs) nanostructures, are attracting increasing interests in the field of biosensing and bioimaging. Due to their remarkable optical, electronic, and thermal properties; chemical and mechanical stability; large surface area; and good biocompatibility, graphene-based nanomaterials are applicable alternatives as versatile platforms to detect biomarkers at the early stage of cancer. Moreover, currently, extensive applications of graphene-based biosensing and bioimaging has resulted in promising prospects in cancer diagnosis. We also hope this review will provide critical insights to inspire more exciting researches to address the current remaining problems in this field. Cancer, the second leading cause of death globally, accounts for 8.8 million deaths and possibly over 21 million by 2030 . Ch. Ch59]. However, constructing biosensors through sandwich strategy attracts more attention because of its higher specificity towards biomarkers detection. Normally, there are graphene-based biosensors and graphene-based probe-amplifying biosensors. The graphene-based biosensor is to employ graphene as an electronic matrix to immobilize recognition units. The first example was developed by Lin group . ChitosaIn Optical biosensing has become a prevailing technology in biomedical diagnostics, which contains fluorescence, colorimetric, interferometric, and surface plasmon resonance (SPR) biosensors. The optical tunability of nanographene and its derivatives are advantageous in optical biosensing. Meanwhile, recognition molecule are involved in a reversible link–delink process on the graphene surface, which is the basis of designing specific optical biosensors. Qu et al. developed a naked-eye readout colorimetric immunoassay for PSA detection based on the intrinsic peroxidase activity of GO . CompareAs for the protein biomarker, many prominent studies by optical biosensors have been reported. One example is demonstrated in Bioimaging enables the morphological features of organs to be correlated with pathological symptoms, which is important for finding biomarkers and subsequently using them for the diagnosis of diseases . GQD andIn-vitro cancer-related bioimaging includes direct cancer cells imaging and targeted cancer cells imaging. Numerous studies have demonstrated that GQD and nanographene with good optical properties are available to get access into cancer cells. Fan demonstrated that boron-doping GQDs gave rise to rich fluorescence owing to their peculiar interaction with the surrounding media. This kind of boron-doping GQD can be further used to detect biomarkers through cellular imaging . Liu et Then there emerged targeted cancer cells imaging. The targeted cancer cells imaging could be achieved by modifying graphene with recognition units with promoted cancer-specific detection. The earliest studies ,153 by DSince targeted cancer cells imaging was achieved, in-vivo imaging has become an alternative method to diagnose cancer. Especially the feasibility of carbon dots as a fluorescence contrast agent for in-vivo optical imaging was successfully reported in 2009 by Yang et al . Just 1 The past few years have witnessed significant developments of graphene-based nanomaterials in cancer diagnosis applications. In this article, we summarized the great potential of graphene-based materials for cancer diagnosis. Three kinds of graphene-based materials have been introduced: 2D graphene films, 3D graphene architectures and GHs. The fabrication of a variety of recognition units including aptamers, antibodies and enzymes enables graphene to identify specific cancers. In addition, graphene in turn makes cancer identification more sensitive, selective and accurate on account of its elegant versatility and outstanding properties. Two main methods of cancer monitoring including biosensing and bioimaging are extensively introduced both in vitro and in vivo. As demonstrated in this article, the research on cancer diagnosis applications of graphene has seen dramatic progress and is expanding rapidly, yet it is still in its infancy.The booming advances made in this area are currently exciting and encouraging; the challenges, however, are also huge and still need to be overcome. Much more attention should be given to the issues of its clinical practicability. The major challenge facing graphene-based cancer diagnosis lies in the huge variations presented in experimental designs among researches. Dawidczyk et al. pointed out in 2014 that the lack of universal rules in preclinical trials have obstructed a systematic comparison of these studies . TherefoFor in-vitro cancer diagnosis, diagnosis of cancer at its earliest stage often requires the detection of biomarkers with very low concentrations in body fluids, which is a complex system including different kinds of interference species. Therefore, biomarkers with low specificities result in a high risk of false–positive signals. Moreover, a single biomarker cannot serve as a perfect cancer screening tool to achieve the purposes of diagnosis, prognosis and therapy. It is urgent to develop effective graphene-based devices with multifunction, which can detect multi-biomarkers simultaneously to promote the accuracy of cancer discovery. Furthermore, design of simpler, faster, smaller graphene-based devices for the monitoring of cancer biomarkers is a thriving research and development area which elevates the potential applications graphene in cancer diagnosis. For in-vivo cancer diagnosis, better understanding of the behaviors of graphene in vivo is remarkably important to investigate their toxicology. Although NGOs, especially PEG-NGO, have been reported to be biocompatible in biomedical applications, its potential long-term toxicity and nonbiodegradable nature are still a major concern for clinical use. More studies on the potential acute and chronic effects of graphene and its metabolism/excretion in the body, are urgently required. In addition, the application in drug delivery and cancer therapy is one of the hottest fields of graphene-based research. Exploitation in multipurpose probes and the integration of both diagnosis and treatment of graphene are extremely promising for elevating the efficiency of diagnosis and therapy.Overall, as has been discussed, graphene with outstanding physical and chemical features defines its prospective progress in the area of cancer diagnosis, appearing to put a solid milestone on the discovery of early cancer. Researchers are continuing to conquer the difficulties above and will eventually develop graphene-based devices capable of clinical application.


Despite a rich choice of two-dimensional materials, which exists these days, heterostructures, both vertical  and in-plane, offer an unprecedented control over the properties and functionalities of the resulted structures. Thus, planar heterostructures allow p-n junctions between different two-dimensional semiconductors and graphene nanoribbons with well-defined edges; and vertical heterostructures resulted in the observation of superconductivity in purely carbon-based systems and realisation of vertical tunnelling transistors. Here we demonstrate simultaneous use of in-plane and van der Waals heterostructures to build vertical single electron tunnelling transistors. We grow graphene quantum dots inside the matrix of hexagonal boron nitride, which allows a dramatic reduction of the number of localised states along the perimeter of the quantum dots. The use of hexagonal boron nitride tunnel barriers as contacts to the graphene quantum dots make our transistors reproducible and not dependent on the localised states, opening even larger flexibility when designing future devices. The possibility to combine planar and van der Waals heterostructures holds great promise for nanoscale electronic devices. Here, the authors report an innovative method to synthesise embedded graphene quantum dots within hexagonal boron nitride matrix for vertical tunnelling single electron transistor applications. At the same time, the transport properties of such quantum dots, which are typically carved out of large sheets of graphene, are dominated by the localised edge states9. Furthermore, arranging tunnelling contacts  to such quantum dots with the specific, reproducible conductivity is a separate challenge.Graphene quantum dots (GQDs) and graphene nanowires have been attracting attention because of the linear spectrum obeyed by the quasiparticles12 and vertical15 heterostructures to mitigate the issues with the localised states both at the edges of the quantum dots and at the edges of the contacts. We propose to form GQDs inside the hexagonal boron nitride (hBN) matrix through catalytic-assisted substitution of boron and nitrogen atoms by carbon16. As the lattices of graphene and hBN are very similar—most of the bonds in a GQD become properly terminated, which minimises the number of such localised states. Thus, the edges of a GQD become effectively passivated with hBN. We would like to stress that our method allows the formation of GQDs with specific size, and can be extended to other structures and devices. We then used hBN tunnelling barrier and graphene electrodes in order to form contacts to such quantum dots, creating single-electron tunnelling transistors. Such method results in very precise, reproducible contact resistance and the graphene-hBN interface free of localised states.Here we use planar2 substrate described in ref. 16. Based on the spatially controlled conversion, the growth of in-plane GQD-hBN heterostructure was achieved on a SiO2 substrate covered by an array of platinum (Pt) nanoparticles (NP), as illustrated in Fig. 17  (PS-P4VP) micelles, with an H2PtCl6 precursor for Pt NPs within their cores, followed by the annealing at 400 °C. Then, the conversion of the hBN sheet to graphene on the array of Pt NPs on the SiO2 substrate was accomplished at ~950 °C in methane/argon atmosphere. During the reaction, the hBN on top of Pt NPs was selectively converted to graphene, with the formation of uniform GQD arrays embedded in the hBN film  and G (1595 cm−1) bands of graphene with an intervening E2g peak (1371 cm−1) of an hBN20. Furthermore, the in-plane graphene domain size (La) was calculated using the ratio of the integrated intensity (ID/IG) according to the Tuinstra–Koenig relation21  spectra of the GQD-hBN in-plane heterostructure  by using dry transfer method32 (see device fabrication in Methods). Here, 30 nm_hBN stands for the hBN layer with an approximate thickness of 30 nm, Gr—for graphene, 2hBN—for 2-layer thick exfoliated hBN, GQD-hBN—for GQD-hBN lateral heterostructure. Schematic structure and the layer arrangement of our devices is presented on the inset to Fig. Such GQDs embedded in an hBN matrix are ideally suited for the formation of van der Waals heterostructures33 for details). Figure G = dI/dVb as a function of the gate (Vg) and bias (Vb) voltages for one of our devices with 13 nm GQDs  to arise owing to tunnelling through the localised electronic state, and the diamond-shaped features—to Coulomb blockade diamonds owing to tunnelling through single-electron states in individual GQDs.We performed tunnelling spectroscopy on our van der Waals heterostructures, by applying a mixed signal of AC and DC bias voltages between the two graphene electrodes, and a gate voltage to the silicon substrate . Currently we don’t have an explanation for this effect. However, it can be speculated that as it is the silicon gate (separated from the layer with GQD by ~ 300 nm of SiO2 and hBN), which provides the most efficient screening (graphene electrodes provide only weak screening)—GQD interact strongly between themselves via Coulomb interaction. It means that the observed Coulomb diamonds are the result of the collective behaviour of several GQDs within the 300 nm radius. This would also explain the different intensities of the conductivity peaks, Fig. In our diagrams Fig. , each se2PtCl6: 1 mL of the PS-P4VP copolymer solution with H2PtCl6 was diluted by 400 mL of pure PS-P4VP. Such mixed solution was spin-coated on the SiO2 substrate, and the micellar film was treated by oxygen plasma to produce Pt NPs. The hBN monolayer was transferred onto the Pt NPs/SiO2 substrate, and the conversion reaction was performed for conversion of hBN on Pt NPs to graphene. After the aqua regia treatment to remove Pt NPs, a GQD-hBN monolayer with a relatively long spacing (0.5–1.5 μm) between GQDs  as a function of the gate and bias voltages is presented in Fig. We used such hBN with a low density of GQDs to prepare single-electron tunnelling transistors 30 nm_hBN/Gr/2hBN/GQD-hBN/2hBN/Gr/20 nm_hBN as it has been described above , where the edges of the diamonds are seen as distinct steps —when it aligns with the Fermi level in the bottom and in the top graphene contacts. The energy positions of these lines can be fitted with very high precision . Note that it has been demonstrated that impurity states in the middle of the barrier contribute the strongest to the tunnelling current43 . Then, the hBN layer transferred on Pt NPs/SiO2 was loaded into the centre of a vacuum quartz tube in a furnace for the conversion reaction16. The tube was pumped down to 0.21 Torr with pure argon gas (50 sccm). Then the furnace was heated to 950 °C in 40 min. During the reaction, methane gas (5 sccm) with argon (50 sccm) was flown as the source for graphene growth. During the reaction, the hBN region on the Pt NPs was converted to graphene, and after 10 min of growth, a uniform GQD array embedded in the hBN film was obtained.The single layer of hBN was synthesised on Pt foil using ammonia borane as a precursor by the CVD method. Experimental details on the synthesis of CVD-grown hBN on Pt can be found in a previous report2 could be transferred to any other substrate via wet-transfer method using HF and an aqua regia solution. First, polystyrene (PS) was spin-coated on the sample, and it was immersed in an hydrogen fluoride solution (5% in deionised water) to detach the GQD-hBN film. The floating PS film was then transferred to the aqua regia solution (3:1 mixture of hydrochloric acid and nitric acid) to remove Pt NPs and then rinsed with copious amounts of DI water. Finally, the film was transferred onto the target substrate and PS was removed with toluene to obtain a GQD-hBN film on the substrate.The GQD-hBN film on Pt NPs/SiOSEM  and atomic force microscopy  were used to determine the surface morphology of the samples. Raman spectra were measured using a micro Raman spectroscope  using 532 nm. The UV-vis absorption spectra of the GQD-hBN samples were recorded on a Cary 500 UV-vis-near IR spectroscope, Agilent. XPS  and Nano-FTIR  were performed to determine the composition of the GQD and confirm the formation of an interface between GQD and hBN. Low-voltage Cs aberration-corrected transmission electron microscopy , operated at 80 kV with a monochromated electron beam, was used for EELS analysis. The spatial and energy resolutions for the EELS measurement are 2 nm and 1.5 eV, respectively.2). Here, the bottom and top layers of hBN were used for the purpose of encapsulation. As the GQDs are embedded in the large area monolayer hBN on Si/SiO2, the vertical heterostructure was assembled in two halves by adopting a mix of dry and wet flake transfer procedure: first, a stack of Si/ SiO2/30 nm_hBN/Gr/2hBN was prepared by standard flake exfoliation and dry transfer procedure. To prepare the other half, a stack of 2hBN/Gr/20 nm_hBN was prepared by the dry pick up procedure using a polymethyl methacrylate (PMMA) membrane. This stack on the membrane was aligned and dropped on the GQD-hBN/SiO2/Si substrate. To release the stack from Si/SiO2, 8% PMMA was spun on the sample and Si/SiO2 was etched using potassium hydroxide (KOH) solution. The floated membrane was thoroughly rinsed with DI water several times to remove KOH residues from the membrane. Finally, to complete the device, this membrane containing GQD-hBN/2hBN/Gr/20 nm_hBN was dropped on the stack prepared in the first half.Optical images of the flakes at different stages of the fabrication process are presented on Supplementary Figure For electrical characterisation of this vertical heterostructure, Cr/Au edge contacts were made on the top and bottom graphene layers using electron beam lithography followed by boron nitride etching, metal deposition and lift-off process. Electrical characterisation of graphene contacts is presented in Supplementary Figure Supplementary Information


Graphene based photocatalysts are highly suitable for the system, but their industrial scale use requires immobilization for improved separation and recovery of the photocatalyst. Therefore for practical purposes, design and fabrication of film type graphene photocatalyst with higher solar energy conversion efficiency is an absolute necessity. As a means to achieve this, we report herein the successful development of a new type of flexible graphene film photocatalyst that leads to >225% rise in visible light harvesting efficiency of the resultant photocatalyst-biocatalyst integrated artificial photosynthesis system for highly selective solar fuel production from CO2 compared to conventional spin coated graphene film photocatalyst. It is an important step towards the design of a new pool of graphene film based photocatalysts for artificial photosynthesis of solar fuels from CO2.Our growing energy demands must be met by a sustainable supply with reduced carbon intensity. One of the most exciting prospects to realize this goal is the photocatalyst-biocatalyst integrated artificial photosynthesis system which affords solar fuel/chemicals in high selectivity from CO This has triggered keen interest in the development of graphene based nanoparticle powder or nanocomposite photocatalysts7. However the industrial scale application of these photocatalysts is largely limited due to difficulty in their filtration and separation. Therefore for practical and economic purposes design and fabrication of film type graphene photocatalyst with higher solar energy conversion efficiency is an absolute necessity.Graphene is as an ideal material for light harvesting applications15. Although easy to formulate, uncontrolled capillary flow and dewetting processes during solvent evaporation result in non-uniform films with random orientation of graphene-chromophore units leading to increased charge recombination and consequently lower photocatalytic activity. Moreover, chromophore coupling to graphene sheet is usually carried out through functional groups such as –COOH present on graphene oxide (GO) or its reduced forms such as chemically converted graphene (CCG)16. They contain a large number of non-conjugated tetrahedral sp3 carbons which lie slightly above or below the graphene plane, further limiting electronic conductivity of the photocatalyst17. Keeping in view these crucial bottlenecks, it is imperative to develop a new design and fabrication strategy for graphene film photocatalysts.Graphene films have been previously fabricated by spin coating method2 compared to conventional spin coated graphene film photocatalyst. The monolayer graphene for this work was supported on a flexible polyimide sheet leading to a practical and easy to use photocatalyst. It also helped to solve the problem of insufficient mechanical strength of monolayer graphene film photocatalyst. The lack of functional groups on graphene monolayer for chromophore attachment presented another major challenge. This was ingeniously overcome by attaching chromophore units to sp2 carbon centers on graphene via 1,3-dipolar cycloaddition reaction. To the best of our knowledge, graphene film photocatalyst for solar fuel production using this approach has not been fabricated prior to this work.Here we report the successful development of a new type of flexible graphene film photocatalyst  collects incident photons as an electronic transition between localized orbital around it (HOMO to LUMO) and conducts via graphene (as a multi electron acceptor) to reduce the rhodium complex Rh ([Cp*Rh(bpy)H2O]2+;Cp* = pentamethylcyclopentadienyl, bpy = 2,2′-bipyridine). Upon reduction, the rhodium complex abstracts a proton from aqueous solution and transfers hydride ion to NAD+, which gets converted into NADH, thus completing the photocatalytic cycle. In this way, Rh behaves as an electron mediator between the graphene film photocatalyst and NAD+ leading to regeneration of NADH cofactor. Finally the enzymatic (Formate Dehydrogenase) conversion of CO2 substrate to formic acid consumes NADH. The NAD+ thus released again acts as a substrate for photocatalytic cycle, leading to photoregeneration of NADH. The photocatalytic-enzymatic cycles thus couple integrally, leading to exclusive formic acid production from CO23.Figure via 1,3-dipolar cycloaddition. More details are provided in the experimental section. The integration of GFPC 1 photocatalyst with biocatalyst (formate dehydrogenase enzyme) afforded the integrated artificial photosynthesis system for carrying out highly selective formic acid production from CO2  with 1,3-Dioxo-1H-dibenzoisoquinoline-2[3 H]-carbaldehyde (hereinafter DdIC chromophore) 18. This blue shift in absorption maxima can be attributed to the 1,3-dipolar cycloaddition of DdIC to GF. This observation indirectly suggested the covalent attachment of DdIC chromophores on the monolayer graphene.The existence of DdIC in GFPC 1 was confirmed by UV-vis spectroscopy. A strong soret band at 433 nm was observed in the absorption spectrum of DdIC chromophore in DMF Fig. . On the −1)19. Moreover in GFPC 1, the C=O stretching was observed at 1772 and 1707 cm−1 while C=C stretching was observed at 1670 and 1597 cm−1  data provided clear evidence for the coupling of chromophore to graphene film Fig. . The GF m−1 Fig. . In comp19. An increased thickness of 1.4681 nm in GFPC 1 indicated functionalization on the GF surface. Apart from increased thickness, the presence of DdIC on the GF surface was further supported by the bloated appearance and roughness of the sample. The difference in the high resolution transmission electron microscopy  and DdIC chromophore23.Raman studies further confirmed the coupling of DdIC chromophore to GF Fig. . The G a24 of DdIC a single new peak at 397 eV ascribed to N atoms of the C-N bond appeared  and GFPC 1 was also studied by thermogravimetric analysis (TGA) Fig. . TGA wasThe most critical part of the work was to evaluate the photocatalytic performance of the GFPC 1 in comparison to the DdIC chromophore, graphene-DdIC powder photocatalyst and its spin coated film sample . Therefore, visible light-driven 1,4-NADH photo-regeneration ability of all these were examined along with GF. While GF failed to carry out any NADH regeneration, DdIC chromophore afforded 21.8% NADH regeneration over a period of 120 minutes. On the other hand, graphene-DdIC powder photocatalyst and GFPC 2 afforded 48.9 and 40.1% of NADH regeneration over a period of 120 minutes, respectively. Since GFPC 2 was obtained by spin coating the graphene-DdIC powder, a lower NADH regeneration by GFPC 2 can be clearly attributed to irregular orientation of the photocatalyst species on the polyimide sheet. In comparison to this 91.8% NADH regeneration was observed when GFPC 1 was evaluated for the photocatalytic activity Fig. . In othe2  and another one without visible light irradiation were also carried out which also failed to show any detectable amount of formic acid. Additionally, no formic acid formation was detected in another set of control experiments carried out in the absence of rhodium complex Rh and/or NAD+ which confirmed their vital role in the functioning of the photocatalyst/biocatalyst integrated artificial photosynthesis system. To evaluate the possibility of rhodium complex Rh functioning as the CO2 reduction photocatalyst a control experiment consisting of rhodium complex and CO2 was performed. However, no formic acid was detected in this case also. This indicated that rhodium complex does not carry out photocatalytic CO2 reduction. Along with the high stability of GFPC 1 under visible light irradiation the various control experiments clearly point towards exclusive visible light driven photocatalytic NADH regeneration followed by enzymatic CO2 reduction as the sole route for formic acid production. This was further confirmed by C-13 isotope labelling experiment which clearly indicated that CO2 is the sole source of formic acid formation in our system H2O]2+; Cp* = pentamethylcyclopentadienyl, bpy = 2,2′-bipyridine) reduction was observed at −0.97 V . The vicinity and potential gradient between the light harvesting GFPC 1 photocatalyst and Rh center enable this efficient electron transfer from former to latter. The resultant reduced Rh species [Cp*Rh(bpy)] upon chemical protonation carries out catalytic regeneration of enzymatically active 1,4-NADH from NAD+ (E = −4.20 eV). This photocatalytic pathway is schematically shown in Fig. To understand the electron transfer pathway, cyclic voltammetry (CV) measurements were carried out. The reduction potential at cathodic peak current of GFPC 1 was observed at around −1.2 V Fig. . On mixi7 V Fig. . This isADH Fig. . From th−2 in response to the on/off simulated sunlight (1 sun) illumination  under identical conditions . The Fermi level of graphene was calculated to be 4.20 eV lower than vacuum level, which lies around the center of bandgap of the chromophore, about 0.6 eV lower than the lowest unoccupied molecular orbital (LUMO) level of chromophore. Although this “divide-and-conquer” method is simple and powerful to deal with a large system without too much computational burden, the two components (DdIC chromophore and graphene monolayer) of the photocatalyst are tightly bonded via strong covalent bond. Thus the electron can be transferred spontaneously to make the Fermi levels equal. So, electronic structure calculations of the whole system were performed, secondly. The optimized atomic geometry and electronic band structure are shown in Fig. To elucidate the origin of the photocatalytic activity and activity enhancement of GFPC 1 theoretically, a series of first principles calculations using plane-wave based density functional theory (DFT) code, VASP were carried out2. We believe that this work would serve as a benchmark example of a new pool of graphene based photocatalysts that can efficiently harvest and transfer solar light and thereby trigger renewed interest in the area of solar fuel/chemical production from CO2. Our strategy may also provide broad contours for application of flexible graphene films in other fields such as solar energy powered wearable electronic devices.In conclusion, the newly designed and fabricated flexible graphene film photocatalyst shows high photocatalytic activity attributed to a regular arrangement of light harvesting units with spatial orientation towards visible light as well as improved delocalization of π electrons and direct electron transfer from covalently attached chromophore to graphene monolayer. The graphene film based artificial photosynthesis system thus obtained shows remarkably higher formic acid production from COThe GFPC 1 photocatalyst for this research work was obtained by coupling CVD grown graphene film with 1,3-Dioxo-1H-dibenzoisoquinoline-2[3 H]-carbaldehyde (DdIC chromophore) via 1,3-dipolar cycloaddition. This was carried out by the following procedure.3. The film was then dried in the oven at 125 °C for 24 hours to obtain GFPC 1 photocatalyst with the loading of one DdIC group per 70 carbon atoms of the graphene film.Graphene film was suspended in 100 ml round-bottom flash via wire in 10 ml of ortho-dichlorobenzene (ODCB) along with sarcosine (2.5 mg), and DdIC (2.0 mg). The solution was stirred at 180 °C under argon atmosphere for 7 days. The GFPC 1 photocatalyst was then washed with ODCB, followed by water and CHCl16. The graphene thus obtained was coupled to DdIC chromophore by following the procedure as outlined for GFPC 1 except for the graphene powder used in this case. This sample is graphene-DdIC powder photocatalyst with the loading of one DdIC group per 68 carbon atoms of graphene.Graphene for this sample was prepared by following a literature method2 polyimide sheet and then dried in an oven at 80 °C for 3 days to obtain GFPC 2.A DMF suspension of the graphene-DdIC powder photocatalyst was spin coated  on 1 × 1 cm+ (1.24 μmol), rhodium complex Rh (0.62 μmol), Ascorbic acid (0.1 mmol) and photocatalyst  in 3.1 mL of sodium phosphate buffer . The regeneration of NADH was monitored by UV-vis spectrophotometer .The photochemical regeneration of NADH was performed within a quartz reactor under an inert atmosphere at room temperature, using a 450 W Xenon lamp (Newport 66921) with a 420 nm cut-off-filter as light source. The photocatalytic regeneration of NADH was carried out as follows. The reaction was performed in a quartz reactor. The reaction consisted of β–NAD2 was also performed within a quartz reactor at room temperature, using a 450 W Xenon lamp with a 420 nm cut-off-filter as light source. The reaction consisted of photocatalyst , β–NAD+ (1.24 µmol), rhodium complex Rh (0.62 μmol) and formate dehydrogenase enzyme (3 units) in 3.1 mL of sodium phosphate buffer  with Ascorbic acid (0.1 mmol) in the presence of CO2 (flow rate: 0.5 mL/min). The amount of formic acid was estimated by GC .The artificial photosynthesis of formic acid from CO2 film of GF, GFPC 1 and GFPC 2 contained 0.5 mg (approx.) of the photocatalyst.Note: The 1 × 1 cmk-points sampling was done with 3 × 3 × 1 grid including Г-point for the integration of the irreducible Brillouin zone. For the molecular system, only Г-point is included. The Kohn–Sham wave functions of the valence electrons were expanded using a plane wave basis set within a specified energy cutoff that was chosen as 400 eV. The positions of the nuclei in the initial structures were first relaxed by the conjugate-gradient algorithm, until the Hellman-Feynman forces on each nucleus were lower than 0.01 eV/Å. After the relaxation, spin-polarized total energy of the electronic structure was calculated using a self-consistent field method that terminated when a change in total energy between two subsequent steps was less than 10−6 eV.All density functional theory (DFT) calculations were performed using the plane-wave approach as implemented in the VASP code within the generalized gradient approximation (GGA) using the Perdew-Burke-Ernzerhof (PBE) exchange-correlation function. Frozen-core projector augmented wave pseudopotentials were used. For the simulation of periodic system (8 × 8 unit cell of graphene), the Monkhorst-Pack scheme Supplementary Information


As a typical 2D carbon material, graphene, that possesses outstanding physical/chemical properties, has revealed great potential for developing soft actuators. Especially, the unique properties of graphene, including the excellent light absorption property, softness, and thermal conductivity, play very important roles in the development of light-responsive graphene actuators. At present, various light-driven actuators have been successfully developed based on graphene and its derivatives. In this mini review, we reviewed the recent advances in this field. The unique properties of graphene or graphene-related materials that are of benefit to the development of light-driven actuators have been summarized. Typical smart actuators based on different photothermal/photochemical effects, including photothermal expansion, photothermal desorption, photoisomerization, and photo-triggered shape memory effect, have been introduced. Besides, current challenges, and future perspective have been discussed. The rapid progress of light-responsive actuators based on graphene has greatly stimulated the development of graphene-based soft robotics. Graphene is a single-atom-thick 2D material with carbon atoms arranged in honeycomb crystal lattice. The unique structure enables graphene to have high electron mobility, high thermal conductivity, large specific area, high transparency, and good flexibility    layer with graphene and gold nanorods composites  , of which mesogenic units can arrange in two separated phases, are inert to light. Macroscopic large-scale deformation can be achieved by the combination of these two materials. However, graphene has only one phase, so that the graphene layer remains unchanged during the photoisomerization process. On the other hand, high photothermal conversion efficiency of graphene also accelerates the photoisomerization progress. Cheng et al. produced a NIR-vis-UV light sensitive actuator by incorporation GO with LC and azobenzene dye  can memorize a specific shape during fabrication. No matter if it is pressed, stretched or folded into any temporary structure, it will recover to the definite shape under certain stimuli, namely shape memory effect (SME). Photo-triggered SMP are also introduced to the actuation systems for their large-scale shape transformation and good mechanical property. Thermal-induced shape memory effect is the most common way to trigger the transformation of the polymer matrix. When combining such SMP with graphene, the actuation performance can be obtained through photothermal effect. Liang et al. proposed an actuator using graphene and thermoplastic polyurethane (TPU) materials, showing excellent light-triggered SME (Liang et al., In conclusion, featuring unique mechanical, physical and chemical properties, graphene is favorable for designing light-responsive actuators. The underlying actuation mechanisms are summarized in this paper, including photothermal expansion, photothermal desorption, photoisomerization, and photo-induced SME. Actuators with fast response, large deflection, and complex shape change can be realized. However, most current studies are focusing on the material composition, fabrication process and fundamental actuation performance. More efforts should be made toward their practical usage in electronics, robotics and medical science, but it is still challenging at present. In addition, actuators can be coupled with various sensing, detecting, monitoring components for developing multifunctional and mature intelligent systems.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Time-resolved photoluminescence demonstrated fast charge transfer and high carrier collection efficiency superior to two-dimensional ultrathin graphite only. Their facile and tunable growth and excellent electrical properties show that the 3D CNTs/G are strongly attractive for various applications such as solar cells, sensors, supercapacitors, photovoltaics, power generation, and optoelectronics.A new way to simultaneously grow carbon nanotubes (CNTs) and ultrathin graphite on copper (Cu) foils has been investigated. This one-step growth process yields three-dimensional networks of CNTs on graphitic layers (3D CNTs/G) on Cu foils. Their synthesis conditions and growth mechanism are discussed in detail taking their structural properties into account. Individual CNTs and the 3D CNTs/G networks by means of an  In particular, 3D carbon nanostructures including stacking of CNTs or graphene oxide, composites of CNTs and graphene enable volumetric modifications combined with conductivity and flexibility8. Especially, hybridization of 3D carbon nanostructures and other active materials has demonstrated its great advantage in solar cell, capacitor, and energy storage applications11. CNTs in perovskite absorbers, for instance, presented excellent photo-generated charge transport9. CNTs and graphene mixtures with electrolytes increased volumetric energy density and resulted in improved supercapacitance performance10. Lithium-sulfur with CNTs and graphene mixtures also enabled high-rate, thermally and chemically stable flexible batteries11. In these hybrid applications, efficient charge transfer, high volumetric area, and interspaces to incorporate active materials and to allow ion exchanges are key factors to maximize charge collection efficiency.Carbon based nanostructures such as carbon based quantum dots, carbon nanotubes (CNTs), graphene oxide, graphite and graphene have a wide structural variety in the range from zero-dimension to three-dimension (3D)12. Though it is a simple and effective technique, their distribution, fraction, and thickness are difficult to control. Recently, alternative growth methods of 3D CNTs on graphene were reported. Aligned CNTs were grown on the underlying graphene layer, and their volumetric area was controlled by the height of CNTs14. Seamless structure without defect-traps between CNTs and graphene provided high carrier transportation15. In addition, high mobility and fast decay behaviour of carriers in 3D carbon structures enabled performance improvements in solar cells, sensors, catalysts, capacitors, and energy storages19.For 3D CNTs and graphene hybrid mixtures, common fabrication methods have been mixing, stacking and coating of CNTs or graphene together with active materials18. It requires numerous process steps involving sequential growth of graphene and CNTs, surface treatments for uniformly distributed catalysts and/or lithographic process on graphene or CNTs. These multiple and complicated procedures rather hamper fast and reliable production of the 3D CNT on graphene and, consequently, limit the implementation of the latter in devices.However, the growth of the aligned 3D CNTs on graphene is still challenging due to the difficulty in tuning the precise growth conditions for both CNTs and graphene, and in controlling the structural properties including the length and the density of CNTs20. The growth mechanism and the controllability of the CNT length and density were investigated. Structural characterizations of the 3D CNTs on ultrathin graphite (3D CNTs/G) networks were carried out by Raman spectroscopy and transmission electron microscopy (TEM). Electrical properties between CNTs and ultrathin graphite were determined by using a conductive atomic force microscope (AFM) integrated in a scanning electron microscope (SEM). Charge transfer ability of the 3D CNTs/G was confirmed by time resolved photoluminescence (TRPL) and compared with ultrathin graphite only. Our results demonstrate that the 3D CNTs/G networks grown by the one-step growth represent an excellent carrier transporting material due to the increased volumetric area and efficient carrier collection through the CNTs. Together with the advantage of the simple growth process, the 3D CNTs/G are highly promising for future carbon-based catalyst, photovoltaic, sensor, electronic and energy storage devices.In this work, we report the one-step growth of the 3D CNT on graphitic layers on Cu foils. Without additional lithography and/or catalyst deposition, vertical CNTs and multilayer graphene were simultaneously grown on Cu foils. These graphitic layers are considered as ultrathin graphite due to multiple stacked (about 20) layers of graphene21. For efficient seeding of CNTs, nanoparticles of 3d transition metals such as iron, nickel, Cu, and their alloys with high carbon adsorption rate are used23. Consequently, so far the 3D CNTs on graphene networks have mostly been obtained in two separate CVD processes for graphene and CNTs, respectively.Compared to the growth of graphene, CNTs require different growth parameters including a lower reactor temperature and metal nanoparticles as catalysts. Especially, the latter plays the most important role for the CNT growth because the nanoparticles enable the catalytic reaction, the confined seeding and finally the formation of CNTs24. As confirmed by X-ray diffraction , the distance between the Cu surface and the AFM tip was approximately 200 nm. When the AFM tip moved close to the CNT, the CNT was attracted to the tip due to the electrostatic interaction and made direct contact, as highlighted in Fig. 19. As illustrated in Fig. 3NH3PbI3. For comparison, also an ultrathin graphite sample with the same active layer was studied. A conventional perovskite optoelectronic device consists of an active layer of CH3NH3PbI3, electron and hole transport layers35. The energy band diagram was drawn on the basis of the work function: carbon nanostructures (−4.4~−4.2 eV), TiO2 (−4.0 eV), and perovskite (−3.93 eV)37. Due to a gradual lowering of the energy level of the carbon nanostructures and TiO2 compared to the active layer, the photo-generated carriers in the active layer easily relax through the vertical CNTs and can be transferred to the FTO electrode19. The lifetime of photo-generated carriers was analyzed from TRPL measurements as shown in Fig. 38. The Raman measurements confirmed that the 3D CNTs/G has the high defect density of 4.4% which is significantly higher than that in ultrathin graphite. Moreover, the XPS results indicated that the presence of C–O groups is 10.6% higher in the 3D CNTs/G. This increased defect density in the 3D CNTs/G can increase a nonradiative recombination rate of the photo-generated carrier and lead to a shorter lifetime38. In addition, the conductive CNTs of the 3D CNTs/G enabled the carrier collection vertically and also provide high surface area. The vertical collection behavior can reduce carrier trapping and increase the carrier lifetime in the active layer35. Both high nonradiative recombination and carrier collection of the 3D CNTs/G are the major reasons of the fast carrier movement, and consequently improved their performance compared to the only- ultrathin graphite based structure as shown in Fig. S519.Obviously, the conductive 3D CNTs/G has the advantage of higher surface area and efficient carrier collection in optoelectronic devicesFor a deeper understanding between the active layer and the 3D CNTs/G structures, systematic investigations are required in order to analyze the device performance including incident photon-to-current behavior, impedance property, and a quantum yield characteristic. Nevertheless, our results clearly show that the conductive 3D CNTs/G networks directly promote the carrier collection by overcoming the drawbacks such as transmittance, surface area, and carrier diffusion issues. We believe that the 3D CNTs/G networks obtained in our single-step procedure are highly promising for further advancement of the 3D carbon based devices in optoelectronics, photovoltaics, sensors, and batteries.2 with a flow rate of 500 and 100 sccm, respectively. After annealing for 5 min, both acetylene and carbon dioxide were introduced into the reactor simultaneously with a flow rate of 15 sccm while keeping the same Ar and H2 flows. After the growth of the 3D CNTs/G, the sample was cooled with a cooling rate of 7 °C/min under the continuous Ar flow of 500 sccm.The 3D CNTs/G was produced on Cu foils by chemical vapor deposition. The Cu foils purchased from Alfa Aesar (0.025 mm thick) were at first cleaned in 0.1 M ammonium persulfate for 30 sec and washed in deionized water. Cleaned Cu foils were annealed on a hot-plate at 190 °C for 10 min to oxidize the Cu surface. The oxidized Cu foil was loaded into the horizontal reactor (Carbolite-Gero), with a quartz tube diameter of 4 cm and a uniform heating region of 30 cm, and annealed at 750 °C under atmospheric pressure in a flow of Ar and H2/Si substrates. Raman spectra were obtained with a green laser of 532 nm wavelength and 0.2 mW power . The crystal structure of Cu and oxide layers were identified by using a Philip X’Pert X-ray diffractometer operating at 40 mA and 40 kV, using a monochromatic Cu Kα1 radiation. X-ray photoelectron spectroscopy (XPS) was performed in a SPECS XPS system with a monochromatic Al source (E = 1486.7 eV) and energy resolution of 2 meV. The in-situ measurement system including the conductive AFM (Semilab) inside a SEM (Carl Zeiss Merlin) was used to measure electrical properties of CNTs and graphene. All samples were mounted on the conductive stub holder with a silver paste, and the conductive AFM tip  was directly contacted to the sample using the DC mode. The current-voltage (I–V) curves were characterized using a Keithley 4200 parametric analyzer.Structural morphologies of the 3D CNTs/G were characterized by SEM (Philips XL30 FEG) operating at 10 kV. For the preparation of the cross-sectional TEM samples, a protective chromium (Cr) layer and epoxy were deposited on the 3D CNTs/G, and focused ion beam  operating at 5~30 kV with Ga ions was used to prepare thin sliced TEM samples. TEM was performed to visualize the inner structure of ultrathin graphite and CNTs by means of a Tecnai G2 F20 (FEI) and an ARM200F (JEOL), both operating at 200 kV. For Raman spectroscopy, Cu foils under the 3D CNTs/G were etched in 0.3 M ammonium persulfate. The remaining 3D CNTs/G was washed in deionized water and then transferred onto SiO2 (purchased from Solaronix) was cleaned by ethanol and deionized water. The synthesized 3D CNTs/G as well as the only-ultrathin graphite samples were separately transferred on the FTO glass after removing the Cu foil by treating with 0.3 M ammonium persulfate. For improving the wettability of organic perovskite active layer, a 50 nm thick mesoporous TiO2 layer was deposited on the transferred ultrathin graphite and the 3D CNTs/G on FTO glass substrates by spin coating and baked at 150 °C for 30 min. CH3NH3I and PbI2 (Aldrich) were dissolved in a mixture solution of dimethylformamide and dimethyl sulfoxide with a volume ratio of 3:1. This organic active layer with a thickness of 700 nm was spin-coated and then annealed at 100 °C for 10 min. TRPL was performed under pulse excitation of ultrathin graphite and the 3D CNTs/G based devices . All decay lifetimes were calculated by using the supplied software of Fluorescence Division.FTO glass with a sheet resistance of 8 ohm/cmSupplementary info


Currently employed cleaning methods cannot completely remove surface impurities since impurity diffusion from the bulk to the surface occurs during growth. We here introduce a new method to remove impurities not only on the surface but also from the bulk. By employing a solid cap during annealing that acts as a sink for impurities and leads to an enhancement of copper purity throughout the catalyst thickness. The high efficiency of the solid-diffusion-based transport pathway results in a drastic decrease in the surface particle concentration in a relatively short time, as evident in AFM and SIMS characterization of copper foils. Graphene grown on those substrates displays enhanced grain sizes and room-temperature, large-area carrier mobilities in excess of 5000 cm Chemical vapor deposition (CVD) on copper foil stands as the most promising method for large-scale production of high quality, single layer graphene. Much effort has been invested on bringing CVD graphene quality to the level of pristine mechanically exfoliated sample3. The largest hurdle to this goal is the polycrystallinity of CVD graphene, for the grain boundaries are the main factor of electron scattering in graphene lattice, which affects carrier mobility4.Graphene is a two-dimensional carbon material that could revolutionize high-speed electronics and sensors7. It has been suggested that their curvature results in local supersaturation of carbon in the growth substrate and subsequent precipitation into a graphene seed9, and some impurities have lower nucleation barrier than Cu and serve as heteronuclei in early growth10.To limit the parallel growth of grains, the nucleation process has to be controlled. Several reports emphasize the role of surface impurities on the copper substrate in the nucleation of graphene15 with varying degrees of success. While such strategies can remove surface contamination, they cannot remove impurities in the bulk substrate that will precipitate to the surface throughout the growth process. Annealing can improve copper morphology due to the evaporation of impurities. However, the presence of impurities even after 3 hours of annealing6 suggests that the evaporation process is too slow to deplete the impurities close to the surface before new ones can diffuse from the bulk. Moreover, impurities can form refractory oxides or alloy with Cu, which are immune to evaporative removal.Therefore, substantial work has been done to clean and smoothen copper substrate, ranging from annealing, wet etching to mechanical and electrochemical polishingWe here introduce a solid-diffusion-based purification process of the copper substrate to overcome these issues. Copper is brought in intimate contact with a solid cap, which allows inter-diffusion at elevated temperatures. Due to the high atomic mass of Cu, it diffuses slower than most of its impurities which results in a purification of the growth substrate.We show that the purification process results in a significant change in the impurity distribution within the copper foil. The diffusion process was found to control the particle density on the copper foil surface and ultralow particle densities could be obtained. Our model of solid diffusion was validated by a comparison of different cap materials. Graphene grown on these copper foils exhibited enhanced quality as confirmed by spectroscopic and carrier-transport measurements.16. Furthermore, under elevated temperatures, surface particles of low melting points such as Al will evaporate. Consequently, an increased annealing time helps reduce particle size and numbers. Unfortunately, even after 12 hour annealing surface particles have not completely disappeared. This issue is due to the limited efficiency of the described removal processes. For example, as the concentration of aluminum decreases, the resulting new Cu-Al alloy will exhibit a higher melting point and thus a lower vapour pressure, slowing down the evaporation.We first examine the effect of annealing on the copper foil surface without conducting graphene growth. Figure 17 and occurs for every impurity species that exhibits a non-zero mobility in the cap material. The high atomic weight of Cu will furthermore limit the solid diffusion of the catalyst and result in selective absorption of impurities in the cap.To overcome this issue, we designed a cleaning process that provides an alternative pathway for impurity particles. By placing a material on top of the copper foil, solid diffusion from the copper surface into the cap could occur. This pathway is similar to the gettering method commonly used to remove metal impurities in solar cell-grade silicon2), which would take normal, uncapped annealing tens of hours to achieve. Fittings of particle densities, obtained from several AFM images for each sample, at different annealing times revealed an exponential decrease whose decay time was 80 times shorter than for uncapped annealing conditions  cut induced less Al on Cu surface Fig.  and cons20 or graphite13. We have previously shown that the gap between substrate and cap alters the fluid dynamics of graphene growth21, whereas Chen et al.22 attributed the confinement to the enhanced redeposition of evaporated Cu, which leads to reduced roughness. While further study needs to be carried out to isolate individual effects, our opinion is that in the case of this study, all the evidences point to the major influence of decreased impurity. In addition to contributing to the surface roughness, impurities with high carbon solubility like Ni23 or Al24 will be highly likely become graphene nucleation sites.It should be noted that beside solid diffusion, capping might cause other effects like strain-induced flattening of copper foil, promoting effect or confinement effect. The promoting effect is precluded from this study by using cap made of inert material (quartz) as opposed to Ni25. A decrease of average ID/IG ratio in Raman spectra mapping also confirms the presence of less defects in the capped annealing setup  were introduced under atmospheric pressure either with or without various caps. The caps used in the study include c-plane sapphire (Al2O3) , frosted and polished quartz (XIDE TECHNOLOGY).Copper foil  was electropolished in 85% H2+ beam of 1 keV to sputter the foil and a Ga+ ion beam of 2.5 keV to analyze the secondary ion distribution at the depth up to 1200 nm.Atom force microscopy (Force AFM Genie E7 model) was used to examine copper foil morphology. TOF-SIMS measurement (by a CAMECA IMF 4 f) was carried out using an O2 and 10sccm CH4 at a pressure of 10 Torr for 20 minutes at 1020 °C, followed by a cooling down to room temperature similar to our previous study28. Raman measurements were performed in a home-built micro-Raman system using 532 nm laser excitation. FET was fabricated by prepared graphene whence carrier mobility was extracted, with channel length of 250 µm.Graphene was grown on the thus treated copper foils with a gas flow of 200 sccm HSupplementary Infomation


The measured resistivity ρxx shows insulating behavior in the sense that ρxx decreases with increasing temperature T over a wide range of T (1.5 K ≤ T ≤ 300 K). The crossover from negative magnetoresistivity (MR) to positive magnetoresistivity at T = 40 K in the low-field regime is ascribed to a transition from low-T quantum transport to high-T classical transport. For T ≥ 120 K, the measured positive MR ratio [ρxx(B) − ρxx(B = 0)]/ρxx(B = 0) at B = 2 T decreases with increasing T, but the positive MR persists up to room temperature. Our experimental results suggest that the large MR ratio (~100% at B = 9 T) is an intrinsic property of ultralow-charge-density graphene, regardless of the carrier type. This effect may find applications in magnetic sensors and magnetoresistance devices.Silicon carbide (SiC) has already found useful applications in high-power electronic devices and light-emitting diodes (LEDs). Interestingly, SiC is a suitable substrate for growing monolayer epitaxial graphene and GaN-based devices. Therefore, it provides the opportunity for integration of high-power devices, LEDs, atomically thin electronics, and high-frequency devices, all of which can be prepared on the same SiC substrate. In this paper, we concentrate on detailed measurements on ultralow-density  Silicon carbide (SiC) is an extremely useful semiconductor material which has already found applications in light-emitting diodes (LEDs) , detecton-type doing to the first conducting layer of EG. To date, n-type EG grown on SiC has been extensively studied in the context of magnetic sensors /B = 0). T ≥ 120 K, the MR ratio decreases with the increasing temperature, suggesting that our ultralow-hole density graphene serves as a better magnetic sensing device at a low temperature (MR ratio around 30% at a T = 120 K). The respectable MR ratio (about 20%) at 2 T at room temperature suggests that our ultralow-hole density graphene may find applications in magnetic sensing. In the low-temperature regime (T = 40 K and T = 50 K), there is a crossover from PMR to NMR; therefore, this device is not suitable for MR applications.In the field of magnetic sensing devices, the magnetoresistivity (MR) ratio is defined as [p-type to ultralow n-type  occurs, as shown by the ρxy data in ρxy approaches a quantized value of h/(2e2) in the high-field regime. Since the data shown in T heating technique only removes gas adsorbate and thus does not vary the graphene structure).Previously, we have shown that, for example, (in our experiment), using the low-pressure gentle heating technique, we are able to remove air adsorbate on epitaxial graphene so that chemical doping can be tuned . By heatn- and p-type graphene in the low-temperature and high-magnetic field regime, as shown in Recently, we have shown that ultralow-electron-density epitaxial monolayer graphene can be used as a magnetic sensing device and its MR ratio is around 100% at 9 T at room temperature . It is tp-type monolayer graphene shows insulating behavior. The most likely transport mechanism is localization in the low-temperature regime. In the low magnetic field regime (−0.3 T ≤ B ≤ 0.3 T), there is a crossover from negative magnetoresistivity to positive magnetoresistivity at T = 40 K, which is ascribed to a transition from quantum to classical transport with increasing temperature. The positive magnetoresistivity is most pronounced at around T = 120 K and becomes weaker at higher temperatures; however, PMR persists up to 300 K, indicating that ultralow-hole-density monolayer graphene may find applications in magnetic sensing and magnetoresistance devices. Simply by gentle heating in a vacuum, we can tune our ultralow-p-type device to an ultralow-n-type one. Interestingly, at room temperature, almost an identical magnetoresistivity ratio is observed on both the hole and electron sides up to 9 T, indicating that the observed large MR is an intrinsic property of an ultralow-density monolayer graphene, regardless of whether it is n-type or p-type. Our experimental results may open the door for future graphene-based CMOS technology on SiC with hexagonal boron nitride as a top dielectric spacer [In conclusion, we have reported extensive magneto-transport measurements on ultralow-hole-density epitaxial monolayer graphene grown on SiC. Interestingly, over the whole measurement range, the resistivity of the c spacer .


Prostate cancer (PC) is the sixth most common cancer type in the world, which causes approximately 10% of total cancer fatalities. The detection of protein biomarkers in body fluids is the key topic for the diagnosis and prognosis of PC. Highly sensitive screening of PC is the most effective approach for reducing mortality. Thus, there are a growing number of literature that recognizes the importance of new technologies for early diagnosis of PC. Graphene is playing an important role in the biosensor field with remarkable physical, optical, electrochemical and magnetic properties. Many recent studies demonstrated the potential of graphene materials for sensitive detection of protein biomarkers. In this review, the graphene-based biosensors toward PC analysis are mainly discussed in two groups: Firstly, novel biosensor interfaces were constructed through the modification of graphene materials onto sensor surfaces. Secondly, ingenious signal amplification strategies were developed using graphene materials as catalysts or carriers. Graphene-based biosensors have exhibited remarkable performance with high sensitivities, wide detection ranges, and long-term stabilities. PC is one of the most common cancers in the world which causes a fatality of approximately 10% in all cancer patients . PC is aTumor markers for early clinical screening and rapid diagnosis cover a wide range of biochemical entities, including, proteins , 14, nucThis review highlights recent graphene-based biosensors for PC protein biomarkers detection. As far as we know, this is the first review that focuses on specific one disease. We reviewed recent progress of graphene-based biosensors for PC protein biomarker detection. Our manuscript clearly stated the advantages and shortcomings of most of the graphene-based when facing PC diagnosis, thus, the manuscript should be valuable for the future application of graphene-based biosensors.Protein biomarkers for cancer diagnosis are usually produced by either cancer cells or other cells in response to cancer –34, whicProstate-specific antigen (PSA) , which iProstate-specific membrane antigen (PSMA)  is a typ2-bonded carbon atoms. GO is chemically produced by oxidation and exfoliation of graphene, causing extensive oxidative modification of the basal plane [Graphene is a two-dimensional (2D) nanomaterial, which plays an important role in the biosensor field –66. The al plane , 75–77. al plane –80. GQDsal plane , 82.Development of protein biosensors based on graphene could be classified into two main groups Fig. : FirstlyGraphene and its derivatives are studied for the construction of novel biosensor interface , which i2) was used for the assembling of antibody on novel biosensor interfaces [2 combining, with the assistant of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and they finally achieved a low detection limit of 2 pg/mL [Traditionally, antibodies are physically adsorbed onto the immune-assay surfaces, such as classic 96-well plates and colloidal gold test strips. However, one of the main obstacles is the affinity and capacity, because the hydrophobic and hydrophilic interaction is relatively weak and the orientation of the antibody molecules is random . As seveterfaces , 91. In  2 pg/mL .In order to realize better-oriented assembling of antibody, Mao et al. applied chitosan as the dispersant to construct an immuno-interface on a glassy carbon electrode Fig. A, which A graphene-modified electrode was also reported in ECL biosensor  for PSA Vg,min) was achieved, indicating the minimum conductivity. Finally, they successfully performed detection of PSA-ACT of femtomolar level.Graphene materials were also applied in FET biosensors, for the construction of 2D nano-FET biosensors –101, witFor interface-based PC biosensors, the DNA capture probe plays a key role, which could recognize and capture the target molecules . The ver4, then SH-labeled DNA aptamer was combined to Au on the electrode surface. Finally, their aptasensor achieved 1.0 pg/mL limit of detection (LOD) by using both DPV and ESI methods. Different chemical reactions were applied for the assembling of DNA onto graphene materials. Branched polyethylenimine (PEI) was applied by Pan et al. to connect thiol-mediated ssDNA onto carboxylated GO for PSA detection [As the first step toward an aptasensing platform, scientists developed several different strategies to assemble the DNA aptamer onto the electrode as the key recognition element –117. In etection . Recentletection .2O2, producing a brown color solution. Thus, they produced an antibody-functionalized GO as the signal tag and developed a sandwich-type colorimetric immunoassay for the detection of PSA. In their work [2) and magnetic bead (MB) with primary anti-PSA antibody (MB-Ab1). After the separation in a magnetic field, the color signal was detected corresponding to the concentration of PSA. Their simple immunoassay can be detected by naked eyes , generating a current signal [3N4) with large surface area, good biocompatibility, and excellent conductivity were used as labels for combining secondary antibodies and amplifying signals. Then secondary antibodies were combined onto this platform and signals were amplified from H2O2 reduction [Han et al. developed a novel signal tag for PSA and free PSA (fPSA) detection, by using onion-like mesoporous graphene sheets (O-GS) as the carrier of different AuNP-based nanohybrids . As the t signal . Feng eteduction .3O4 nanoparticles together with antibody onto GO sheets to produce a multi-function nanocomposite  were used in PC biosensor development and most of them were combined with other nanomaterials like nanoparticles. We have also summarized various strategies and approaches which can be used for graphene-based biosensor development. Graphene-based materials were used not only for novel biosensor interfaces construction but also as excellent carriers for the construction of novel nanocomposites for signal amplification. In most of the cases, graphene-based biosensors have exhibited satisfactory biocompatibility towards the bioactive species and remarkable performance with high sensitivities, wide linear detection ranges, low detection limits and long-term stabilities Table . As otheAlthough tremendous progress has been made in the past a few years of graphene-based biosensors for PC detection, there still remain some challenges. Firstly, PSA has been demonstrated not a specific biomarker in prostate cancer early screening. As a result, detection of multiple biomarkers is crucial for precise diagnosis and prognosis of PC , 131. Mo