The cases were divided into two groups: those without pTDP-43-positive neuronal cytoplasmic inclusions in the hippocampal dentate granule cells , and those with such inclusions . Furthermore, the Type 2 cases were divided into two subgroups based on semi-quantitative estimation of pTDP-43-positive dystrophic neurites (DNs) in the temporal neocortex: Type 2a  and Type 2b . Clinico-pathologic analysis revealed that cognitive impairment was a feature in patients with Type 2a and Type 2b, but not in those with Type 1, and that importantly, Type 2b is a distinct subtype characterized by a poor prognosis despite the less severe loss of lower motor neurons, the unusual subcortical dendrospinal pTDP-43 pathology, and more prominent glial involvement in cortical pTDP-43 pathology than other two groups. Considering the patient survival time and severity of motor neuron loss in each group, transition from Type 1 to Type 2, or from Type 2a to Type 2b during the disease course appeared unlikely. Therefore, each of these three groups was regarded as an independent subtype.Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are types of major TDP-43 (43-kDa TAR DNA-binding protein) proteinopathy. Cortical TDP-43 pathology has been analyzed in detail in cases of FTLD-TDP, but is still unclear in cases of ALS. We attempted to clarify the cortical and subcortical TDP-43 pathology in Japanese cases of sporadic ALS (The online version of this article (doi:10.1186/s40478-016-0335-2) contains supplementary material, which is available to authorized users. Amyotrophic lateral sclerosis (ALS), an adult-onset, fatal neurodegenerative disorder, is the most common type of motor neuron disease (MND). Most cases 90-95 %) appear to occur randomly without a family history (sporadic ALS) , 2. The  % appearSince the identification of a nuclear protein, 43-kDa TAR DNA-binding protein , as the major component of the ubiquitinated inclusions in frontotemporal lobar degeneration with ubiquitin-positive, tau-and α-synuclein-negative inclusions (FTLD-U) and ALS , 12, manTypes A, B, C and D) of FTLD-TDP pathology [Type D being associated with mutations of the VCP  gene [Type B, being characterized by moderate numbers of NCIs and very few DNs throughout all cortical layers. However, one of the above studies demonstrated that 7 of 10 cases that were sporadic and exhibited MND in addition to dementia had cortical ubiquitin pathology characterized by a mixture of numerous NCIs and frequent small DNs [Type A of the new classification system mentioned above [Cases of FTLD-U were originally divided into three types by two independent groups using their own classification systems based on the morphology and anatomical distribution of cortical ubiquitin neuronal lesions, including NCIs and DNs , 22. Aftathology , the newin) gene , 25. In ed above ; this fiUsing a phosphorylation-independent anti-TDP-43 antibody, we have previously demonstrated that immunopositive NCIs and glial cytoplasmic inclusions (GCIs) can occur in many brain regions in ALS, and that cases can be classified into two types – type 1 and type 2–based on the distribution pattern of NCIs in the CNS and hierarchical cluster analysis of the pattern . Type 2 In the present study, we attempted to reevaluate the cortical and subcortical TDP-43 pathology in cases of sporadic ALS using the above monoclonal antibody, which never recognizes endogenous non-phosphorylated TDP-43 in nuclei, thus allowing unambiguous identification of pathologic structures. The results obtained eventually allowed us to classify the examined cases into three pathologic groups, whose clinical, pathologic and biochemical features were then analyzed.The present study was conducted within the framework of a project, “Neuropathologic and Molecular-Genetic Investigation of CNS Degenerative Diseases”, approved by the Institutional Review Board of Niigata University. Informed consent was obtained from the patients’ families prior to genetic analyses.We retrieved all cases of pathologically confirmed ALS from our institutional autopsy files covering the period between 1975 and 2013, reviewed the medical records and identified 128 cases of clinically sporadic ALS without any family histories of similar neurological disorders. All of the patients were of Japanese ancestry, and their clinical information was obtained retrospectively by reviewing their medical records.Among these 128 cases, the tissue samples were of poor quality due to complications of infarction, etc. and/or sampling in 26 cases, pathologic features indicative of complications arising from other major neurodegenerative diseases affecting the cerebral cortex and basal ganglia were evident in 4 cases , and no TDP-43-positive inclusions were detected in the CNS, including the lower motor neurons, in 2 cases. Accordingly, a total of 32 cases were excluded, leaving 96 cases  for analysis. Seven cases were found to have only a few Lewy bodies, with α-synuclein-positive NCIs and DNs confined to the brainstem. These cases were considered to be incidental Parkinson’s disease and were included in the present study. All of the studied cases showed loss of upper and lower motor neurons as well as ubiquitin-positive skein-like inclusions in the remaining lower motor neurons, and Bunina bodies were evident in the remaining lower motor neurons in 91 of the 96 cases.Multiple formalin-fixed, paraffin-embedded CNS tissue blocks for all cases were available for the present study. For the motor cortex, frontal cortex , temporal cortex (including the hippocampus), basal ganglia, hypoglossal nucleus, and cervical and lumbar anterior horns, 4-μm-thick sections stained with hematoxylin-eosin (H-E) were used for semi-quantitative analysis employing a 4-point scale  of neuronal cell loss , frontal and motor cortices and basal ganglia for immunohistochemical studies. The sections were autoclaved at 120 °C in 10 mM citrate buffer, pH 6.0, for 10 min, and then immunostained with a mouse monoclonal antibody against phosphorylated TDP-43  . Selected sections were also immunostained with a rabbit polyclonal phosphorylation-independent anti-TDP-43 antibody . Immunolabeling was detected using the peroxidase-polymer-based method using a Histofine Simple Stain MAX-PO kit  with diaminobenzidine (DAB) as the chromogen. To estimate the neuropathological staging of changes associated with Alzheimer’s disease, we performed Gallyas-Braak silver impregnation, and immunohistochemistry using mouse monoclonal antibodies against hyperphosphorylated tau protein  and β-amyloid . Then, we evaluated the Braak stages of neurofibrillary tangles and amyloid deposits , 27, andn = 63) and the other with such inclusions   and Type 2 accompanied by abundant DNs   Fig. . We then11) Fig. .Fig. 1PrA double-labeling immunofluorescence study was performed to assess the anatomical localization of pTDP-43 deposits forming granular and dot-like DNs. Sections of the temporal lobe and basal ganglia, including the neostriatum and globus pallidus from three representative cases of Type 2b were examined using rabbit polyclonal anti-pTDP-43 (phospho Ser409/410)  and mouse monoclonal anti-neurofilament H (non-phosphorylated) , as well as rabbit polyclonal anti-pTDP-43 (phospho Ser409/410) and mouse monoclonal anti-synaptophysin . The second antibodies used were Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 555 goat anti-mouse IgG . The sections were treated with an Autofluorescence Eliminator Reagent , mounted under glass coverslips using VectaShield mounting medium with 4,6-diamidino-2-phenylindole (DAPI) nuclear stain , and analyzed using a Carl Zeiss confocal laser scanning microscope (LSM700).n = 22), Type 2a (n = 12) and Type 2b (n = 7) among cases logged after 1990. In the Type 1 cases, a significant number of pTDP-43-positive NCIs and GCIs were seen in the motor cortex. Therefore, from the Type 2a and Type 2b cases, we selected cases in which a larger number of pTDP-43-positive NCIs and GCIs were evident in the motor cortex than in the temporal or frontal cortex.The numbers of neurons and glial cells possessing cytoplasmic pTDP-43-positive inclusions (NCIs and GCIs) were assessed in the motor cortex using a double-labeling method with in situ hybridization (ISH) and immunohistochemistry, and compared between the three groups. For this study, we selected representative cases of Type 1 (neurofilament 3 (hNF: 150-kDa medium) (GenBank accession number: BC002421) was used. As a result, the sections from 11 cases were found to be inadequate for ISH, leaving 30 cases  logged between 1990 and 2012 for the subsequent immunohistochemical study.ISH was performed in these three groups using newly prepared paraffin-embedded 10-μm-thick sections from the motor cortex, as described previously , with mi2, covering the cortical layers II-VI. The numbers of hNF-positive cells with nuclei, hNF- and pTDP-43-positive cells with nuclei, and hNF-negative and pTDP-43-positive cells with nuclei were counted manually.The hybridized sections were immunostained using mouse monoclonal anti-pTDP-43 , and then counterstained with Nuclear Fast Red solution . In each case, 10 sequential images of the motor cortex were taken through a × 20 objective lens using a single ISH-labeled and immunostained section. The total area taken from a section from each case was 1.5 mmhttp://www.r-project.org/). Differences were considered statistically significant at P <0.05.To compare clinical and pathologic features between the three groups, we used Kruskal-Wallis test with post-hoc Steel-Dwass test for non-parametric analysis of independent samples, multiple regression analysis to determine whether independent variables can predict the value of the dependent variable, Kaplan-Meier plots and a log-rank test to compare survival distributions, and Fisher’s exact test with Bonferroni-corrected multiple comparisons or Ryan’s multiple comparison test for comparison of categorical data. These statistical analyses were performed using GraphPad Prism version 5.0 , SPSS Statistics version 12.0  and R version 3.1.2 (n = 4) cases and from the temporal cortex of Type 2a (n = 4) and Type 2b (n = 4) cases, as well as from the frontal cortex of FTLD-TDP Type A, B and C (two each), as described previously [Fractionation of frozen brain tissues and TDP-43 immunoblotting were performed on selected cases. Protein lysates were generated from the motor cortex of Type 1 . We amplified all the exons of the TARDBP (NM_007375.3) gene using a series of primers, followed by sequence reaction [C9ORF72 (NM_018325.2), repeat-primed PCR was performed on an ABI 3130xl genetic analyzer  using Peak Scanner software v1.0 (Applied Biosystems), as described previously [The presence or absence of reaction . For scrBP NM_0075.3 gene eviously .P = 0.024), and that the survival time from disease onset was shorter for Type 2b than for Type 1 (P <0.0001) and Type 2a (P = 0.003). Multiple regression analysis using age at onset and ALS subtype as independent covariates and the survival time from disease onset as a dependent variable, a significant correlation was found between ALS subtype and the survival time from disease onset (P = 0.039), whereas the correlation for age at onset was not statistically significant (P = 0.106). Comparison of initial symptoms between the three groups showed that limb weakness was more frequent whereas bulbar and other symptoms were significantly less frequent in Type 1 than that in Type 2a (P <0.0001) and Type 2b (P <0.01). Cognitive impairment was present in 15 (16 %) of the 96 patients, the incidence being similar to that (10-15 %) of overt cognitive impairment meeting the criteria for frontotemporal dementia reported previously in patients with ALS [P <0.001) and Type 2b (P <0.00001). There were no evident differences in the age at death between cases with (n = 15) and those without (n = 81) cognitive impairment .The demographics and clinical features of the studied patients are summarized in Table t P = 0.0, whereasP <0.001 and P = 0.001, respectively) or in Type 2a . No neuronal loss in the basal ganglia was evident in any of the cases. None of the present cases showed hippocampal sclerosis characterized by neuronal loss and gliosis in the hippocampal formation, which is found often in FTLD-TDP but rarely in ALS [The pathologic features of the studied cases are summarized in Table y in ALS , 41. They in ALS , 93 and P <0.001) or in Type 2b . In around half of the cases in both the Type 2a and Type 2b groups, pTDP-43-positive NCIs showed a temporal cortex-predominant distribution pattern, in comparison with the motor cortex. No pTDP-43-positive neuronal intranuclear inclusions were evident in any of the cases examined.In each group, a proportion of cases showed FTLD manifested by frontotemporal atrophy and frontotemporal cortical neuronal loss with gliosis; the rate of occurrence of FTLD was only 6 % in Type 1, being much lower than in Type 2a  and Type 2a (P <0.0001), respectively, than in Type 2b. These inclusions were also detectable with the phosphorylation-independent anti-TDP-43 antibody  Table . The ratody Fig. .Fig. 2DeSMI-32 and synaptophysin are known to be dendritic and presynaptic markers, respectively. In the temporal cortex and globus pallidus, pTDP-43-positive granular and dot-like structures were observed along the SMI-32-positive dendrites Fig. . In the hNF (mRNA) and immunohistochemistry using an antibody for pTDP-43 (protein). Brain cells positive for hNF were recognized as neurons   for Type 1, 573.7 (516.7-619.7) for Type 2a, and 575.4 (533.3-650.3) for Type 2b, P = 0.316. The density of GCI-bearing glial cells was higher in Type 2b than that in Type 1 (P = 0.027) and Type 2a (P = 0.018)    Fig.  i, altho18) Fig.  ii. The 04) Fig.  iii, alt14) Fig.  i.Type B, and different from those in FTLD-TDP Type A, showing no evident differences between the three ALS groups.The data obtained by immunoblot analysis of sarkosyl-insoluble fractions are shown in Fig. TARDBP) gene in the 83 cases examined, there were no mutations in the coding regions except for one case with a known synonymous mutation in exon 6 [C9ORF72 repeat expansion was observed.With regard to the sequencing of the TDP-43  [Type A in the classification system mentioned above . Furthermore, immunoblot analysis of sarkosyl-insoluble pTDP-43 revealed that the band patterns of Type 2b and Type 1 were indistinguishable and differed from those of FTLD-TDP Type A  [Comparison of the ubiquitin- or TDP-43-related cortical pathology of FTLD described by many investigators suggested that Type 2a apparently corresponded to . (2011) , 40, 42,e A Fig. . By contis (PLS) . TherefoFrom a clinico-pathologic viewpoint, cognitive impairment was much less frequent in patients with Type 1 as we suggested previously ; in otheIn the present study, clinical information was limited to that obtained retrospectively from the medical records of the patients for whom details of some clinical symptoms were unavailable. This was one of the limitations of the present study. It has been shown that classification of patients with ALS based on clinical assessment provides information for better understanding the wide spectrum of this motor and cognitive disorder . MoreoveThe results of double-labeling immunofluorescence using anti-pTDP-43 antibody and dendritic SMI-32) or presynaptic (synaptophysin) markers in the temporal cortex and basal ganglia of patients with Type 2b suggested that deposition of pTDP-43 might occur in neuronal dendritic spines  repeat expansion in a non-coding region of C9ORF72 is the major genetic cause of FTD and ALS (c9FTD/ALS) in the Caucasian population [None of the patients in the present study had mutations in pulation , but extpulation , 50, 51.pulation , being mpulation .In conclusion, we have demonstrated heterogeneity of cortical pTDP-43 pathology in sporadic ALS, allowing the disease to be classified into three groups: Type 1, and Type 2a and Type 2b. Several significant clinical and pathologic correlations among these groups were evident. It was re-confirmed that dementia is not a feature of Type 1. Furthermore, the distinct subtype, Type 2b, accompanied by dendrospinal accumulation of pTDP-43, was shown to be characterized by a poor prognosis despite the less severe loss of lower motor neurons, the unusual subcortical dendritic pTDP-43 pathology, and more prominent glial involvement in cortical pTDP-43 pathology than other two groups. Further studies will be needed in order to establish the character of this subtype, Type 2b. Recognition of the heterogeneity of the anatomical and cellular localization of pTDP-43 will be important for clarifying the pathomechanisms involved in pTDP-43 aggregation and propagation in sporadic ALS, and eventually for developing future strategies of treatment and prevention.ALS, amyotrophic lateral sclerosis; DN, dystrophic neurite; FTLD, frontotemporal lobar degeneration; GCI, glial cytoplasmic inclusion; MND, motor neuron disease; NCI, neuronal cytoplasmic inclusion; pTDP-43, phosphorylated 43-kDa TAR DNA-binding protein


Monte Carlo simulations and tight-binding calculations shed light on the properties of three-dimensional nanographene, a material composed of interlinked, covalently-bonded nanoplatelet graphene units. By constructing realistic model networks of nanographene, we study its structure, mechanical stability, and optoelectronic properties. We find that the material is nanoporous with high specific surface area, in agreement with experimental reports. Its structure is characterized by randomly oriented and curved nanoplatelet units which retain a high degree of graphene order. The material exhibits good mechanical stability with a formation energy of only ∼0.3 eV/atom compared to two-dimensional graphene. It has high electrical conductivity and optical absorption, with values approaching those of graphene. Recent efforts in graphene research include the realization of three-dimensional (3D) bulk materials with graphene nanoplatelets (nanoribbons) as their building blocks –4. These2 geometries of the graphene plane, which may possibly alter the anticipated electronic conduction and optical properties of these materials.The reported up-to-date 3D graphene structures, produced by a variety of experimental techniques, such as chemical vapor deposition (CVD) , assemblThis inherent disorder, therefore, raises a number of questions about the stability and the microstructure of these porous networks, and how this disorder influences the optoelectronic properties of 3D nanographene. Some key issues to be tackled include the rigidity and robustness of the networks, their high specific surface area, whether and in what degree they preserve graphene order and bonding percolation, and if conduction and optical absorption are strongly influenced by the deformations of the nanoplatelets.Here, we report what is presumably the first attempt to simulate and study theoretically the structural, mechanical, and optoelectronic properties of realistic 3D nanographene (3D-NG) materials. We achieve this through a combination of atomistic Monte Carlo simulations, to generate the networks in a random unbiased way, and tight-binding calculations to relax them properly and extract their basic properties. The outstanding finding of our studies is that 3D-NG exhibits good mechanical stability and high electrical conductivity and optical absorption, approaching those of 2D graphene.We construct and characterize the 3D-NG networks in two steps, following a procedure we used earlier to construct carbon nanofoam networks . In the s and p atomic-like orbitals and distance- and environment-dependent parameters for transferability between different structures. The successful description of various carbon phases by this model was previously demonstrated [Γ point for the calculation of energies and optoelectronic quantities.In the second step of our procedure, the 3D-NG networks generated by the MC method are fully relaxed using tight-binding molecular dynamics (TBMD) simulations in the  canonical ensemble. We first anneal extensively the networks at 2000 K in order to bring the structures out of any local energy minima, and then we relax the volume/density at 300 K, where the structural, mechanical, and electronic properties are inferred. The calculations are carried out within the TB framework developed at the Naval Research Laboratory (NRL) . This isnstrated , 13. DueThe model also describes nicely the well-known , 15 inst2 atoms and the formation of new, both large and small, atomic rings. These have a profound effect on the electronic properties, as shown below. The density of the network is 0.5 g/cm 3, making the material mechanically very robust. This should be considered as an upper limit to the density of 3D-NG, in view of the rather small size of the computational box used here and the possibility of larger pore sizes that inevitably lower the density. Biener et al. [3 for 3D-NG prepared by pyrolysis and etching of sol-gel organic polymers. Ultra-low density (of the order of mg/cm 3) foamy structures might also be possible to achieve, but these should be less stable.We first begin with the microstructure of 3D-NG. Panel  of sp 3 sites, some of them located at the junctions between the units as linking geometries, while the rest (9 %) are sp 1 sites, found both at the junctions and decorating the internal pore surfaces. The curved nature of the nanoplatelets is reflected in the appearance of both large and small atomic rings, other than the dominant (70 %) six-membered (6 m) rings forming the ideal graphene layer. We find, for example, a large fraction of 9-m rings (9 %), 7-m rings (5 %), and 8-m rings (4 %). There are also small 3-m rings (5 %) and 5-m rings (8 %). Non-6-m rings and non-sp 2 sites put their signature on the electronic density of states to be discussed below.As expected, the main bonding hybridization in our model in Fig. 22/g, compared to 2636 m 2/g for ideal monolayer graphene. Our values are in accord with the experimental value of 3000 m 2/g reported by Biener et al. [We calculate the specific surface area (SSA) of our networks to be in the range of 3000–3200 m r et al. . Most li3) amorphous carbon (a-C) network (shown in the inset), which was generated with the same TBMD methodology. There are some striking differences between the G(r) of 3D-NG and a-C. One comes from the feature between the second and third peak, at ∼2.8 Å. This extra peak corresponds to the third-nearest-neighbor distance in the graphene hexagon, and is absent in a-C. Another notable difference is that peaks beyond remain intense in 3D-NG, while they vanish in a-C. These differences show that not only short-range order but also medium-range order correlations, and thus graphene order, are highly preserved in 3D-NG despite its distorted nature. It is evident that the sharp peaks in the G(r) of graphene are broadened into the peaks of 3D-NG at exactly the same radial distance. Overall, we may characterize 3D-NG as a continuous random network (CRN) at the microlevel, in the sense that the units percolate throughout but they are randomly oriented, while high order prevails at the nanolevel.Figure 2B0 of our networks by fitting their energy versus volume curves to the Murnaghan’s equation of state [B0 of 60 GPa. All networks studied have values of B0 in the range of 55–65 GPa despite their porous nature. This is lower than values for typical 3D carbon materials with low density, such as graphite-like a-C (B0∼200–250 GPa), but still high enough to make 3D-NG durable for potential applications. Again, our values should be an upper limit since for networks with larger pores and smaller density we expect lower moduli. Work is in progress to estimate the hardness of the material.As a stringest test of the mechanical stability of 3D-NG, we calculated the bulk modulus of state . Such a Further evidence for the stability of 3D-NG networks is provided by calculating their formation energies relative to the undulated graphene plane at 300 K. We find values ranging from 0.2 to 0.5 eV/atom. For example, the total energy of the network shown in Fig. 2εF arising from the disorder due to the ripples. The EDOS of 3D-NG, on the other hand, follows more or less the shape of the graphene EDOS, which is in accord with the experimental work by Biener et al. [εF, it is completely gapless. As these states around εF are π orbital states, and given the deformations induced by crosslinking, one wonders whether their π orbitals are properly aligned so as to keep their delocalized nature, as in graphene, and give a metallic character to the material.We now proceed to the study of the electronic structure of 3D-NG. Figure 2r et al. , but exhci are the coefficients in the expansion of the eigenstates in terms of the local orbitals, and which is a measure of the localization of the electronic states in the system. The localized states and their IPR are denoted by vertical spikes in Fig. εF region is practically free of localized states. This is in sharp contrast to a-C where the π and π∗ states near εF are localized, due to misalignment of the π orbitals, causing the low conductivity of the material [1-bonded atoms at the internal surfaces, while sp 2 and sp 3 sites contribute minimally.We investigate this issue by calculating the inverse participation ratio (IPR) defined by material . The IPRπ orbitals in the presence of deformations can be explained by noting that the large-membered rings in the network induce locally a planar geometry, causing the proper alignment of π orbitals. This is significant because many of the atoms contributing significantly at εF are sp 2-bonded atoms located at large 8- and 9-m rings. Otherwise, such atoms would contribute localized states at εF, as defect atoms do. We observed a similar phenomenon of large-ring-induced local planarity in our previous studies [The proper alignment of π states in 3D-NG are delocalized at εF is expected to have a strong effect on the conductivity. In its standard frequency-dependent form, this is given by We now proceed to the calculation of the optoelectronic properties of the material. We first examine the electrical conductivity. Our finding that the Ei and Ef are the energies of the initial occupied valence eigenstates |i〉 and the final unoccupied conduction eigenstates |f〉, respectively. The rigorous relation i〉 and |f〉 eigenstates into atomic states  |a〉 and |b〉 where )〈f|r|i〉  has beenj and all atomic orbitals in the basis set. The basis sets of the atomic orbitals |a〉 and |b〉 are in principle different. Considering the LCAO expansions in Eq.  [ω (or E) → 0.where the index |r|l〉=0) . Using tσDC of the graphene layer is ∼ 2×10−4μΩ−1cm−1. This agrees with several experimental measurements in graphene materials [−3μΩ−1cm−1. This agreement provides us with a benchmark and shows the reliability of our approach for the calculation of the transition matrix elements. For 3D-NG, our calculations predict that σDC is of the order of ∼ 5×10−5μΩ−1cm−1, which underlines that 3D nanographene retains in a large extent the conductivity of 2D graphene. For comparison, the experimental work of Worsley et al. [−6μΩ−1cm−1, while Chen et al. [−5μΩ−1cm−1, which is closer to ours. Obviously, the conductivity of covalently bonded 3D-NG will depend on the connectivity and percolation of units, the pore size, and the density.The results of our calculations of the conductivity as a function of the energy are given in Fig. 6aterials –21. For y et al.  on 3D-NGσDC of graphite-like a-C, which is highly sp 2-rich, and more than eight orders of magnitude higher than sp 3-rich tetrahedral a-C [−8μΩ−1cm−1 for the former and practically zero for the latter. As discussed above, this is due to the strong localization of π states in the a-C phase due to the misalignment of π orbitals.Note that, as shown in Fig. 6dral a-C . Our comε2(ω) is the imaginary part of the dielectric function and n is the refractive index of the material [The 3D-NG materials are also highly absorbtive, as demonstrated in Fig. 6material , revealsIn conclusion, we reported here the first simulational and theoretical study of the structural, mechanical, and optoelectronic properties of realistic 3D nanographene materials, through Monte Carlo simulations and tight-binding calculations. We found that 3D-NG is characterized by nanoporosity and high specific surface area. It is a random network at the macrolevel but with high graphene order at the nanolevel, within the nanoplatelet units. It exhibits good mechanical stability and high electrical conductivity and optical absorption, approaching those of 2D graphene. These characteristics and the calculated relevant quantities are in good agreement with experimental measurements. Actually, some of them, such as the electrical conductivity, are even higher than what is reported experimentally. This may indicate that there is room for improvement of these materials at the lab through better unit connectivity and percolation, and pore size and density control.


Graphene is composed of a single atomic layer of carbon which has excellent mechanical, electrical and optical properties. It has the potential to be widely used in the fields of physics, chemistry, information, energy and device manufacturing. In this paper, we briefly review the concept, structure, properties, preparation methods of graphene and its application in lithium ion batteries. A continuous 3D conductive network formed by graphene can effectively improve the electron and ion transportation of the electrode materials, so the addition of graphene can greatly enhance lithium ion battery’s properties and provide better chemical stability, higher electrical conductivity and higher capacity. In this review, some recent advances in the graphene-containing materials used in lithium ion batteries are summarized and future prospects are highlighted. However, these materials have some drawbacks that limit their use. For example, carbon materials have good cycle performance but low initial charge and discharge efficiency; tin-based oxides have good cycle but high irreversible capacity loss in the first cycle ), outstanding thermal conductivity (3000 W/(m · K)) 3. These materials have electronic conductivities of 10-4 S/cm  are LiCoOExisting studies show that pure graphene can’t become a direct substitute for current carbon-based commercial electrode materials in lithium ion batteries due to its low coulombic efficiency, high charge–discharge platform and poor cycle stability  and poor lithium ion diffusion (10- 14 -10- 16 cm2/S) lead to capacity fade quickly under high rate charge and discharge  nanoparticles, mixed them with graphite oxide in solution. The mixed solution was then underwent a spray-dried and sintered process to obtain the LiFePO4/graphene composite (LFP/G) shown in Figure 4, with a thickness of approximately 2 nm (about 3–5 layers of graphene) and formed a continuous lamellar structure; LiFePO4 nanoparticles had uniform size at 2–5 nm. The obtained material displays a more regular morphology and structure, which can potentially lead to a large enhancement of conductivity. In addition, this material can be further carbon coated to obtain carbon coated LiFePO4/graphene (LFP/(G + C)), which presents superior rate and cycle performances.When used as an electrode material, LiFePO4, Li3V2(PO4)3 is an attractive cathode material for LIBs, because its average extraction/reinsertion voltage is about 4.0 V, and its theoretical capacity is 197 mAh/g 3 forms a monoclinic structure and has a high operating voltage and shows a good performance at high discharge currents. However, its intrinsic low electronic conductivity (240 nS/cm at 25°C) limits its rate capacity, so graphene is added to improve its electrochemical performance. Li3V2(PO4)3/graphene cathode material has been prepared by sol–gel, solid state and spray-drying synthesis methods , high surface-to-volume ratio, ultra-thin thickness which can shorten the diffusion distance of ions, structural flexibility that paves the way for constructing flexible electrodes, thermal and chemical stability which guarantee its durability in harsh environments.Graphene has opened new possibilities in the field of lithium ion battery materials due to its light weight, high electrical conductivity, superior mechanical flexibility, and chemical stability  can cause large volume changes. This leads to the pulverization of the particles and the electrical disconnection of the electrode. In order to circumvent this, new anode materials with graphene have been examined in many recent studies. For example, the performance of anode electrode was improved when tin nanoparticles embedded in grapheme was used , the actual capacity with Co doped can only reach a maximum of 400 mAh/g. When the composite with graphene formed through a two-step liquid method, followed by hydrothermal synthesis, the delithiated ratio capacity of this compound is about 900 mAh/g at low current density (40 mA/g), closing to the theoretical capacity. When current density reaches 1600 mA/g, the specific capacity of this compound maintains 390 mAh/g .MnCuO has low band gap energy and high catalytic activity. However, as an anode material, it has a low conductive performance and large volume expansion effect. These shortcomings can be improved by forming CuO/graphene composites  and low price (Choucair et al.2O3/graphene composites by a hydrothermal method. The addition of graphene prevented Fe2O3 from aggregation and also buffered material’s volume expansion. At l160 mA/g current density, the reversible capacity of the composite remains 660 mAh/ g after 100 cycles.Fe2 with graphene is another nanocomposite that can be synthesized by a facile gas/liquid interface reaction (Cai et al.-1 at a current density of 100 mAg-1. This specific charge capacity drops to 150 mAhg-1at a high current density of 3000 mAg-1. The advantage of this nanocomposite is that the oxygen-containing groups on the graphene sheets can be reduced after the heat treatment and residual functional groups including the OH and COOH on the surface of graphene sheets can strongly interact with the metal ions during the synthesis process .The random hybridization of TiO2 nanoparticles and ultrathin graphene sheets form a three-dimensional porous structure of the TiO2-graphene nanocomposite. The small nanoparticles provide a short mean-free-path for electron and lithium ion to travel during the lithium ion insertion/extraction process, resulting in excellent rate capability.TiO4-graphene, Tin-based, Si-based and transition metal based electrode materials with graphene have been extensively studied in this paper. The composite materials’ advantages can be summarized as following. Firstly, graphene’s flexibility makes it an ideal material to buffer metal electrode’s volume expansion and contraction during the charge–discharge process. This improves the electrode material’s cycle life performance. Further, the excellent electrical properties of graphene can enhance the conductivity of metal electrode material. Moreover, the addition of graphene can control the growth of metal oxide particles. Smaller particles means the diffusion distance of lithium ions and electrons is reduced, this improves the material’s rate performance. Finally, the lithium storage capacity for most metal oxide composite materials with graphene has improved greatly.Compared to traditional carbon electrode materials, graphene, due to its low initial coulombic efficiency, high charge and discharge platform and other shortcomings, cannot directly replace traditional graphite as an anode material. However, graphene does demonstrate high electrical conductivity, good mechanical strength, excellent flexibility, great chemical stability and high specific surface area. This is especially noticeable when graphene is chemically converted with a greater proportion of functional groups, proving that it is suited for use as a base composite electrode material. When used as electrode material, graphene can effectively reduce the size of the active material, prevent agglomeration of nanoparticles, improve electrons and ions transmission capacity, as well as enhancing the electrode’s mechanical stability. As a result, graphene-containing electrode materials have high capacity and good rate performance. Lithium metal oxide-graphene, LiMPOIn addition, various approaches are taken to prepare graphene. These vary from simple mechanical mixing to the well controlled in situ reactions and interfacial reactions, which result in better graphene morphology and structure. Developing easily replicable methods for producing graphene is the key to future application of graphene in lithium ion batteries.


The size-controllable and ordered Au nanostructures were achieved by applying the self-assembled monolayer of polystyrene microspheres. Few-layer graphene was transferred directly on top of Au nanostructures, and the coupling between graphene and the localized surface plasmons (LSPs) of Au was investigated. We found that the LSP resonance spectra of ordered Au exhibited a redshift of ~20 nm and broadening simultaneously by the presence of graphene. Meanwhile, the surface-enhanced Raman spectroscopy (SERS) of graphene was distinctly observed; both the graphene G and 2D peaks increased induced by local electric fields of plasmonic Au nanostructures, and the enhancement factor of graphene increased with the particle size, which can be ascribed to the plasmonic coupling between the ordered Au LSPs and graphene. Graphene is the first two-dimensional carbon atomic crystal which is constructed by several layers of honeycomb-arrayed carbon atoms. This promising material is highly attractive for the fabrication of high-frequency nanoelectronic and optoelectronic devices due to its exceptional optical and electrical properties, such as extreme mechanical strength, ultrahigh electrical carrier mobility, and very high light transmittance –3. Unfor2O3 spacer layer inserted between the graphene and nanoparticles Im(α). The calculated results of Qabs for a single 30-nm Au nanosphere with and without graphene coating show that the absorption peak of the Au nanosphere exhibits a slight redshift (about 20 nm) after graphene coating, which is in good agreement with the experimental data. The discrepancy of the absorption results between calculation and experimental results can be ascribed to the simplified assumptions during the calculation such as the semi-infinite substrate, the dipole approximation, the homogeneous particle size, and the crystal perfection of the Au nanosphere [Theoretical calculation based on the dipole approximation (results not shown here) has been conducted to understand the redshift of Au LSPs after graphene coverage. In the calculation, the Au nanosphere is utilized as a representative of the Au nanostructures and is placed above a transparent substrate. The thickness of the substrate is assumed to be semi-infinite, and absorption of the substrate is completely omitted. The dielectric constant of graphene is based on an assumption that the optical response of every graphene layer is given by optical sheet conductivity , 9, and itten as 1\documenectively . The absnosphere , 10.−1), G peak (1590 cm−1), and 2D peak (2690 cm−1). The G peak originates from the first-order Raman scattering process, while the 2D peak is due to a double-resonance intervalley Raman scattering process [2/Si substrate. It is apparently revealed that the intensities of graphene G and 2D peaks are significantly enhanced with increasing size of the ordered Au nanostructures from 100 to 140 nm. With increasing size of the ordered Au nanostructures to 140 nm, the Raman peak intensities of graphene on top of Au display an enhancement factor of ~3-fold for the G peak and 2.5 for the 2D peak. Therefore, the SERS of graphene caused by the ordered Au nanostructures was distinctly observed, and the underlying mechanism will be discussed in detail later.To shed light on the effects of plasmonic ordered Au nanostructures on the Raman properties of graphene, the Stokes Raman spectra of pristine graphene and graphene transferred on differently shaped and sized Au nanostructures were measured and the corresponding results are shown in Fig.  process , 18. TheISERS is the increase in Raman intensity with respect to its original intensity I0, σ is the relative cross-sectional area of the nanoparticle, Q(ω) is the plasmonic enhancement from the Mie theory with ωs representing the Stokes Raman frequency, α is the particle radius, and h represents the separation between the particle center and the surface of the graphene sheet. From Eq. Q(ω) of the nanoparticle. The shapes of the particles vary with different initial thicknesses of the Au film, although majority of Au nanostructures can be treated as spheres or triangle prisms. Experiments of the absorption spectra have shown that the plasmonic resonance positions for the ordered Au nanostructures with various size ranges from 100 to 140 nm lie around the excitation laser wavelength  in Eq. ΔISERS will be distinctly increased which can be simply estimated from Eq. Generally, both the LSP and charge transfer can play a role in the SERS of graphene modes, since the SERS electromagnetic enhancement results from the Raman excitation and coupling with the LSP of nanostructures, while the metal-induced charge transfer can lead to the chemical resonance enhancement . In our mentclasspt{minima−1 for graphene that was transferred on the surface of the ordered Au nanostructures while the 2D peak position almost remains constant. It has been reported that the G peak of graphene is blueshifted for both electron and hole doping, while the 2D peak is redshifted for electron doping and blueshifted for hole doping [Now we turn towards the nature of the chemical interaction between the ordered Au nanostructures and graphene, which is another physical mechanism for the SERS especially for the graphene-nanoparticle hybrid system , 19. Froe doping , 21. In In summary, the coupling between graphene and LSPs of ordered and size-controllable Au nanostructures has been investigated systematically by directly transferring graphene on the surface of the Au nanostructures. The absorption spectra of Au exhibit a redshift of ~20 nm after graphene coverage, which can be ascribed to the plasmonic coupling between the Au LSPs and graphene. On the other hand, the graphene SERS is significantly observed, and intensities of the G and 2D peaks increase with increasing size of the ordered Au nanostructures. The electromagnetic plasmonic effect rather than the charge transfer mechanism is considered to be the dominant mechanism for the SERS effect of graphene. We believe the results are beneficial not only for further understanding the coupling mechanism between graphene and the ordered metallic nanostructures, but also for developing plasmonic graphene-based optoelectronic devices.


We report theoretical study of the effects of energy relaxation on the tunneling current through the oxide layer of a two-dimensional graphene field-effect transistor. In the channel, when three-dimensional electron thermal motion is considered in the Schrödinger equation, the gate leakage current at a given oxide field largely increases with the channel electric field, electron mobility, and energy relaxation time of electrons. Such an increase can be especially significant when the channel electric field is larger than 1 kV/cm. Numerical calculations show that the relative increment of the tunneling current through the gate oxide will decrease with increasing the thickness of oxide layer when the oxide is a few nanometers thick. This highlights that energy relaxation effect needs to be considered in modeling graphene transistors. Two-dimensional graphene has a zero band gap and linear energy-momentum dispersion. Graphene’s linear energy − momentum dispersion causes its charge carriers to behave as massless Dirac fermions that can travel at a speed of 10observed . The val devices .m* = 0.012 m0 (m0 is the free electron mass) that is magnetic field independent in monolayer graphene has been obtained from the temperature dependent amplitude of SdH oscillation. It has reported that in a graphene device, the energy relaxation time is about 1 ps for carrier-dense samples [Hot electron phenomena have become important for understanding all modern semiconductor devices. In hot electron transport, elastic collisions dominant its momentum relaxation, whereas inelastic interaction with phonons determines its energy relaxation. Electro-thermal analysis is used for predicting the heat generation of semiconductor devices. The governing equations of electro-thermal analysis consist of the continuity equation, momentum conservation equation, and energy conservation equation . The non samples .2 and SiO2 stacks also needs quantum coupling effects correction [2/SiO2 gate dielectric stacks in experiments is still higher than predictions from motion-coupled quantum model which neglects the energy relaxation of hot electrons [The parabolic band effective mass theory is used to investigate the quantum feature of electrons in the inversion layer of metal-oxide-semiconductor field-effect transistors (MOSFETs), quantum well, and superlattices. And one can notice that most studies assume that electron motions in three spatial directions are described by decoupled Schrödinger equations. It has been pointed out that this decoupled motion approximation results in a large error in evaluating the tunneling current and quantization of the inversion layer when the channel electron velocity is higher than the thermal velocity , 11. It rrection . Howeverlectrons . This im2 layer at 3.5 V, which is the maximal gate-source voltage, is found to be 3 μA/cm2 [2 at the same gate bias of 3.5 V [For graphene FETs, the tunneling current through a 50-nm SiO3 μA/cm2 . This gaof 3.5 V ). Proceeof 3.5 V , 17, whiThe three-dimensional full time independent Schrödinger equation can be written as 1\documenϕ represents the potential energy, m⊥ and mz denote the mass in and perpendicular to the plane of the graphene/gate oxide interface plane at the bottom of the conduction band of graphene, respectively, z is the tunneling direction which is perpendicular to the graphene/gate oxide interface, p⊥ and pz represent the electron momentum operators perpendicular to and parallel with the graphene/gate oxide interface, respectively, and E is the total energy of an electron. To calculate the tunneling current in a graphene FET, the Schrödinger equation for the electron motion in the xy plane keeps unchanged; thus, we need to solve the Schrödinger equation along the z direction. At the same time, one can note that m⊥ and mz (in and perpendicular the plane) change along the z direction because a graphene FET is a multi-layer structure, which has layer dependent effective electron mass along z direction. Therefore, Eq. ϕ(z) represents the potential energy along z axis. Note that x-y plane). According to the conservation of the total energy and transverse momentum of a tunneling electron, the longitudinal energy of a tunneling electron in the graphene is E is the total energy of a tunneling electron). Thus, in the active material (graphene) and the gate oxide  applied to the tunneling electron in the gate oxide mainly depends on the temperature of channel electrons, energy relaxation rate, electron mobility, and effective electron mass.where perature . Usuallyperature . Under tre TL is 5\documenCox and tox are the capacitance and the thickness of the gate oxide, respectively, V is the voltage across the gate oxide layer, ε0 is vacuum permittivity, and ε the dielectric constant of the gate oxide. With setting the bottom of the conduction band of the graphene as zero energy point, the electrons density in the graphene layer of a graphene FET can be obtained asFor graphene FETs, electron or hole density in the channel induced by the voltage across the gate oxide can be written as 7\documenf is the Fermi-Dirac distribution function, N(E) the density of states, T the transmission probability, and v(E) the electron velocity along the tunneling direction.At last, an electron current will form because of the imbalance between the Fermi level of the gate and that of substrate induced by the voltage across the gate oxide, and it can be written as 9\documenN partial sub-barriers. When a voltage across the gate oxide is applied, the potential barrier shape of the gate oxide will change from square to triangular. Thus, the shape of all sub-barriers is trapezoid as this triangular potential barrier is divided into N partial sub-barriers. For trapezoid potential barrier, Airy functions secure an analytical and exact expression of the transmission probability. This is why we use Airy function approach to solve one-dimensional Schrödinger equation to calculate the transmission coefficient of tunneling electrons. Considering the boundary conditions that the wave function and quantum current density are continuous at graphene/gate oxide interface, the transmission probability can be calculated according to the following equation [S11 represents the row 1 and column 1 element of the transfer matrix S which isAfter obtaining the effective potential barrier of the graphene transistors, we can divide the potential barrier into equation , 2410\dowhereMj is a (2 × 2) product matrix, mj* the effective electron mass for jth trapezoid barrier, Fj the electric field across the jth trapezoid barrier, V(xj) the voltage at the position of xj, and Ai (Ai’) and Bi (Bi’) are Airy functions and their corresponding derivatives, respectively.where The abovementioned method has been applied to study high-energy (hot) electron distribution in the graphene layer induced by its linear energy-momentum dispersion and the gate leakage current in graphene FETs . Firstly2 has an electron affinity of 0.9 eV and relative dielectric constant of 3.9. The barrier height used in this study is 3.5 eV. Electron mass of 0.012 m0 in 2D graphene according to [m0 in the gate oxide, was used in all calculations. Because graphene is a 2D crystal, there is no band-structure in the tunneling direction which is perpendicular to the graphene plane. This is the reason why we use free electron mass along such a direction in all calculations. The energy relaxation time of 1 ps [2 V−1 s−1 [In this work, dielectric constant of 2.4 has been used in graphene field-effect transistor . The thirding to , and 0.5How the tunneling current through the gate oxide with and without considering the energy relaxation of channel electrons changes with the gate voltage at different electron mobility is illustrated in Fig. Figure The effects of the electron mobility on the gate leakage current of a graphene field-effect transistor are given in Fig. The reduction in the barrier height caused by the channel electric field and the electron mobility was detailed shown in Fig. The electron energy relaxation time impacting on the tunneling current through the gate oxide at different channel electric fields is illustrated in Fig. The reduction in the barrier height as a function of the electron energy relaxation time at different channel electric fields is depicted in Fig. Figure Figures Figure It is well known that metal contacts  are crucial in the creation of high-performance graphene FETs . The impIn conclusion, energy relaxation effect on the tunneling current through the gate oxide of a graphene field-effect transistor has been theoretically investigated. The quantum coupling among the three electron spatial motion directions can result in a larger reduction in the barrier height. Note that the energy relaxation of electrons may largely increase electron temperature. The energy relaxation due to electron thermal motion along the graphene channel will lower the effective barrier height which enhances the gate leakage current. This study points out that the increase in the electron mobility, channel electric field, and the energy relaxation time always leads to higher leakage current in a graphene field-effect transistor. Based on quantified results, this paper also details how to secure high-performance graphene device and ICs. Devices with energy relaxation of time shorter than 0.1 ps or a working channel field of less than 1 kV/cm must be secured. It is also worth studying how electro-thermal effects affect the performance of multi-stack gated 2X nano-meter or 1X nano-meter MOSFETs and relative study will be reported later.


Long interspersed element type 1  is a mobile genetic element comprising about 17% of the human genome, encoding a newly identified ORF0 with unknown function, ORF1p with RNA-binding activity and ORF2p with endonuclease and reverse transcriptase activities required for L1 retrotransposition. L1 utilizes an endonuclease (EN) to insert L1 cDNA into target DNA, which induces DNA double-strand breaks (DSBs). The ataxia-telangiectasia mutated (ATM) is activated by DSBs and subsequently the ATM-signaling pathway plays a role in regulating L1 retrotransposition. In addition, the host DNA repair machinery such as non-homologous end-joining (NHEJ) repair pathway is also involved in L1 retrotransposition. On the other hand, L1 is an insertional mutagenic agent, which contributes to genetic change, genomic instability, and tumorigenesis. Indeed, high-throughput sequencing-based approaches identified numerous tumor-specific somatic L1 insertions in variety of cancers, such as colon cancer, breast cancer, and hepatocellular carcinoma (HCC). In fact, L1 retrotransposition seems to be a potential factor to reduce the tumor suppressive property in HCC. Furthermore, recent study demonstrated that a specific viral-human chimeric transcript, HBx-L1, contributes to hepatitis B virus (HBV)-associated HCC. In contrast, host cells have evolved several defense mechanisms protecting cells against retrotransposition including epigenetic regulation through DNA methylation and host defense factors, such as APOBEC3, MOV10, and SAMHD1, which restrict L1 mobility as a guardian of the human genome. In this review, I focus on somatic L1 insertions into the human genome in cancers and host defense mechanisms against deleterious L1 insertions. HBV integration into the human genome was found in most HBV-related HCC and it has been implicated in the development of HCC. An initial study proposed that HBV integration occurs randomly without preferred integration site   including IFNα and IFNβ have been involved in innate immunity against viruses. In this regard, a recent study reported that L1 induces IFN1 and IFN1, in turn, inhibits L1 retrotransposition, suggesting that IFN1 controls propagation of L1 as well as maintenance of genomic integrity  silence L1 during genome reprogramming in the embryonic male germ line , by the Research Program on Hepatitis from Japan Agency for Medical Research and Development, AMED, and by Takeda Science Foundation.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here we demonstrate that OH groups can efficiently induce robust magnetic moments on the basal plane of the graphene sheet. We show that the inducing efficiency can reach as high as 217 μB per 1000 OH groups. More interestingly, the magnetic moments are robust and can survive even at 900°C. Our findings highlight the importance of OH group as an effective sp3-type candidate for inducing robust magnetic moments on the basal plane of the graphene sheet.Inducing robust magnetic moments on the basal plane of the graphene sheet is very difficult, and is one of the greatest challenges in the study of physical chemistry of graphene materials. Theoretical studies predicted that introduction of a kind of  For the edge-type approach, it mainly includes ion irradiation to create point vacancies47891011121415etc. The biggest weakness of this approach is that it can only induce limited edge magnetism at the vacancy or edge sites of the graphene sheet. For example, although a single isolated vacancy can theoretically induce magnetic moment μ ~ μBMagnetic properties of graphene are gaining increasing interest recently for its potential application in spintronics, since its weak spin-orbit interaction and long spin-relaxation time provide ideal conditions to manipulate the spins1234sp3-type defects on the basal plane of the graphene sheet and, thus more defects can be introduced. Therefore, higher spin density is expected by this approach, and which is considered as hitherto the most promising approach to induce robust magnetic moments on the graphene sheet81819202122568181920242525sp3-type alternative that can effectively induce robust magnetic moments on the basal plane of the graphene sheet.Quite differently from the edge-type approach to create defects only at vacancy or edge sites, the basal-plane approach is to introduce μB6276862930632343536Recently, theoretical studies confirmed that the chemisorption of a single OH group on the basal plane of graphene can introduce magnetic moment ~1 μB per 1000 OH groups) and high stability (surviving even at 900°C). Our results demonstrate that OH group is an effective candidate for inducing robust magnetic moments on the basal plane of the graphene sheet. These results have important implications for the synthesis of high-magnetization graphene for future applications of spintronics. In particular, the robust magnetic moments we observe in OHG provide the prerequisite to induce long-range magnetic ordering. Conversely, the ferromagnetic coupling between these basal-plane magnetic moments still needs to be addressed for the realization of ferromagnetic graphene with high magnetization.In this study, we prepared hydroxylated graphene (OHG) with a very high magnetization of 2.41 emu/g by annealing of graphene oxide (GO) to remove the unstable oxygen groups and leave the stable OH groups. We demonstrate that OH groups can effectively induce robust magnetic moments on the basal plane of the graphene sheet. Our results highlight the two great superiorities of OH groups: high magnetic inducing efficiency  oxygen groups present on GO mainly in forms of epoxy, OH, carbonyl, and carboxyl groups; and (ii) most epoxy and OH groups lie on the basal plane of the graphene sheet, and carbonyl and carboxyl groups can only sit at the edge or vacancy sites3815404042μB on the basal plane of the graphene sheet627343536It is known that (i) epoxy is nonmagnetic for it creates equal defects in A and B sublatticeThe atomic force microscope (AFM) image of aGO shows that the heights of most of them are ~0.7 nm see , indicatsp3-type defects  measurements were carried out. As shown in 3434ca. 34%. Therefore, one can conclude that many new OH groups were created with the decomposition of other unstable oxygen groups during annealingsp3-type defect, is predominant among all the oxygen groups in all the OHG samples, and (ii) the dominance increases with the increase of the annealing temperature. Group A samples have a high OH coverage ~8 at.%, while in Group B, the coverage decreases to a steady value ~3 at.%, implying that a number of OH groups decomposed at high temperatures. Even so, the OH coverage of OHG-900 still maintains 2.96 at.%, confirming the high thermal stability of OH group343536ca. 3 to 10 at.% by changing the annealing temperature.According to the O contents and C–OH ratios in oxygen bondings, we calculated the OH coverage (OH/C) of aGO and the OHG samples . The OH M of OHG-500 on temperature T and it fits well with Curie law χ = C/T. χ is the susceptibility and χ = M/H. H is the applied magnetic field. Inset is the corresponding 1/χ – T curve, which demonstrates a linearly, purely Curie-like paramagnetic behavior. The M – T dependences of aGO and other OHG samples were also measured, and they are all Curie-like paramagnetic just like OHG-500. The M – H dependences were also measured. At 300 K, all the samples are linearly diamagnetic , Bk is the Boltzmann constant, N is the number of present magnetic moments, S is the spin angular momentum number, and g is the Landau factor assumed to be 2. The fitted values for all the samples are summarized in sM of 1.27 emu/g, much higher than the value observed in GO with long oxidation duration  located at the vacancies and/or edges. Generally speaking, some factors, such as isolated vacancies814etc, may only contribute to the edge magnetism. However, the vacancies and edges generated by annealing GO are disordered662728We now consider the magnetic source for the robust magnetic moments observed in aGO and the OHG samples. As mentioned above, our OHG is a kind of defective graphene with sM. The C contents are calculated based on the XPS results of C and O atomic ratio. In short, if the O/C atomic ratio of the sample is x and the OH coverage is y, its chemical formula can be represented as COxHy. It is found that the spin density is ~4.42 μB per 1000 C for aGO, which increases up to ~6 μB per 1000 C for the OHG samples of Group A. In view of the fact that the weight loss of aGO by annealing aGO at 200°C is ca. 34%, one can make a reasonable assumption that magnetic contribution of the newly annealing-created OH groups is low. The reason may be that the newly created OH groups prefer to lie at edges or vacancies6μB per 1000 C, this may result from both the decrease of the OH coverage  and inducing efficiency (71.5 μB per 1000 OH groups). It indicates that the magnetic moments are robust despite that some of them vanished with the unavoidable decomposition of original ‘magnetic’ OH groups on the basal plane of the OHG sheet.To take a deeper insight into the magnetic properties of aGO and the OHG samples, we calculated their spin densities  accordincoverage  and the coverage . Surpris7 clusters on the basal plane of the graphene sheet477 cluster can induce a large magnetic moment of 5 μB7 cluster configuration inducing 5 μB was sketched in Bμ ed in μB , close tS = 2 are generated by 1000 C atoms, so on average, a 25 × 25 or 26 × 26 graphene supercell (~666 C atoms) contributes a large magnetic moment. Assuming that the graphene sheet is flat and the magnetic moments distribute uniformly, we obtain that the average distance is at least 6 nm. Since the OH group interaction is negligible when the space is larger than 3 nmInducing high-density magnetic moments on graphene is usually the prerequisite to induce long-range magnetic orderingμB per 1000 OH groups, it is still lower than the theoretical value of 1 μB per single OH group. Namely, most of the OH groups on the aGO and OHG sheets do not contribute magnetic moments. It may attribute to the analogous clustering of OH groups, similar to the case of fluorine on graphene sheet8In addition, we should note that even the highest efficiency observed in aGO is as high as 217 μB per 1000 C by reducing the oxidation duration, and have observed a very high magnetic inducing efficiency of 217 μB per 1000 OH groups. By annealing of aGO to remove the ‘non-magnetic’ unstable oxygen groups and leave the ‘magnetic’ stable OH groups, we have obtained OHG with very high magnetization of 2.41 emu/g. Our results provide the solid experimental evidence that OH groups can induce robust magnetic moments on the basal plane of the graphene sheet. More importantly, we demonstrate that the magnetic moments are highly stable and can survive even at 900°C. Our results provide an effective sp3-type functional group for generating robust magnetic moments in graphene and, thus represent an important step for the potential applications of spintronics.In summary, we have obtained aGO with high spin density of 4.42 3, 48 g KMnO4, and 384 ml condensed H2SO4 was stirred for 1.5 h at 0°C, and then followed by another 2 h stirring at 35°C. Thereafter, 320 ml H2O was added into the mixture within 15 min at a steady flow. Without any delay, the premix of 800 ml H2O and 40 ml H2O2 was added within 6 min at a steady flow. During the process, the temperature of the solution fixed at 35°C. To avoid deeper oxidation, the mixture was immediately transferred to other container and diluted with 2000 ml H2O. The obtained solution was washed for 13 times by repeated 30 min centrifugation at 13000 rpm. The sediment was redispersed into water and centrifugated at 6000 rpm for 20 min, and only the supernatant was left to get GO sheets with high few-layer ratio. Finally, the lightly oxidized aGO was obtained by drying the GO solution in vacuum freeze drier at 50°C. To avoid other extra source which may induce magnetic moments, no ultrasonication was adopted. The OHG samples were synthesized by annealing aGO in Ar atmosphere at different temperatures for 1 h.The as-prepared GO was synthesized by chemical exfoliation of 8 g natural flake graphite powder (500 mesh)−8 emu , and all data have been corrected for the diamagnetic contribution by subtracting the corresponding linear diamagnetic background at room temperature. The 3 d impurity elements of all the samples are measured by inductively coupled plasma (ICP) spectrometry .The morphologies of the samples were investigated by TEM  and AFM . XPS measurements were performed on PHI5000 VersaProbe  using Al Ka radiation. Raman images were performed on inVia confocal Raman microscope  using a laser excitation of 532 nm. The magnetic properties of the samples were measured using SQUID magnetometer with a sensitivity less than 10Our calculation was performed by density function theory as implemented in the SIESTA codeN.J.T. proposed and supervised the project, T.T. and N.J.T. designed the experiments, T.T. and Y.L. carried out the experiments, Y.P.Z. performed the DFT calculations, T.T., X.G.W. and N.J.T. analyzed the data and wrote the manuscript. Y.P.Z., X.G.W., Y.L., F.C.L., Q.H.X. and Y.W.D. had valuable discussions and edited the manuscript. All the authors participated in discussion of research.Supplementary Information


Onchocerca volvulus transmission by Simulium guianense s.l. in 16 previously hyperendemic Yanomami communities in southern Venezuela after 15 years of 6-monthly and 5 years of 3-monthly mass ivermectin treatment.The World Health Organization (WHO) has set goals for onchocerciasis elimination in Latin America by 2015. Most of the six previously endemic countries are attaining this goal by implementing twice a year  mass ivermectin (Mectizan®) distribution. Elimination of transmission has been verified in Colombia, Ecuador and Mexico. Challenges remain in the Amazonian focus straddling Venezuela and Brazil, where the disease affects the hard-to-reach Yanomami indigenous population. We provide evidence of suppression of Baseline and monitoring and evaluation parasitological, ophthalmological, entomological and serological surveys were conducted in selected sentinel and extra-sentinel communities of the focus throughout the implementation of the programme.S. guianense flies tested by PCR none had L3 infection (heads only). Prevalence of infective flies and seasonal transmission potentials in 2012–2013 were, respectively, under 1 % and 20 L3/person/transmission season. Serology in children aged 1–10 years demonstrated that although 26 out of 396 (7 %) individuals still had Ov-16 antibodies, only 4/218 (2 %) seropositives were aged 1–5 years.From 2010 to 2012–2015, clinico-parasitological surveys indicate a substantial decrease in skin microfilarial prevalence and intensity of infection; accompanied by no evidence  of ocular microfilariae in the examined population. Of a total of 51,341 We report evidence of recent transmission and morbidity suppression in some communities of the focus representing 75 % of the Yanomami population and 70 % of all known communities. We conclude that onchocerciasis transmission could be feasibly interrupted in the Venezuelan Amazonian focus.The online version of this article (doi:10.1186/s13071-016-1313-z) contains supplementary material, which is available to authorized users. Onchocerca volvulus Leuckart and transmitted among humans through the bites of blackfly species of the genus Simulium Latreille. The embryonic stages of the parasite  migrate through the skin and cause severe itching, skin disease, and ocular lesions, with the severity of the clinical manifestations depending on the length of exposure to blackfly bites and the density of mf in the skin [Onchocerciasis is a chronic and cumulative skin and ocular disease caused by infection with the filarial nematode the skin , 2. Visuthe skin .In the Americas, the infection was formerly prevalent in 13 endemic foci distributed in 6 countries , where 565,232 persons were considered at risk of infection . In VeneSimulium guianense sensu lato (s.l.) Wise, S. incrustatum Lutz, and S. oyapockense s.l. Floch and Abonnenc are the main vectors in the Southern Focus of Venezuela, with the former species being the most competent for O. volvulus and the predominant human-biting blackfly in most of the hyperendemic areas of the focus [Simulium incrustatum, with a lower vector competence than S. guianense s.l., contributes to the transmission of onchocerciasis in mesoendemic and some hyperendemic areas, whereas S. oyapockense is the main vector in hypoendemic communities with low intensity of transmission [he focus , 10. Simsmission .O. volvulus, and interruption of transmission by 6-monthly mass administration of ivermectin , delivered by mobile teams with a therapeutic coverage ≥85 % of eligible population in all the endemic communities of the region, including hypoendemic areas [The strategy adopted by the Onchocerciasis Elimination Program for the Americas (OEPA) since its commencement in 1993 has included elimination of new (ocular) morbidity caused by ic areas , 14.  communities localized in different geographical areas of the southern Venezuelan focus after 15 years of 6-monthly and 5 years of 3-monthly mass drug administration (MDA) of ivermectin.The present work reports on the progress towards onchocerciasis elimination in southern Venezuela, according to the protocols proposed by the World Health Organization (WHO), which include in-depth parasitological, entomological and serological surveys, as well as guidance on operational thresholds . SpecifiThe parasitological, clinical, entomological and serological studies received ethical clearance from the Ethics Review Committee of CAICET . All the participants signed an informed consent form before undergoing any examination, testing, or agreeing to help as human attractants for entomological collections. Additionally, there was active participation of Yanomami volunteers (and their organization “Horonami”) in the identification of new communities and distribution of ivermectin across the focus.Text S1. Geographical and environmental characteristics of the Venezuelan part of the Amazonian onchocerciasis focus). The main onchocerciasis transmission seasons (regardless of the Simulium vector species present), occur during the dry to rainy (February–April) and rainy to dry (September–November) transitions [Onchocerciasis transmission in southern Venezuela occurs in the lowlands  and uplands 500–1200 m asl) of the Upper Orinoco, Upper Siapa and Upper Caura River basins (in the Amazonas and Bolivar States), which are part of the ancient Guayana Shield of northern South America, the oldest (3600 million years) region of the world .The Venezuelan part of the focus encompasses 12 geographical areas: Padamo; Ocamo; Mavaca; Platanal; Guaharibos; Orinoquito; Parima; Chalbaud; Ventuari; Uasadi; Caura, and Siapa. Within these 12 main areas, 31 geographical sub-areas have been described  Yanomami at-risk population has been estimated as 13,231 people, living in 241 ‘shaponos’ scattered deep in the forest, and practicing shifting cultivation, hunting, fishing and gathering of forest products . Their sAccording to OEPA guidelines , those cS. guianense s.l. in most of the localities [The pre-treatment levels of endemicity across all geographical areas were very heterogeneous Table , with socalities , 22.The criteria for selection of sentinel and extra-sentinel communities for regular monitoring and evaluation activities, consisting of in-depth epidemiological evaluations included: a) hyperendemic status, b) relative ease of accessibility by the mobile teams that conduct such evaluations, c) existence of historical, baseline epidemiological data prior to wide-spread ivermectin distribution; d) illustrative of the simuliid species composition of the focus. Accordingly, eight communities were selected as sentinel communities, namely : (1) Hasupiwei  in Guaharibos–Unturán; (2) Awei  and (3) Pashopëka  in Upper Ocamo–Shitari; (4) Koyowë (= Coyowë) , (5) Waharafitha—previously Fubalema— and (6) Matoa  in Orinoquito; and (7) Kanoshewë  and (8) Niayopë—previously Niyayowë— in Parima–Shamatari. Since the size of Yanomami communities is generally small, to increase sample size an additional 8 villages were selected as extra-sentinel communities. These were: (9) Yaurawë  in Guaharibos–Unturán; (10) Masiriki  and (11) Toumawei  in Parima (A); (12) Arokofita  and (13) Okiamo  in Parima (B); (14) Warapawë  in Parima (C); and (15) Kakarama  and (16) Pokoshiprare  in the Parima–Shamatari sub-area within the Parima area, the latter two communities derived from Yoreshiana A and Yoreshiana B  iliac crests were taken from each examined individual with a 2-mm Holth corneoscleral punch, and incubated for 24 h in buffered saline solution; the emerging mf were counted under an inverted microscope and snips were weighed to express results as mf/mg [Parasitological surveys were carried out in the sentinel and extra-sentinel communities of the focus at baseline 1981), pre-ivermectin MDA , and during ivermectin (twice a year and subsequently quarterly) MDA . The skin snip method was used to determine the prevalence and intensity of 1, pre-ivas mf/mg . When itas mf/mg , 26.Text S4. Calculation of parasitological indices).Ophthalmological evaluations were carried out during similar periods . The prevalence of microfilariae in cornea (MFC) and/or in the anterior chamber (MFAC) was determined by an ophthalmologist experienced in conducting onchocerciasis ocular evaluations for OEPA (HM). Ocular examinations were conducted with a slit-lamp in a darkened area after patients were asked to sit with their head between their legs for 5 min to allow any mf present in the anterior chamber to settle in a visible position in order to determine MFAC , 27. TheO. volvulus L3 larvae and the mean number of L3 larvae per fly were evaluated using classical dissection methods, examining the abdomens, thoraces, and heads of flies [S. guianense s.l. were calculated according to [Detailed entomological evaluations were carried out between 1982 and 2000 in two sentinel communities (Koyowë (=Coyowë) and Niayopë (=Niyayowë) from the hyperendemic geographical areas of Orinoquito and Parima, respectively. In these communities, the proportion of flies with of flies . The basrding to .S. guianense s.l. females were collected throughout the high O. volvulus transmission seasons (January–March/February–April or September–November) by using collecting protocols adjusted to the local transmission conditions previously established in the area and known periods of highest biting activity by parous flies [In a selection of sentinel  and extra-sentinel (Arokofita) communities, and during several consecutive collection days , host-seeking us flies , 30. AllO. volvulus-specific DNA probes has been generally applied to examine pools of flies in the OEPA region [O. volvulus using a species-specific PCR assay [HBR), seasonal biting rate (SBR), seasonal transmission potential (STP) and annual transmission potential (ATP) .Polymerase chain reaction (PCR) using A region . TherefoCR assay  . According to the results of the most recent epidemiological evaluation (2008–2009 for Awei and 2013–2015 for the remainder), 8 out of 16  had 0 mf in skin (and eyes), and 7 out of the 8 remaining communities had CMFL <1 mf/ss. A striking decline was also observed in the prevalence of MFC , with prevalence of MFC as high as 12 % Fig. . Of thesS. guianense s.l. in two sentinel communities prior to ivermectin MDA. Biting rates were high, particularly in Koyowë, where the monthly biting rate (MBR) reached values up to 23,997 bites person−1 month−1 and the monthly transmission potential (MTP) up to 576 L3 person−1 month−1. Seasonal biting rates (during the higher transmission months of January through March/April plus October through November) were in excess of 100,000 bites per person per transmission season. As these flies were processed by manual dissection, it was possible to determine both the proportion of infective flies and the number of L3 in the flies, with the latter ranging from 0.001 to 0.036 L3/fly  by dissection and 0.38 % (95 % CI 0.19–0.69 %) by PCR are in agreement with the value of 0.37 % for the entire baseline study period , in contrast with the 7.4 (0.37 %) infective flies per 2000 dissected flies recorded at baseline . In Hasupiwei, Pashopëka and Arokofita, after 2–3 years of quarterly ivermectin treatment, all 8085, 6464 and 12,793 flies respectively tested in 2012–2013 were PCR negative (with the upper 95 % CI for STP of 3–4 L3/person/transmission season).Results of the entomological evaluations carried out from 2006 to 2013 in Hasupiwei, Pashopëka, Koyowë and Arokofita are presented in Table O. volvulus Ov-16 for this age group  in 2009 to 100 %  in 2015. By 2015, CMFL and MFAC have become, respectively, negative in 7/16 44 %) and 5/7 (71 %) of the communities examined for these indicators. Communities of the Orinoquito sub-area  are still positive for 4 % and 5O. volvulus infection  in children have been the WHO criteria followed to certify focal interruption of parasite transmission [O. volvulus transmission by the most competent vector of the focus, S. guianense s.l., in areas formerly hyperendemic to holoendemic. The dramatic decline in the seroprevalence among children and the lack of evidence of parasite-vector contact suggest that four times per year treatment has successfully suppressed transmission in many of the endemic communities. It will now be necessary to maintain pressure on the parasite population, continuing to suppress transmission, until the existing adult female parasites are either killed or rendered sterile by repeated ivermectin treatments [In the OEPA region, absence or near absence of L3 larvae in the head of blackfly vectors , a 99 % reduction in the intensity of transmission , and the absence of detectable smission . Prior tsmission , 34. Hereatments .S. oyapockense s.l. and/or S. incrustatum [R0 ≥ 1 [O. volvulus, but also on the human blood index  of the various blackfly species and populations therein. At present, this parameter is unknown for the simuliid species prevailing in the Amazonian focus, but field and theoretical studies on S. damnosum s.l. in West Africa indicate that the HBI can be highly variable [SBRs) of S. guianense s.l. lower than 50,000 bites/person/transmission season after at least 4 rounds of treatment with coverage ≥85 % of eligible population. In these communities 133 children under 5 years of age from Pashopëka (Jénita–Putaco); Hasupiwei (Guaharibos); Arokofita, Kanoshewë, Niayopë and Okiamo (Parima B); Warapawë (Parima C); Masiriki and Toumawei (Parima A), and Kakarama (Shamatari) were negative for Ov-16 , where 4/85 (5 %) children aged 1–5 years were seropositive in 2013.Ivermectin is expected to have a faster impact in those areas of the Amazonian focus with lower vector competence blackfly species . These variable  and possvariable . Given tModelling studies conducted with the EPIONCHO transmission model in African savannah settings  have sugOnchocerca parasites along the reticular nature of the Yanomami use of space may indeed be very diffuse, necessitating intensive treatment in all sub-areas; however, if particular networks could be identified as being responsible for most transmission, a more targeted approach could be beneficial towards the goal of achieving elimination.More likely, in the Amazonian focus, and due to the Yanomami network of kinship ties, alliances and hostilities , 22, oncThe semi-nomadic characteristics of the human population, the remoteness of the Yanomami territory, the holoendemic status of some areas, and the ongoing identification of new endemic communities in the Venezuelan part of the Amazonian focus constitute the main challenges for the elimination of onchocerciasis in the Amazonian focus. From 2009, ivermectin treatment frequency has been increased to four times per year in 80 % of the hyperendemic communities in an attempt to hasten interruption of transmission in areas showing slow progress or in communities recently identified and incorporated into the programme at later stages. Sustaining a high geographical and therapeutic coverage for each treatment round is essential. Only the first round of the 2012–2014 quarterly distributions reached the ≥85 % goal. Besides, since the sentinel and extra-sentinel communities included in this study were partly selected because of relative facility of access, there is the possibility that more remote communities received a lower treatment coverage and/or frequency. Currently, there is a total of 72 communities identified as being remote , with a population of 3359 individuals . This represents approximately 25 % of the total population. Most of these communities are receiving regular treatment, and many of them have received more than 8–12 treatment rounds . However, there are communities located in the Upper Siapa and Upper Ocamo-Parima sub-areas that are only accessible by helicopter . This represents only 11 % of the total population and 12 % of all the communities under treatment. Treatment is delivered to these communities when helicopter support is available. Depending on their endemicity status and their connectedness with well-controlled areas, these less accessible communities could pose a risk of infection re-introduction. The use of high spatial-resolution satellite data to identify remote communities in the rainforest is a strategy currently used in an attempt to delineate the extent of the focus, the distribution of transmission zones , and theOur results contribute to the ongoing success of the OEPA strategy , 37, 38.


Two-dimensional (2D) atomic crystals and their heterostructures are an intense area of study owing to their unique properties that result from structural planar confinement. Intrinsically, the performance of a planar vertical device is linked to the quality of its 2D components and their interfaces, therefore requiring characterization tools that can reveal both its planar chemistry and morphology. Here, we propose a characterization methodology combining (micro-) Raman spectroscopy, atomic force microscopy and time-of-flight secondary ion mass spectrometry to provide structural information, morphology and planar chemical composition at virtually the atomic level, aimed specifically at studying 2D vertical heterostructures. As an example system, a graphene-on-h-BN heterostructure is analysed to reveal, with an unprecedented level of detail, the subtle chemistry and interactions within its layer structure that can be assigned to specific fabrication steps. Such detailed chemical information is of crucial importance for the complete integration of 2D heterostructures into functional devices. The properties of 2D materials such as graphene can vary according to the quality and, for vertical devices, the interfaces between materials. Here, the authors report a method using TOF-SIMS, micro-Raman spectroscopy and atomic force microscopy to give high levels of detail of vertical 2D heterostructures. Our characterization approach is able to detect, identify, localize and quantify the various contaminants introduced during fabrication, thus leading to improved manufacturing processes and, therefore, better-performing devices.Here, TOF-SIMS is applied, for the first time to our knowledge, to a 2D atomic crystal heterostructure fabricated by common sequential layer transfer, to study its planar chemical composition at the atomic level, with significant implications for understanding the influence of manufacturing and transfer processes. As a system of choice, the conductor-on-insulator stacking of graphene on h-BN shows promise as a base for high-quality nanoelectronics11132 substrate that was partially covered by mechanically exfoliated h-BN flakes, was first characterized by (micro-) Raman spectroscopy (−1 and AFM height topography confirmed large (tens of microns), continuous graphene patches extending over the h-BN flakes and SiO2 substrate . However, atop SiO2 the graphene D peak is disentangled from any substrate-related signal and presents a relatively low intensity compared with the G peak , thus confirming a relatively small defect density of the graphene domains .To evaluate its final configuration, the 2D system, composed of a CVD-grown, wet-transferred graphene onto a SiOtroscopy  and AFM troscopy . Initialrate  sputtering. As deposited  that is better or comparable to micro-Raman (−1), we can progressively study the outer adsorbed species, the top graphene layer, the interfacial contaminants and the underlying h-BN and SiO2., before  and afte, before  ∼60 s ofeposited , the traro-Raman , TOF-SIM2 interfaces and (ii) estimate sputtering rates for the species of interest, the surface topography of the 2D heterostructure was investigated by AFM before (+ at 500 eV energy. Both height distributions (histograms) shown in 2 substrate (Z≈−65 nm) and h-BN flake surface (Z≈150 nm). An obvious change in shape from Gaussian to Lorentzian and a significant reduction of the width (FWHM) for the two main histogram peaks suggest that Cs+ sputtering substantially decreases the surface corrugation of both the SiO2 substrate and h-BN flake surface , both the acquired data (discrete markers) and their 1-point spline interpolations (continuous curves), representing single-layer graphene, adventitious organic material chemisorbed at the h-BN surface and the h-BN flake, respectively. Owing to the discontinuous nature of the transferred graphene (tens-of-microns-sized patches), the depth profiles on both the graphene/h-BN and graphene/SiO2 systems were reconstructed from the initial data using regions of interest that ensured graphene coverage . For depth profiling, polyatomic species such as C3− and 10BB− were chosen to avoid the intrinsic artefacts that monoatomic species such as C− and B− have due to residuals from oxides or other chemisorbed adventitious species. An estimation of the atomic mixing length between graphene and h-BN or SiO2 can be obtained by applying the so-called mixing–roughness–information (MRI) model3− profile , we use the forward calculation procedure3− data points. The fitting procedure searches for the minimum deviation between the simulated profile and the C3− profile points . This is a result of the chemisorbed species (identified below) accumulated in graphene during the transfer process and which are chemically interacting with the SiO2 substrate 2S2O8  equates to about 0.4% of the total mass (<0.08 atomic %). However, given its depth localization, the copper residue appears to be spatially decoupled from the graphene overlayer and thus may not contribute to its defect density. Moreover, it is virtually undetectable by other spectroscopic techniques, for example, XPS, accounting for <0.03% of the mass (<0.006 atomic %) in the first 10 nm of the graphene/h-BN surface 2S2O8 . An oxide system . The cor− and CB−) are a result of chemisorbed adventitious sulfur or carbon, respectively, with h-BN and are formed before the addition of graphene. Most of the physisorbed adventitious species  at the graphene, h-BN and SiO2 surfaces are likely diffused to the sides of the graphene domains by the capillary forces exerted at the graphene/h-BN and graphene/SiO2 interfaces or uniformly spread at the graphene surface− species that appears to be located in two layers, one at the very surface corresponding to the physisorbed water (due to air exposure) and another seemingly chemisorbed in the graphene overlayer  recorded in high lateral resolution (∼200 nm) mode with a Bi3+ (30 keV) analysis ion beam. These maps are recorded after ∼0.3 nm of the surface has been removed by Cs+ sputtering (500 eV ion energy) and represent the main species of interest related to the graphene overlayer (C3−), chemisorbed copper solvent residues (C2N−), chemisorbed water (OH−), PMMA/acetone residues partial underlayer (C2O−), chemisorbed adventitious organic material at the h-BN flake surface (CB−) and the SiO2 substrate (SiO2−). The dark areas visible in the C3− maps correspond to the space between the graphene islands that are inherent to the transfer process. Having the same in-plane localization, the C3−, C2N−, S− , within the transferred graphene. In addition, as the depth profiles of C2N− and S− extend over the depth profiles of C3− and C2O− , respectively. As a clear indication of being a PMMA/acetone marker, the C2O−map follows both the C3− and SiO2− maps, as expected for a solvent that was used after the deposition of the PMMA/graphene system onto the h-BN/SiO2 substrate. Given that the physisorbed species (∼0.3 nm) were previously removed by sputtering, the secondary ion map of the OH− species represents the chemisorbed water that appears uniformly distributed within the plane of the graphene overlayer and the exposed SiO2 substrate. Additional contaminants, if present, were below the detection limit or their signal was too low to produce a reasonable image, as in the case of Cu−. Albeit impossible to spatially map in plane, the copper residue is most probably isotropically chemisorbed at the bottom of the graphene overlayer in the lower ammonia persulfate residual layer, as inferred in C2N−, S−  and C2O−and C2O− , we conc2 substrate, and (ii) consists of three atomic-like layers: a single layer graphene and two partial atomic underlayers that combine residues from the solvents used in the transfer process and the initial copper foil support for the CVD graphene. All residues detected as a part of the graphene system are chemisorbed in or at the bottom of the graphene overlayer, thereby having a passivation effect and possibly acting as an electronic decoupling layer from the substrate. Located in the lower organic residual (passivation) layer, the copper residue (< 0.08 atomic %) appears spatially decoupled from the graphene overlayer thus not contributing to the actual graphene defect density. Finally, traces of water are identified both physisorbed at the very surface of the 2D system and chemisorbed in the graphene overlayer, confirming previous SPM observationsTo summarize, in depth AFM and TOF-SIMS analysis show that the transferred graphene system (i) is chemically inert with respect to h-BN but interacts with SiOAs shown above, all detected surface and interface species can be traced back to the transfer or CVD growth processes, thus TOF-SIMS profiling can directly measure the effects of synthesis, handling, and fabrication processes on the final 2D materials and heterostructures. Such detailed information could be critical towards understanding their device function. TOF-SIMS can also be applied to other ultra-thin layered structures, providing detailed, vertical chemical analysis at the atomic level . We thus2 (285 nm thick) on a Si wafer4)2S2O8); Sigma-Aldrich) solution2/Si substrate and the top PMMA was removed using acetone, giving the final graphene/h-BN/SiO2 configuration. We deliberately used common growth, processing and transfer techniques to produce 2D heterostructures that are typical of other published results.The substrate was prepared by depositing mechanically exfoliated h-BN flakes from h-BN powder  atop SiO2 system. The instrumentation consisted of a Raman spectrometer WITec alpha300 using a laser wavelength of 488 nm, an AFM Asylum Research MFP3D and a TOF-SIMS ION-TOF GmbH TOF.SIMS 5 configured with 500 eV Cs+ sputtering and 30-keV Bi1+/Bi3+ analysis ion beams. Depending on the acquisition mode, the TOF-SIMS analysis ion gun was set in either high-current bunched mode (Bi1+ at 30 keV ion energy and 18 ns pulse duration) for depth profiling or burst alignment mode (Bi1+/Bi3+ at 30 keV ion energy and pulse duration of 100 ns without or 18 ns with bursts) for high lateral resolution. The raster area, beam sample currents and specific settings for both the analysis and sputtering beams are indicated in the main text and supporting information. The high-current and burst alignment (with bursts) modes allowed a mass resolution better than 5,000 (m/δm) for all analysed masses. All detected secondary ions had negative polarity. The TOF-SIMS data were acquired at a base pressure of ∼7.5 × 10−10 Torr.Raman spectroscopy and mapping, AFM and TOF-SIMS were used in conjunction to characterize the structure, morphology and layered chemical composition, respectively, of the graphene/h-BN/SiOHow to cite this article: Chou, H. et al. Revealing the planar chemistry of two-dimensional heterostructures at the atomic level. Nat. Commun. 6:7482 doi: 10.1038/ncomms8482 (2015).Supplementary Figures 1-10, Supplementary Notes 1-7 and Supplementary References