The PERSEIDS study aimed to estimate incidence/prevalence of interstitial lung diseases (ILDs), fibrosing interstitial lung diseases (F-ILDs), idiopathic pulmonary fibrosis (IPF), systemic sclerosis-associated ILD (SSc-ILD), other non-IPF F-ILDs and their progressive-fibrosing (PF) forms in six European countries, as current data are scarce.This retrospective, two-phase study used aggregate data (2014–2018). In Phase 1, incident/prevalent cases of ILDs above were identified from clinical databases through an algorithm based on codes/keywords, and incidence/prevalence was estimated. For non-IPF F-ILDs, the relative percentage of subtypes was also determined. In Phase 2, a subset of non-IPF F-ILD cases was manually reviewed to determine the percentage of PF behaviour and usual interstitial pneumonia-like (UIP-like) pattern. A weighted mean percentage of progression was calculated for each country and used to extrapolate incidence/prevalence of progressive-fibrosing ILDs (PF-ILDs).5 person-years ranged between 9.4 and 83.6 (ILDs), 7.7 and 76.2 (F-ILDs), 0.4 and 10.3 (IPF), 6.6 and 71.7 (non-IPF F-ILDs), and 0.3 and 1.5 (SSc-ILD); and prevalence/105 persons ranged between 33.6 and 247.4 (ILDs), 26.7 and 236.8 (F-ILDs), 2.8 and 31.0 (IPF), 22.3 and 205.8 (non-IPF F-ILDs), and 1.4 and 10.1 (SSc-ILD). Among non-IPF F-ILDs, sarcoidosis was the most frequent subtype. PF behaviour and UIP-like pattern were present in a third of non-IPF F-ILD cases each and hypersensitivity pneumonitis showed the highest percentage of progressive behaviour. Incidence of PF-ILDs ranged between 2.1 and 14.5/105 person-years, and prevalence between 6.9 and 78.0/105 persons.In 2018, incidence/10To our knowledge, PERSEIDS is the first study assessing incidence, prevalence and rate of progression of ILDs across several European countries. Still below the threshold for orphan diseases, the estimates obtained were higher and more variable than reported in previous studies, but differences in study design/population must be considered. The PERSEIDS study provides updated and detailed epidemiological data for ILDs across six European countries. Prevalences ranged between 26.7–236.8 (F-ILDs), 2.8–31.0 (IPF), 22.3–205.8 (non-IPF F-ILDs). A third of non-IPF F-ILD cases showed PF behaviour.https://bit.ly/3wLH7pF Interstitial lung diseases (ILDs) display varying degrees of inflammation, fibrosis or both, and a wide range of clinical courses and prognoses. Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive-fibrosing ILD (PF-ILD), characterised by a usual interstitial pneumonia (UIP) pattern on high-resolution computed tomography (HRCT) or lung biopsy . Its pulDespite their major impact on mortality, quality of life and resource use, in Europe the epidemiological data available for ILDs and particularly their PF forms is scarce , 11. TheA retrospective, database study with a two-phase design  was condThe main objective was to investigate the annual incidence and prevalence of ILDs and their time trends over the study period. The focus was placed on F-ILDs as these show a greater risk of progression, but the epidemiology of non-F-ILDs was also assessed to provide a broad picture of ILDs in the participating countries. The primary outcome was crude incidence/prevalence of ILDs, F-ILDs, IPF, non-IPF F-ILDs and SSc-ILD by country and overall, for the whole study period and annually. Secondary outcomes were: a) crude incidence/prevalence of non-IPF F-ILD subtypes (other than SSc-ILD), including ILDs associated with rheumatoid arthritis (RA-ILD), mixed connective tissue diseases (mixed CTD-ILDs) and other connective tissue diseases (other CTD-ILDs), idiopathic nonspecific interstitial pneumonia (iNSIP), unclassifiable idiopathic interstitial pneumonias (uIIPs), hypersensitivity pneumonitis (HP), exposure-related ILDs, sarcoidosis and other F-ILDs; b) relative percentage of each non-IPF F-ILD subtype; and c) crude incidence/prevalence of non-fibrosing ILDs (non-F-ILDs).supplementary material Section A). These codes/keywords were searched in all centres following a common algorithm, specifically designed to classify cases in ILD subtypes and subsequently, in overarching ILD categories while avoiding duplicates . The methodology for obtaining prevalence/incidence estimates (minimum and maximum), as well as alternative methods and adjustments applied due to centre/country particularities, are detailed in the supplementary material Section B. The relative percentages of non-IPF F-ILD subtypes were obtained by dividing the prevalent cases of each subtype by total prevalent cases of non-IPF F-ILD.To identify incident/prevalent cases of ILD, 10 of the 13 centres participating in Phase 1 conducted a systematic search of the institution's clinical database , and the remaining three in other clinical databases  . The seaThe aim was to further characterise non-IPF F-ILDs. The primary outcome was the relative percentage of cases within each non-IPF F-ILD subtype presenting PF behaviour alone (without UIP-like pattern), UIP-like pattern alone (without PF behaviour), both PF behaviour and UIP-like pattern, and none of them, overall for all countries. Secondary outcomes included: a) crude prevalence/incidence of PF-ILDs by country and overall, for the whole study period and annually; b) positive predictive value (PPV) of the algorithm used in Phase 1, by centre and country; and c) adjusted (by PPV) values for incidence/prevalence estimates obtained in both study phases.Each of the 10 centres participating in Phase 2 reviewed the medical records of their first 100 prevalent non-IPF F-ILD cases identified in Phase 1, sorted by date of healthcare encounter from January 1, 2016 onwards.supplementary material Section C). If necessary, centres could review additional cases to the first 100 until reaching this number. These additional cases were considered when calculating the relative percentages of the primary outcome.The review encompassed the pulmonary function tests and HRCT results available in each patient's file, and either confirmed (true positive) or ruled out  the presence of the fibrosing (non-IPF) condition. The primary outcome was determined on true positives, and it was calculated that at least 80 were required to reach enough precision  within the 2-year period following the healthcare encounter, or between 5 and <10% but with any of the following: ≥1 ILD-related hospitalisation (excluding emergency visits), increasing extent of fibrosis on HRCT, starting or increasing oxygen use, or death due to respiratory event. Owing to the lack of uniformly accepted criteria for PF-ILD, the use of relative decline in FVC was based on previously published expert recommendations . The defTrue and false positives obtained during the Phase 2 review were also used to assess the specificity of the search algorithm at each centre, by calculating its PPV. The PPV was obtained by dividing true positives by the sum of true positives and false positives. Only the first 100 prevalent cases reviewed at each centre  were considered. PPVs of centres in each country were averaged to obtain a country PPV. Simple (not weighted) means were calculated under the assumption that each centre's PPV contributed equally to the country mean.supplementary material Section D).Finally, to account for possible overestimation of incidences/prevalences arising from the use of codes and/or keywords in the systematic search, crude estimates for each country were multiplied by the country PPV to obtain adjusted estimates. Some exceptions to this methodology were made based on country particularities . Categorical variables were summarised as absolute and relative frequencies, and 95% confidence intervals. Pairwise comparisons were conducted to assess differences in the percentage of progression between non-IPF F-ILD subtypes and centre levels (secondary versus tertiary). Significance was set at the 0.05 level.Sample size considerations are presented in the supplementary material Section B, Alternative methods and adjustments). When not, estimates were obtained based on available data.Missing values were completed when possible . The centre/country PPVs are provided in the 5 person-years) ranged between 20.0 and 42.5 (ILDs), 17.9 and 38.3 (F-ILDs), 2.1 and 6.3 (IPF), 14.7 and 33.9 (non-IPF F-ILDs), and 0.5 and 1.0 (SSc-ILD) . The annual prevalences of ILDs in each country are presented in supplementary material sFigure 2. The overall prevalence (cases/105 persons) ranged between 72.1 and 164.2 (ILDs), 66.8 and 152.6 (F-ILDs), 7.8 and 24.3 (IPF), 56.2 and 132.0 (non-IPF F-ILDs) and 2.8 and 5.7 (SSc-ILD). Throughout the study period, incidence/prevalence were generally stable within each country, with few exceptions in Finland, Belgium and Greece .In 2018, the latest year assessed, the lowest (minimum adjusted by PPV) incidence estimates for ILDs, F-ILDs, non-IPF F-ILDs and SSc-ILD were obtained in Portugal, and for IPF in Denmark. The highest (maximum crude) incidence estimates for ILDs, F-ILDs and non-IPF F-ILDs were obtained in Belgium, for IPF in Denmark and for SSc-ILD in Norway. The overall  incidence .supplementary material sTable 6.In 2018, sarcoidosis was the most frequent non-IPF F-ILD subtype in most countries , except for Belgium  and Norway . In these two countries, however, sarcoidosis was the second most common subtype . In the remaining countries, the second most common subtypes were other F-ILDs (24.3% Denmark), exposure-related ILDs (21.2% Finland) and HP . The most prevalent conditions within the other F-ILD category were only assessed in Denmark; in 2017, they were unspecified ILDs  and other ILDs with fibrosis . Relative percentages annually and for the whole study period are provided in the supplementary material sTable 7.The minimum adjusted/maximum crude incidence of non-F-ILDs by country and overall for the whole study period and annually is shown in supplementary material sTable 8. Of total non-IPF F-ILD cases, approximately a third showed PF behaviour; 43% of these had a UIP-like pattern , but not between tertiary and secondary centres (p=0.6181).Absolute numbers and relative percentages of non-IPF F-ILD subtypes are shown in  pattern . Of note5 person-years and prevalence between 15.8 and 40.0/105 persons. Supplementary material sTable 10 (incidence) and sTable 11  show the widest variability observed in the primary and sensitivity analyses, annually and for the whole study period. Within each country, the results of the sensitivity analyses were very similar, and the results for the primary analysis were within the range defined by sensitivity analyses. The I2-statistic obtained in the random effects model for Method 3 (90%)  revealed substantial heterogeneity in PF behaviour among countries.The incidence in 2018 across the participating countries, assessed with the primary methodology, is show in PERSEIDS addressed the paucity of detailed and updated incidence/prevalence data for ILDs and its PF forms. The study has various strengths: the parallel implementation in six countries and the participation of both rheumatology and pulmonology units provides a broad, contemporaneous picture of the burden of ILDs across countries and grants a study population large enough to obtain sufficiently precise initial estimates. Also, the period of 5 years considered was wide enough to observe temporal trends and between-country differences.5 persons/year in 1998) [5 persons/year in 2004) [5 person-years between 1995 and 2005, 3.8–6.6 per 105/year between 2003 and 2009) [5 persons/year in 2009) [5 and 17.3/105, respectively) [5/year), Greece (0.93/105/year), Denmark (7.27/105 person-years between 1995 and 2005 and 1.3/105/year between 2003 and 2009) and the Nordics (0.4–10/105/year) were lower than in PERSEIDS, as was IPF prevalence reported previously for Belgium (1.25/105) and Greece (3.38/105) [5 persons between 2005 and 2012 and the prevalence at 13/105 persons in 2013 [5 persons [The few epidemiological studies on ILDs conducted so far in countries participating in PERSEIDS show quite homogeneous estimates. Incidence data were available for Belgium 1.0/10in 1998) , Greece in 2004) , Denmarknd 2009) , 18, and98 , Greectively) , 16. In .38/105) –19. A No persons ).supplementary material Section B). The extent of overestimation and the strictness of the adjustment applied may explain the highest/lowest estimates compared to other countries.Highest maximum crude estimates for ILDs, F-ILDs and non-IPF F-ILDs were consistently found in Belgium, and lowest in Portugal. Multiple factors may explain these findings. In Belgium, Liege Hospital was the only centre providing data for all ILDs in Phase 1. As rheumatological ILDs are referred to this centre from all over the country, we adjusted the reference population in these ILDs only. However, non-rheumatological referrals cannot be ruled out, so there may have been overestimation of these conditions and of the overarching ILD categories. Other contributing factors may be the concentration of heavy industries in the province of Limburg  and the Incidence of ILDs was relatively high compared to prevalence. This may point to a high mortality rate; but there may be other factors contributing, such as the aged population structure in Europe  and a besupplementary material. The expected bias was reduced by using wider reference populations for calculating incidence/prevalence, applying a different PPV to rheumatological ILDs (Liège) and estimating F-ILDs incidence/prevalence based on data from surrounding countries (Norway), but some bias may have remained. The high percentage of exposure-related ILDs in Finland may due to its prominent ship-building industry [The relative distribution of non-IPF F-ILDs subtypes observed may help clinicians discern which patients should be screened for ILDs, to achieve prompt diagnoses and improved outcomes; and for the most prevalent subtypes, should trigger the development of proper recommendations for management, as they are currently lacking except for SSc-ILD . In mostindustry , with itindustry . The expindustry , 29, botindustry –32. The industry  may haveSarcoidosis was the most frequent non-IPF F-ILD subtype in Phase 1. Though rarely fibrotic in early stages, sarcoidosis was included among non-IPF F-ILDs in PERSEIDS because fibrosis ultimately affects 20% of patients with stage IV disease . The cod2 statistic revealed a considerable heterogeneity among them.Differences in the relative distribution of non-IPF F-ILD subtypes among countries and in their percentage of progression explain the variability in incidence/prevalence estimates of PF-ILDs, and reinforce the primary methodology chosen (weighted mean percentage of progression). Also, primary and sensitivity analyses gave similar results in most countries, further supporting the strength of results. Nevertheless, the primary approach had some limitations, as the weighted percentage was calculated by pooling progression data from all countries, and the Ie.g. Portugal, Norway) did not provide incident/prevalent cases for the whole study period, precluding comparisons among countries for missing years and possibly affecting overall estimates. Between-country differences in ILD management, previously highlighted [Despite the efforts to reduce bias, the study has other limitations. It was not always possible to use a homogeneous methodology due to centre/country particularities, which may limit the comparability of results. Coding particularities/issues may explain some of the variability found between countries, as it may have influenced the ability to capture incident/prevalent cases and may have led to more misclassification in specific countries. Some centres . However, as ≥100 consecutive cases were reviewed per centre, the sample is expected to be representative.Other limitations were not country-specific. ILDs are often rare and diagnosis can be difficult, leading to over/underestimation and misdiagnosis. Also, the use of ICD codes for ILDs has known limitations , but the5 person-years and 6.9–78.0/105 persons, with differences between countries probably explained by the relative distribution of non-IPF F-ILD subtypes and their different rates of progression.To our knowledge, PERSEIDS is the first study assessing the epidemiology and progression of ILDs in parallel across several European countries. The incidence and prevalence of ILDs was higher and more variable between countries than previously reported, but below the threshold for orphan diseases. Overall, approximately a third of non-IPF F-ILDs cases showed a PF behaviour, and nearly half of those (43%) had a UIP-like pattern. The prevalence and incidence of PF-ILDs ranged between 2.1–14.5/1010.1183/23120541.00597-2021.Supp1Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.00597-2021.SUPPLEMENTSupplementary material


As the application of graphene nanomaterials gets increasingly attractive in the field of tissue engineering and regenerative medicine, the long-term evaluation is necessary and urgent as to their biocompatibility and regenerative capacity in different tissue injuries, such as nerve, bone, and heart. However, it still remains controversial about the potential biological effects of graphene on neuronal activity, especially after severe nerve injuries. In this study, we establish a lengthy peripheral nerve defect rat model and investigate the potential toxicity of layered graphene-loaded polycaprolactone scaffold after implantation during 18 months in vivo. In addition, we further identify possible biologically regenerative effects of this scaffold on myelination, axonal outgrowth, and locomotor function recovery. It is confirmed that graphene-based nanomaterials exert negligible toxicity and repair large nerve defects by dual regulation of Schwann cells and astroglia in the central and peripheral nervous systems. The findings enlighten the future of graphene nanomaterial as a key type of biomaterials for clinical translation in neuronal regeneration. They include graphene and its derivatives, such as graphene oxide (GO), reduced GO, graphite oxide, and ultrathin graphite, and are highly stiff, elastic, thermally, and electrically conductive6. These qualities make them increasingly important and popular in different areas of life, such as bioelectronics, energy conversion, and biomedicine9. In the field of tissue engineering, the incorporation of graphene and its derivatives into polymer has become an important way of manufacturing artificial compound scaffolds for tissue repair12. In this way, GBM improve the physical and mechanical characteristics as well as the general manifestation of the polymeric substrate by imparting significant cues such as electrical conductivity and mechanical reinforcement of synthetic scaffolds. This is very vital to electro-responsive tissue regeneration, such as nerves, cardiac, and skeletal muscles16. In addition to the unique properties, the biocompatibility of biomaterials should be considered carefully.The graphene-based materials (GBM) are a class of two-dimensional carbon nanomaterials with incredible physical, chemical, and mechanical properties17. The GO displayed antibacterial ability by disrupting E. coli bacterial cell membranes, decreasing the viability by over 90%, and elucidating strong oxidative stress18. It further impaired membrane integrity by degrading phospholipids19. Polyvinyl-N-carbazole-GO (containing 3% GO) could even reduce the metabolic activity and induce cellular apoptosis by encapsulating bacterial cells20. In lung epithelial cells and fibroblasts, 50 μg mL−1 GO nanoparticles might induce minor toxicity and insignificant cell death21. Graphene, especially few-layer derivatives, exerted varied effects on neuronal cells. The expression level of caspase 3 was significantly increased in PC12 neuronal cells at the presence of 10 μg mL−1 few-layer graphene (FLG) that led to mitochondrial dysfunction and oxidant insults22. In contrast, another group found that FLG was quite biocompatible in three cell species, PC12 cells, oligodendroglial cells and osteoblasts23. These researches reported preliminary findings in cellular levels and resulted in huge deviations in GBM biocompatibility and other biological effects.The biosafety profile of GBM has caught huge attention and remains a controversy in some studies. Different evaluation systems may yield different outcomes. Some claimed that graphene and its derivatives possessed huge potential in biological and medical fields. Previous literature reported that graphene could regulate cell attachment, migration, differentiation, and viability24. Nevertheless, 1% graphene solution caused lung damage and inflammatory reactions with bioaccumulation and granuloma deposition in major functioning organs25. It seems more controversial in terms of in vivo application of GBM. Yang et al.26 found that polyethylene glycolylated nanographene sheets caused insignificant toxicity at the tested dose (20 mg kg−1) to the experimental mice during 3 months and were gradually cleared by both renal and fecal excretion. In contrast, Krajnak et al.27 reported that pathological changes and dysfunctions occurred after exposure to 40 μg graphene nanoparticles in the vascular/renal function in a load and form-dependent style in a mouse study. Therefore, it is vital and urgent to investigate the biological profiles of GBM comprehensively, especially in the nervous system. Our group previously reported the effects of polydopamine and RGD modified single-layered, multilayered graphene on improving Schwann cell (SC) proliferation and inducing myelination and axonal extension in a lengthy rat sciatic nerve defect model at 18 weeks after injury28. These findings preliminarily indicate that GBM promisingly facilitate neuronal repair after surface modification. However, the real biocompatibility of GBM alone is not clear in a long-term evaluation (over 1 year). Moreover, graphene can increase glial cell and SC viability in the central nervous system (CNS) and peripheral nervous system (PNS), respectively; Nevertheless, it has not been investigated whether GBM affect both nervous systems simultaneously because the CNS shows responsive neural plasticity after PNS injury. It seems exceptionally vital to dig into the deeper correlation in the neuronal regulation between CNS and PNS in peripheral nerve regeneration.Even for in vivo evaluation, previous researches discussed the GBM biosafety via inhalation or intravenous injection, and found no prominent short-term toxicityTo solve these issues, the present study will lay emphasis on the long-term biological characteristics of graphene-based nanoscaffolds (GBN) in the PNS. In addition, interactions between PNS and CNS will also be investigated thoroughly to reflect the influence of GBN on neuronal activity and the microenvironment.We investigated the toxicity effect of GBN in a lengthy sciatic nerve defect model over 18 months in vivo. The histological results of the major functioning organs reflected that no prominent morphological changes occurred in any of the heart, liver, spleen, lung, or kidney due to toxic insults from GBN  group Fig. . Except Myelin basic protein (MBP) expression levels were higher in the 4% and 2% layered graphene-loaded PCL scaffold groups than the PCL scaffold control group. Nevertheless, the MBP expression level was markedly higher in the autograft nerve Fig. . The immWe evaluated the endothelial cell activity by measuring CD34 and vascular endothelial growth factor (VEGF) protein expression levels. The angiogenesis status is significantly higher in the 4% and 2% graphene-based PCL scaffolds than PCL groups. The CD34 expression level was relatively higher in the 4%GBN@PCL group than autograft Fig. .Fig. 4AnIn graphene-loaded PCL scaffolds, a higher value was seen in the muscle fiber area, though the muscle structure was the best in the autograft group Fig. . The resThe electromyography (EMG) was positioned on the injury side and then we evaluated nerve conducting velocity (NCV) and distal compound motor action potential (DCMAP) values, respectively. The 4% graphene-loaded PCL group showed a remarkably higher NCV level than other scaffold groups and comparable to autograft Fig. . DCMAP l29. PCL is one of the most common polymer materials used for neural substrates31. It is biocompatible and its biodegradation products are also toxic-free in the living body. Moreover, PCL provides certain mechanical stability with its high stiffness and relatively low degradation rate33. Consequently, the composite scaffold guarantees long-term in vivo support of structural integrity.The strong C–C bond determines the excellent mechanical characteristics of graphene and its derivatives due to the perfect planar nature. The addition of graphene to polymeric materials is a great reinforcement to the compound scaffold46. Graphene may cause different levels of tissue damage in organs (such as the lung)37. In the nervous system, previous researches showed widely divergent outcomes in various settings. Bianco et al.38 reported that graphene fabricated by chemical vapor deposition induced apoptotic effects, and an increased level of lactate dehydrogenase, and the formation of reactive oxygen species in neurons. Agarwal et al.39 found that single-layered graphene crosslinked collagen cryogel at 0.5% concentration was beneficial to the proliferation of bone marrow mesenchymal stem cells in vitro. However, the potential toxicity of GBN was rarely discussed in previous studies in vivo. Therefore, it is important to understand the real situation of the long-term performance of graphene nanomaterials in vivo for potential clinical application. In the meantime, the PCL degrades slowly in the physiological environment, from several months to years40. Consequently, we investigated the toxicity effect of GBN in a lengthy sciatic nerve defect model over 18 months in vivo. The findings indicate that relatively low concentration and loading of GBN (4% in the PCL scaffold in this study) may be biocompatible as it exerts no appreciable toxicity to the liver, kidney, heart, lung, or spleen in the long-term repair of peripheral nerves in vivo. The toxicity of nanomaterials, especially GBN concerns researchers due to the harm on major functioning organs and consequent hepatic and renal failure. It prevents the possible translational application of GBN materials in future clinical work. Our findings preliminarily confirm the relative biosafety profile of GBN in a long-term murine model.The biological application of GBM has resulted in huge attention on their biocompatibility and tissue toxicity both in vitro and in vivo. Like other carbon-based materials, graphene is not biodegradable under most conditions. However, it may be degraded at the presence of activated, degranulating neutrophils, by erythrophagocytosis in liver Kupffer cells, or by photodegradation in normal (HEK-293) and cancerous (HeLa and HepG2) human cells41.Then, the biologically regenerative effects of GBN were thoroughly investigated for peripheral nerve injury (PNI) because these effects were also representative for evaluating bioactive materials in nerve tissue engineering. Axon and myelin were repaired well after graphene-based scaffold implantation. It implies a certain reparative capacity of GBN in a lengthy nerve defect. Although autologous nerve transplantation is the gold standard for treating injured peripheral nerves, its therapeutic effect is limited on the large defect so that additional alternative therapies are necessary42. The MBP expression level was positively associated with the development of the major dense line, and the compacted myelin sheath failed to form without the anchoring of the lipophilin particles to the myelin sheath43. In addition to myelin maturation evaluation by MBP, Tuj1 is a major neuronal protein and reflects axonal viability and regeneration after sciatic nerve injury44. The myelin sheath enwraps the axon tightly and these two important structures contribute to the healthy and functional peripheral nerve. The MBP and Tuj1 expression levels imply that GBN repair myelin and axons by recreating a pro-healing condition of the injured nerves.MBP is an important marker for myelin formation from SCs in the PNS45. The spinal cord neurogenesis after PNI was measured and indicated by nestin and GFAP expression. Nestin is a class VI intermediate filament protein, which is expressed both in neuronal and glial progenitors and their common precursors46. The nestin-positive cells appear in the spinal cord following different injuries to the local site and distal peripheral nerves. GFAP is a class III intermediate filament protein and is widely expressed in astrocytes in the CNS47. Although previous researches have shown the reparative potential of GBN in CNS and PNS injuries, respectively, this study evaluates the multiple manipulations of central and peripheral neuronal activities by GBN in the severe PNI. The findings indicate a notable GBN-dependent increase in glial expression in the dorsal root ganglion, spontaneous ectopic activity in primary afferent fibers, neuronal sprouting, and rearrangement of axonal termination in the spinal cord after severe PNI. GBN can improve myelin extension and axon outgrowth by inducing a dual activation of central and peripheral neurons after PNI. Traditionally, the activation of astrocytes was considered as a pathological response to injury. However, many studies have shown that the mutual interaction between CNS and PNS is a potential sign of regeneration and is beneficial to neuronal proliferation, migration, and differentiation50. The simultaneous regulation of graphene-based scaffolds on PNS and CNS contributes to a better understanding on regenerative mechanisms of remyelination and axonal restoration.In addition, the feedback of PNI on CNS was also investigated in detail. The spinal cord is directly influenced by PNI and starts to degenerate and form pathological plasticity immediately53. However, their effects could not match the autograft nerves, at either in vitro settings or a relatively short-term in vivo period. In this study, we confirm that GBN may alleviate injury-related environment by restoring nutrient supply and stimulating vessel regrowth within regenerated areas.An ideal microenvironment requires persistent support and transport of nutrients and energy. In PNI, the vessel regrowth and angiogenesis are of primary significance. A number of studies claimed a pro-angiogenic effect of graphene in different types of tissue damage54. The gastrocnemius muscle morphology and viability were extensively analyzed after PNI. In addition, muscle viability and proliferative status were evaluated using fast and slow myosin protein staining. These proteins represent proliferative and apoptotic muscle cells, respectively55. The findings show that muscle structure atrophy is alleviated and muscle viability is increased significantly. As to the electrical transduction evaluation, we evaluated and confirmed improved NCV and DCMAP values from the GBN scaffold, respectively. Based on the results, we think GBN significantly restore the locomotor and electrophysiological functions of the hind limb after severe PNI.The neuronal function is evaluated by both locomotor and electrophysiological activities56. Graphene nanofamily has long been discussed and evaluated as to its potential biological applications, especially in the biomedical field. However, the real biocompatibility of GBN is uncertain57. There are many researches about the in vitro cytotoxicity of GBM in different disease settings. Nevertheless, this cannot mimic the real scenario of disease progression in the living system. Therefore, we performed the preclinical assessment of layered graphene-loaded PCL scaffold in the long-term regeneration of severe PNI in vivo. In addition to confirming potentially low toxicity and immune insult of the implants to the local site and main functioning organs , we further analyzed the regenerative capacity of GBN on rebalancing and restoring a pro-healing microenvironment for injured neurons and peripheral nerves.The appropriate evaluation of the biosafety of biomaterials is of huge significance, because the complex materials are positioned inside living tissues or organs and will stay for a lengthy term in the body. “Biocompatibility” refers to a biomaterial that is compatible with a living system by inducing insignificant toxicity or injuries. In addition, the biomedical scaffold is expected to induce a beneficial cue for tissue–biomaterial reaction in the bodyGBN scaffold implants will be useful in many clinical situations. Their interaction with different vital supporting cells should be analyzed carefully in nerve regeneration. This will help to increase understanding of the mechanisms by GBN-dependent repair. In addition, researchers should put more efforts on evaluating potential biological and biocompatible profiles of GBN in large animal models in the future before clinical practice. In summary, the graphene-loaded PCL scaffold provides a biocompatible and biologically regenerative microenvironment for severe PNI after 18 months in a murine model. It exerts multiple pro-healing effects on myelination and axonal regeneration, possibly via dual regulation and activation of SCs and astroglia in the central and peripheral nervous systems. Furthermore, the graphene-loaded PCL scaffold significantly enhances the motor function of lower limbs and electrophysiological performances for nerve electrical transduction. This study implies that GBN may be advantageous for neuronal regeneration and will be useful for clinically translational application in the future.The layered graphene and PCL were purchased from Suzhou HENGQIU Technologies Co., Ltd. (China) and Perstorp (UK), respectively. The GBN was manufactured using a layer-by-layer casting technique. In brief, the layered graphene nanoparticles and PCL were dissolved in the dichloromethane and were injected onto a tubular mold from the inner to the outer layer. Then, microneedles were applied for adding aligned pores in the scaffold surface.n = 5/group, Shanghai Xipuerbikai Laboratory Animal Company) were used for in vivo evaluation of the biological profiles of GBN. SD rats were randomly allocated into four groups: 4% layered graphene-loaded PCL scaffold, 2% layered graphene-loaded PCL scaffold, PCL scaffold, and autograft. They were kept in the specific pathogen-free animal house and were provided with enough water, food, leisure music, and free activity. The rats were anesthetized using sodium pentobarbital  intra-peritoneally. The right thigh was exposed and the 18-mm sciatic nerve was dissected. The proximal and distal nerve stumps were reconnected by a scaffold or an autologous nerve segment. None of the animals suffered from infection, foot ulcer, death, or other complications after model establishment. Animal care and use were authorized by the Animal Ethics Committee of Shanghai Jiao Tong University .Sprague Dawley (SD) rats , the third toe to the heal (PL), and the second toe to the fourth toe (IT). Experiment side (E) and normal side (N) were compared according to the formula, SFI = (−38.3 × (EPL − NPL) ÷ NPL) + (109.5 × (ETS − NTS) ÷ NTS) + (13.3 × (EIT − NIT) ÷ NIT) − 8.8. SFI values are positively correlated with functional performance. As to sensory performance, withdrawal latency was evaluated using a paw withdrawal apparatus .Electrophysiological evaluation was carried out at 18 months after surgery under proper anesthesia with sodium pentobarbital  of the rats. The bipolar electrodes were fixed at the distal and proximal nerve stumps in addition to another electrode at the belly of the gastrocnemius muscle for EMG under electrical stimulation. NCV was used to measure the conducting velocity between the two electrodes and DCMAP was used to reflect the excitability of the nerve fibers.All reagents were purchased from Sigma unless specified. All rats were sacrificed at 18 months post-injury. The major functioning organs, including heart, liver, spleen, lung and kidney, regenerated sciatic nerve, gastrocnemius muscle, and dorsal root of spinal cord tissues were harvested carefully from each group. After euthanasia using an overdose of sodium pentobarbital  for the rats, we shaved the surgical area and incised the injured site to expose the nerve and the scaffold. Then, we removed the scaffold and the regenerated nerve inside the scaffold by cutting off two ends, harvested the dorsal root of the spinal cord, and then rinsed the samples in the phosphate buffer saline (PBS) three times. Next, we dissected the gastrocnemius muscle from the injured leg with a mosquito clamp and scalpel without tearing muscle fibers, and rinsed the muscle sample in the PBS three times. Afterward, we incised the abdominal and thoracic cavity to separate and fetch the liver, spleen, kidney, lung, and heart carefully, and rinsed the samples in the PBS three times.All reagents were purchased from Sigma unless specified. The sciatic nerves, gastrocnemius muscle, and organs  were fixed in 4% paraformaldehyde fix solution overnight, and then were rinsed, and dehydrated in 75%, 80%, 95%, 95%, 100% and 100% ethanol solution for 1 h, respectively. Then, the samples were waxed at around 56 °C, embedded in paraffin, and sliced in order. The paraffin sections were dewaxed using dimethylbenzene and ethanol, respectively, and then they were immersed in hematoxylin for 8 min, and in 0.6% ammonia before running-water rinsing. Then, sections were stained in the eosin for 3 min and were dehydrated using ethanol before mounting.All reagents were purchased from Sigma unless specified. The sciatic nerves were fixed in 4% paraformaldehyde fix solution overnight, and were rinsed in PBS. Then, sections were dewaxed, and immersed in TB solution for 30 min at 50 °C, and were dehydrated in 75%, 90%, and 100% ethanol solution, respectively. The samples were processed using dimethylbenzene before mounting.All reagents were purchased from Sigma unless specified. The sciatic nerves, spinal cord, and gastrocnemius muscle were fixed in 4% paraformaldehyde fix solution overnight, and then were rinsed, and dehydrated in 75%, 80%, 95%, 95%, 100%, and 100% ethanol solution for 1 h, respectively. Then, the samples were waxed at around 56 °C, embedded in paraffin, and sliced in order. Then, the slices were blocked by 5% bovine serum albumin  and incubated with primary antibodies overnight and secondary antibodies for 2 h at room temperature, respectively. Primary antibodies included anti-GFAP , anti-nestin , anti-CD34 , anti-VEGF , anti-Tuj1 , anti-MBP , anti-fast type myosin , and anti-slow type myosin .All reagents were purchased from Sigma unless specified. The ultrathin sciatic nerve samples were immersed in 2.5% glutaraldehyde solution for 2 h at 4 °C, rinsed in PBS for three times, and fixed in 1% osmic acid for 2 h. Then, the samples were rinsed in PBS again and underwent dehydration by acetone  for 1 h. After that, the samples were embedded in Epon812 at 37 °C overnight. Finally, after double staining with 3% uranium citrate, the ultra-structure of sciatic nerve sections was evaluated.Sections for HE and TB assays were observed under an optical microscope  and random vision fields were pictured from evaluation. Sections for immunofluorescence assays were observed under an immunofluorescence microscope  and random vision fields were pictured from evaluation. The ultra-structure of sciatic nerve sections was observed under a transmission electron microscope  at 80-kV voltage. Different magnification was chosen for observation, ×1000, ×3000, and ×8000 by selecting random vision fields. The number and area of myelinated axons were observed by HE and TB staining and calculated by identifying positive areas using image pro plus 6.0 software. The diameter of the myelinated fiber and myelin sheath thickness were observed by TEM calculated by identifying axon fiber and myelin sheath structures using image pro plus 6.0 software. The average muscle fiber area was observed by HE staining and calculated using image pro plus 6.0 software. Relevant expression levels of different proteins were calculated based on the immunofluorescent intensity measured using image pro plus 6.0 software.p < 0.05 by one-way analysis of variance (ANOVA) was statistically significant in this study.The statistical analysis was carried out by GraphPad 7.0 Software ; the results were displayed as the mean ± standard deviation. A Further information on research design is available in the Reporting Summary


Intercalation experiments on epitaxial graphene are attracting a lot of attention at present as a tool to further boost the electronic properties of 2D graphene. In this work, we studied the intercalation of Pb using buffer layers on 6H-SiC(0001) by means of electron diffraction, scanning tunneling microscopy, photoelectron spectroscopy and in situ surface transport. Large-area intercalation of a few Pb monolayers succeeded via surface defects. The intercalated Pb forms a characteristic striped phase and leads to formation of almost charge neutral graphene in proximity to a Pb layer. The Pb intercalated layer consists of 2 ML and shows a strong structural corrugation. The epitaxial heterostructure provides an extremely high conductivity of  The outstanding optical and electronic properties of graphene were intensively studied in the past ,2. The nIn this respect, epitaxial graphene grown by sublimation on SiC is an interesting system. Currently, almost defect-free graphene is grown on semi-insulating SiC(0001) substrates, such that quantum transport experiments are directly performed without the need for any complex transfer processes ,8,9,10. Intercalation of heavy atoms are expected to introduce spin-orbit coupling (SOC) to the 2D electron gas of graphene. In case of Au intercalation, the SOC was seen for the Au-induced interface states, but not in graphene . For epiIn contrast, Pb monolayer and submonolayer structures on Si surfaces were intensively studied in the past. For densely packed Pb monolayer structures, the so-called striped incommensurate phase (SIC)  and the ctively, . The depctively, ,28,29,30Recently, the Pb intercalation of buffer layer (BL) structures on SiC(0001) came into the focus of research . DFT calectively .In this work, we studied the Pb intercalation of buffer layer structures on 6H-SiC(0001). In agreement with previous investigations, we also found striped phases, however, formed with a considerably larger amount of intercalated Pb. Angle resolved photoemission clearly revealed almost charge neutral graphene coming along with an extremely low resistivity.Buffer layer (BL) samples were prepared by heating nitrogen doped 6H-SiC(0001) substrates (Pam Xiamen Semiconductor). After wet-chemical cleaning the substrates were first flattened by hydrogen etching in a 1000 mbar hydrogen atmosphere at a temperature of 1425 out 3 µm .−1 onto the BL sample kept at 200 In order to obtain percolated phases after intercalation, multiple cycles of adsorption and subsequent annealing were mandatory. First, 5 monolayers (MLs) of Pb  were deposited at a rate of 1/3 ML minα radiation (1486.6 eV) was used and the photoelectrons were analyzed with a Specs Phoibos 150-MCD analyzer. ARPES measurements were performed with HeII radiation (40.82 eV) using a Specs UVS 300 He-lamp in conjunction with a Specs TMM304 monochromator and a Specs Phoibos 150 analyzer with a 2D-CCD-detector. The sample investigated by XPS and ARPES had to be exposed to air. Annealing at 320 °C in UHV resulted in the desorption of contaminants, e.g., oxygen.The structure of the samples was controlled by scanning electron microscopy (SEM), high resolution low energy electron diffraction (SPALEED) and scanning tunneling microscopy . Although the intercalated samples turn out to be reasonably stable against oxidation, i.e., all structural and spectral features are fully recovered after mild annealing in vacuum, the samples were, when possible, transferred between the UHV systems  . IronicaE and in our case for a SiC step height of The samples were sputtered at ion energies of 1 keV, at a emission current of 10 µA for 10 min at a Ar-pressure of 4 × 10 d=2.5 Å . As mentBy covering the BL/SiC sample with a shadow mask (Si wafer), as sketched in STM measurements were performed on the high-intercalation sample, discussed in context of ions cf. b–d. As iions cf. d. This pThe average distance of these stripes is 2.3 nm and fits nicely to the additional diffraction spots at around 13% SBZ, discussed in context of In ref.  the authIn order to estimate the Pb coverage and check the bonding at the interface XPS measurements were performed. All measurements were made after an additional annealing step. This annealing step was mandatory for cleaning the surface, however, at the expense of a slight desorption of intercalated Pb (around 10%).Two further components can be seen in the C1s spectrum. The asymmetrical component labeled G (284.5 eV) is an evidence of successful intercalation. Such an asymmetrical component was also observed in case of the formation of a QFMLG via hydrogen intercalation ,15,47. TC (101.8 eV) belongs to the Si atoms of the substrate which are bonded to the BL. Two further components can be seen, whereby the component SiC’ (100.4 eV) refers to Si atoms of the SiC substrate beneath intercalated areas of the sample. Finally, the component SiPb (99.8 eV) can be attributed to the Si atoms which are bond to the intercalated Pb atoms. The energy shift between the SiC and SiC’ components of both, the Si2p and the C1s spectrum, is comparable. Furthermore, the intensity ratio of the SiC’ and SiC component is the same for the C1s and the Si2p spectrum.A closer look to the Si2p core level in 7/2-state which is in good agreement of the bulk value measured for pure Pb [ pure Pb . The con pure Pb . From STDE is less than 50 meV above the the Fermi level FE, yielding a low charge (p-type) carrier density of FE, thus reminiscent of n-type doped monolayer graphene (MLG) [In addition, the Pb intercalation was characterized also by ARPES around the K-point of graphene cf. d. Apparene (MLG) . The BL p in graphene, determined by ARPES (see above), is attributing to the transport. For epitaxially grown graphene on Ge/Si(100) similarly high charge carrier mobility values were found [Moreover, in situ four-point probe surface transport experiments were performed. As shown in re found . Despitem2V−1s−1 ,50. NeveRotational square measurements, shown in panel b performed with an average tip distance of 100 µm reveal an extremely low anisotropy  substrate is electrically insulating at these temperatures [In fact, a material . For an eratures ,52, thus as well ,54. AppaTemperature dependent transport measurements for a fixed collinear probe geometry with stem cf. e. The AnkBD(EF)) ,56, wherAs seen, charge neutral graphene is formed where intercalation takes place. Thereby, Pb grain boundaries are easily overgrown by graphene, resembling an extended band transport channel in parallel to a hopping channel across the Pb islands. In contrast, extended BL areas in between are electrically insulating, thus providing no transport channels. At 300 K we found a highly conductive path for electrons across the surface. Thereby, most likely both channels along the graphene and Pb islands are involved. The Anderson temperature for the fit shown in 2, i.e., at lower temperatures the proximity coupling may become more severe.The low temperature regime below 70 K can not be captured by disorder along the intercalated path. The Arrhenius analysis in this temperature regime reveals an activation energy of around 10 meV, as shown in In summary, we have comprehensively studied the intercalation of Pb on BL structures on SiC(0001). Triggered by structural defects, up to 70% of the areas was intercalated coming along with the formation of epitaxial graphene on top. In contrast to previous studies, the amount of Pb is around 2 ML, thus the striped phase resemble a structural modulation of the interface rather than a Moiré effect. By means of in situ surface transport the conductivity within these films were determined. Assuming only transport across the graphene, the charge neutrality of the delaminated graphene would come along with charge carrier mobilities of around


The corresponding sensor exhibits a greatly improved humidity sensitivity (~34.3 Hz/%RH) compared with the original pure GO-based QCM sensor (~4.0 Hz/%RH). In addition, the sensor exhibits rapid response/recovery times (7 s/6 s), low hysteresis (~3.2%), excellent repeatability and good stability. This research is conducive to understanding the mechanism of GOM-based humidity sensors. Owing to its good humidity-sensing properties, the HGO&GO&Mg2+ membrane-based QCM humidity sensor is a good candidate for humidity sensing.The application of graphene oxide (GO)-based membranes combined with a quartz crystal microbalance (QCM) as a humidity sensor has attracted great interest over the past few years. Understanding the influence of the structure of the GO membrane (GOM) on the adsorption/desorption of water molecules and the transport mechanism of water molecules in the membrane is crucial for development of applications using GOM-based humidity sensors. In this paper, by investigating the effects of oxygen-containing groups, flake size and interlayer spacing on the performance of humidity sensing, it was found that humidity-sensing performance could be improved by rational membrane-structure design and the introduction of magnesium ions, which can expand the interlayer spacing. Therefore, a novel HGO&GO&Mg There is an increasing demand for high-precision and sensitive real-time humidity detection, which is important not only for traditional industries, such as agriculture , grain sThe materials coating a QCM are the key to determining the detection performance of QCM-based humidity sensors ,11. Vari2+ can not only prevent GO from dissolving but also expand the interlayer spacing of GO. Our results show that the introduction of magnesium ions remarkably improved the sensor performance of GO- and HGO-based humidity sensors. In addition, a reasonable composite membrane structure (composed of HGO and GO) can further balance the humidity sensitivity and response/recovery rate. Eventually, the obtained structure (HGO&GO&Mg2+) exhibited excellent humidity-sensing properties, showing vast potential for practical applications in the area of humidity sensors.In this work, considering the complex structure of GO ,25, it i2+-based QCM sensors, characterization was performed by atomic force microscopy (AFM), scanning electron microscopy (SEM), energy dispersive X-ray (EDX) spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). Humidity-sensing properties, such as sensitivity, repeatability, stability and response/recovery time, were investigated by a QCM. In addition, to understand the mechanism of the high-humidity-sensing performance of HGO&GO&Mg2SO4), potassium persulfate (K2S2O8), phosphorus pentoxide (P2O5), potassium permanganate (KMnO4), hydrogen peroxide (H2O2), magnesium chloride (MgCl2) and hydrochloric acid (HCl) were used in this research. All chemical regents were purchased from Sinopharm Chemical Reagent Co.  and used with ultra-pure deionized (DI) water with 18.2 MΩ·cm−1 in all experiments. All the reagents were utilized as received, without further purification.Graphite powder (325 mesh), sulphuric acid  water. After vacuum drying at 60 °C overnight, oxidized graphite was obtained. The obtained oxidized graphite was further oxidized in concentrated H2SO4 and KMnO4 at constant temperatures of 6 and 60 °C, respectively. The solutions were stirred at 35 °C for 2 h, and then they were diluted with 250 mL DI water at 40 and 80 °C, respectively. The graphene oxide prepared at higher temperatures (oxidized at 60 °C and adding DI water at 80 °C) was named HGO, while the other one was named as GO. The solutions were further stirred at room temperature for 2 h, and H2O2  was added immediately after dilution with DI water (700 mL). The product was centrifuged and sequentially washed with a 1:10 aqueous HCl solution and DI water to remove impurities. Finally, the GO and HGO aqueous suspension with a concentration of about 5 mg/mL was prepared. The mixed solution of GO and HGO (GO&HGO) suspension was obtained by mixing equal volumes of HGO and GO solutions with same concentration (5 mg/mL).GO aqueous suspension (5 mg/mL) was prepared according to the modified hummers method, as previously reported ,28. Grap−1 onto the Pt surface of the QCM electrode and drying at 70 °C for 2 h, as illustrated in 2 at 25 °C for 2 h and was measured after drying at 70 °C for 1 h to improve the adhesion properties of the GOM on the Pt electrode.QCM devices  consisting of AT-cut quartz crystals (8 mm diameter) with a fundamental resonance frequency of 9 MHz and Pt electrodes  on both sides were employed in this research. Before the preparation of thin sensing membranes, all QCM devices were washed successively with diluted hydrochloric acid, deionized water and ethanol, and each process was followed by a drying procedure at 70 °C for 30 min. The GO, HGO and GO&HGO sheets were deposited by drop-casting 10 µL of the corresponding suspension with a concentration of 0.1 mg mL3COOK, MgCl2, K2CO3, Mg(NO3)2, NaCl, KCl and K2SO4 at 25 °C provided 11%, 23%, 33%, 43%, 52%, 75%, 85% and 97% RH levels [0) of the sensor exposed to 11% RH was used as the baseline to evaluate the performance of the QCM sensors. The recording periods for response and recovery times were fixed at 30 s.The schematic of the experimental system used for relative-humidity (RH)-sensing measurement is illustrated in H levels , respect0 are the response frequency at the corresponding tested RH level and 11 %RH, respectively. The humidity hysteresis is an important criterion for practical application, defined as:max is the maximum hysteresis error and Ftotal is the total response-frequency-shift output from minimum humidity level (11%) to maximum humidity level (97%).The response-frequency shift was defined as: −1 using a Nicolet iS50 FTIR spectrometer , and the XPS results of free-standing GO and HGO membranes were characterized by a Thermo Fisher ESCALAB Xi+ . XRD patterns of membranes were obtained by an X-ray diffractometer system . XRD spectra of membranes under different relative humidity levels were tested on a glass substrate with a cylindrical groove in the middle. Saturated solutions of different salts were added to the groove, as previously described. These saturated solutions can control the humidity. Meanwhile, the sample was placed on a glass supported by a rubber cement above the liquid level. The whole groove was sealed with plastic wrap during the entire test process. GO and HGO flakes were measured by atomic force microscopy . SEM images were collected using a Gemini SEM 500  series field-emission scanning electron microscope operated at an accelerating voltage of 15 kV.The FTIR spectra were collected in the mid-infrared range of 4000–1000 cm2+. It can be clearly seen that the framework is composed of GO and HGO flakes with different lateral sizes. The AFM images in 2+, HGOM&Mg2+ and HGO&GOM&Mg2+ are very similar to those of GOM, HGOM and HGO&GOM, it is clear that magnesium was uniformly distributed in these membranes, according to EDX spectroscopy  groups, carboxyl (C=O) groups, aromatic ring (C–C/C=C) and epoxy (C–O–C) groups [−1, 1730 cm−1, 1622 cm−1 and 1116–1228 cm−1, which shows that the oxygen content of the former is lower than that of the latter. It can be concluded that a high reaction temperature is beneficial for further intercalation of oxidized groups, which also promotes the exfoliation of graphite oxide into smaller GO sheets. This is identified with the results of AFM. The number of oxygen-containing groups in GO has a significant impact on the humidity sensitivity of GO-based humidity sensors . To expl) groups , respect2+ under different humidity levels  are 7.5 Å, 7.9 Å, 8.5 Å, 9.7 Å and 11.7 Å, respectively. It can be easily observed that the interlayer spacing of HGO&GO&Mg2+ increases with increasing humidity. The interlayer spacing at 97% RH is ~4.2 Å greater than that at 11% RH, while the interlayer spacing of HGO&GOM completely immersed in MgCl2 solution is 15.3 Å. According to a previous report, the interlayer spacing of GO controlled by MgCl2 solution is 13.6 Å, which is less than that of the HGO&GO controlled by MgCl2 solution obtained in this work. From the full-width half maximum (FWHM) of the characteristic peak, MgCl2-solution-controlled GOM exhibits a greater degree of lamellar orientation, which is consistent with our previous report [2-solution-immersed HGO&GOM. In summary, HGO&GO&Mg2+ possesses a large interlayer spacing and a greater degree of lamellar orientation, which may be beneficial for the adsorption capacity of water molecules and the transport rate of water molecules inside the membrane. It is easily understood that the interlayer spacing of GOMs may have a direct influence on their ability to adsorb water molecules . As shows report . Since t2+- and HGO&Mg2+-based sensors both exhibit significant strengthening in response, ΔF, compared with the GO- and HGO-based sensors. Although the response, ΔF, of the GO&Mg2+-based sensor (19.6 Hz/%RH) is not as high as that of the HGO&Mg2+-based sensor (52.7 Hz/%RH), the former still increased to approximately 10 times that of the original GO-based sensor. Because the size of GO flakes is too large and the oxygen-content is low, it is difficult for water molecules to enter the deep area when GO is not completely wet. Therefore, the interior of GO membranes may not be fully utilized. The introduction of Mg2+ increases the interlayer spacing of the GO and makes it easier for water to enter, while this has a relatively smaller effect on HGO. This is in agreement with the XRD results. In addition, the evenly distributed Mg2+ ions have a certain degree of hydrophilicity [The response-frequency shifts (ΔF) of the GO- and HGO-based sensors with increasing RH levels in the range of 11–97% RH are shown in hilicity ,36,37, wres and trec) in this work is defined as the time for the response, ΔF, to achieve 90% of the total ΔF amount between two different humidity levels, respectively, during the process of adsorption and desorption. As shown in 2+-based sensor presents a shorter tres/trec (5 s/3 s) than the HGO&Mg2+-based sensor (11 s/10 s). The reason is that on one hand, the response, ΔF, of GO is relatively smaller, and on the other hand, the large area of sp2 hydrophobic channels in the GO membrane can help oxygen-containing groups and Mg2+ ions more quickly absorb and desorb water molecules [2+ can greatly improve the response-frequency shift and GO with Mg2+ can decrease response/recovery time of the QCM sensor, respectively, a more practical sensor may be prepared by combining HGO, GO and Mg2+.To assess the dynamic response performance of the sensors, the response and recovery times between 11% RH and 97% RH were also measured. Response and recovery time  decreased compared to that of HGO&Mg2+ (52.7 Hz/%RH), while the tres and trec of the former were reduced to 7 s and 6 s, respectively. 2+ can effectively limit the swelling of GO [The response, ΔF, humidity hysteresis and tng of GO . These r2+-based QCM sensor is better than most GO-based QCM sensors not only in terms of sensitivity but also in response/recovery time. The HGO&GO&Mg2+-based QCM sensor is also excellent, even compared to other materials used for humidity sensors.2+-based sensor for different humidity levels: 11% to 33% RH and 52% to 97% RH. There is no clear variation in the response, ΔF, under the same humidity conversion conditions, suggesting that the HGO&GO&Mg2+-based sensor has excellent repeatability. In addition, as observed in 2+-based sensor at three humidity levels  was tested every two days for 10 days, and a minimal variation in the response, ΔF, appeared at different humidity levels. This obviously means that the HGO&GO&Mg2+-based sensor has good stability.Stability and repeatability are two important criteria to measure the practical value of humidity-sensor performance. 2+-based sensor represents a remarkable enhancement compared with that of GO and HGO. Previous work [2+ can further enlarge the interlayer spacing of HGO&GOM in different humidity states. Moreover, the interlayer spacing at 97% RH is ~4.2 Å larger than that at 11% RH, which means that HGO&GO&Mg2+ can attract and hold more water molecules. Compared with that of GO or HGO, the reason why the humidity-sensing performance of the HGO&GO&Mg2+-based sensor represents a remarkable enhancement can be summarized by four aspects:The introduction of magnesium ions can increase the interlayer spacing of GOMs, which is beneficial for attracting and holding more water molecules. This can be proven by XRD results.2+ can prevent the swelling of GOMs [Mg of GOMs , which i2+ has a certain degree of water-absorption capacity [Mgcapacity ,36,37. M2 hydrophobic regions in the large layer of GO is conducive to the rapid transmission of water molecules inside the membrane. These are both conducive to improving humidity-sensing performance. In short, a mixed structure of GO and HGO has the ability to balance response sensitivity and response/recovery time.For the composite structure of GO and HGO, on one hand, a large number of oxygen-containing groups in HGO provides many adsorption sites for water molecules; on the other hand, a large number of spAccording to the aforementioned experimental results, the humidity-sensing performance of the HGO&GO&Mgous work  has repo2+ and a reasonable structural design. The obtained HGO&GO&Mg2+ sensor exhibits a high response-frequency shift (34.3 Hz/%RH), short response/recovery times (7 s/6 s), low humidity hysteresis (3.2%) from 11% RH to 97% RH and good repeatability/stability. These two improved strategies are based on the fact that Mg2+ not only expands the interlayer spacing of GOMs but also attracts more water molecules into the interior of GOMs, while the unique structure composed of HGO and GO still ensures a rapid response/recovery time when humidity sensitivity is vastly improved. Most importantly, the findings in this work contribute to further understanding of the transport mechanism of water molecules in GOMs, which promotes the application of not only QCM humidity materials but also fuel-cell, water-desalination and nanofluidic devices.In summary, the humidity-sensing properties of GO-based humidity sensors can be significantly enhanced by introducing Mg


In order to influence the adhesion properties of graphene, the wettability properties of the target substrates were investigated in this study. To increase the adhesion of the graphene on the isolating surfaces, they were pre-treated with oxygen plasma prior the transfer process of graphene. The wetting contact angle measurements revealed the increase of the hydrophilicity after surface interaction with oxygen plasma, leading to improved adhesion of the graphene on 200 mm target wafers and possible proof-of-concept development of graphene-based devices in standard Si technologies.One of the limiting factors of graphene integration into electronic, photonic, or sensing devices is the unavailability of large-scale graphene directly grown on the isolators. Therefore, it is necessary to transfer graphene from the donor growth wafers onto the isolating target wafers. In the present research, graphene was transferred from the chemical vapor deposited 200 mm Germanium/Silicon (Ge/Si) wafers onto isolating (SiO Dry graphene transfer is based on the employment of the intermediate polymer layer (e. g. PDMS stamp10) or by usage of release tape11 to avoid the direct contact of graphene with the wet solution; however, this method suffers from polymer residuals and defects like cracks, folds, and wrinkles after the transfer procedure due to the strong graphene-substrate interaction. In order to weaken it, several treatments have been investigated in the literature to ease the release of graphene from the donor wafer. Intercalation at the graphene-metal interface has also been studied in order to tune the interaction strength and to oxidize the metal surface below graphene before the graphene transfer process. For example, water intercalation12 process, where water penetrates into the graphene-metal interface for easier graphene transfer from the donor to target wafer was reported. The disadvantage of such a method is long interaction times (16 h to up to 3 days) and low process control for wafer-scale graphene transfer. On the other hand, the intercalation with carbon monoxide13 or chemical modification14 of the graphene-metal interface to achieve higher transfer speeds has also been reported in the literature.One of the limiting factors of graphene integration into electronic and photonic devices is the unavailability of large-scale graphene, directly grown on isolators. Therefore, for the development of graphene-based devices, graphene transfer process from the growth wafer (donor) onto the isolating (target) wafer needs to be performed. Different graphene transfer methods have been described in literature, which can be classified as dry15. The alternative method of chemical etching is the wet electrical delamination process, where graphene is released from various growth substrates16. This method is based on the water electrolysis process, where H2 formation at the Gr-metal interface detaches graphene from the growth wafer17 and is a faster method compared to the chemical etching process. However, the electrical delamination process also involves chemicals  and poly(methyl methacrylate) (PMMA) as a support layer for graphene transfer procedure; therefore, the precise cleaning and polymer removal steps have to be understood and optimized in order to prevent the extrinsic doping of graphene. The optimized delamination process itself does not guarantee the successful transfer process, as surface contamination, hydrophilicity and roughness of target wafers also may influence the adhesion properties and respectively quality of transferred graphene.Another group of graphene transfer methods is based on chemical etching of the catalytic substrates on which graphene is grown. The main problem of chemical etching is metal cross-contamination, which alters the electrical properties of the transferred graphemex18. At this point, the hydrophilicity-hydrophobicity of the target wafers is one of the most critical parameters for the adhesion of 2D graphene; however, only very few literature works deal with this phenomenon. Kim et al.19 showed the relation between the SiO2 surface wettability and graphene quality transferred by wet transfer to reduce the content of wrinkles in graphene after the transfer on the target wafer. However, the organic solvent (instead of DI water) was used in their work, making the transfer process more complex. Another study involved aluminum nitride (AlN) surfaces, as they were investigated as a target wafers for graphene transfer20. Different surface treatments lead to increased surface wettability as detected by measured wetting contact angle (WCA) measurements between solid surface and water drop. This clearly shows the need for further investigations and understandings of the relation between surface wettability properties and therefore the adhesion of graphene on the target wafers.Indeed, the 2D nature of graphene, where the ratio between the surface area to volume area is increased, leads to strong surface interactions affecting the adhesion capability compared with 3D materials. S. Das et al. reported the adhesion energy increase after thermal annealing procedures to improve the adhesion of graphene on SiO2/Si and Si3N4/Si target wafers by changing their hydrophilic behavior with an additional surface treatment to achieve low-defect and up-scaled graphene transfer process. The electrochemical delamination process was selected since wafer-scale graphene transfer through wafer bonding or etching of Ge under graphene is still not achieved for Gr/Ge/Si system. In the graphene growth-transfer process, no metals  were involved; therefore, metal cross-contamination problems were avoided. In the present research, the surface wetting contact angle measurements of the target wafers and surface composition and roughness measurements by XPS and AFM were performed, respectively. The obtained results allowed us to determine the relationship between surface composition, roughness, wettability, and graphene’s adhesion ability on the target wafers with respect to additional surface treatment by oxygen plasma. Finally, the improved graphene adhesion was achieved for possible future fabrication of proof-of-concept graphene-based devices in standard Si technologies.In this paper, graphene was transferred by the electrochemical delamination process from 200 mm Ge/Si donor wafers on 200 mm, Si technology standard SiO2  and one type of Si3N4 (PE-Si3N4) films were employed as target surfaces on standard Si wafers. To be precise, HDP stands for high-density plasma deposition using silane (SiH4) precursor at 650 °C deposition temperature, TEOS is the deposition of SiO2 by using the tetraethyl(ortho)silicate (TEOS) precursor and plasma at 400 °C, whereas thermal SiO2 is produced at a temperature of 1000 °C. Si3N4 films were grown by plasma enhanced (PE) chemical vapor deposition at 400 °C by using silane and NH3/N2 gasses as precursors. For all SiO2 and Si3N4 target surfaces the graphene transfer and post-transfer processing steps were identical in order to observe the effect of target surface and dependences with respect to certain changes.In the present research, four different target surfaces were selected for the transfer experiments of graphene. Three types of SiO2, Si3N4 target surfaces was investigated by measuring the wettability contact angle (WCA) with the sessile drop method21. By this method, the interaction and spreading of a small volume (1µL) water droplet on a solid surface is investigated. The contact angle (θ) for a flat surface is described by Young equation22 cos (θ) = (γSV−γSL)/γLV, where (γ) is the surface tension of solid–vapor (SV), solid–liquid (SL) and liquid–vapor (LV) interfaces, respectively. Theoretically, surface is hydrophilic if the contact angle between the solid surface and water is below 90°. On the hydrophilic surfaces, water tends to spread out on the material surface due to higher adsorption as binding energies between water molecules. The observed contact angles revealed the hydrophilic behavior of SiO2 and Si3N4 surfaces independently on the deposition conditions—contact angles were below 90°  was detected, related to high process temperature (1000 °C) and therefore higher stability towards adsorption from the ambient  doping of the surface. In the case of thermal-SiO2, the functional –C=O group was completely removed from the surface after plasma treatment , and it seems that the surface of thermal oxide is more stable compared with other oxides, leading to less C-contaminated surface and lower surface modification after oxygen plasma treatment at 250 °C. However, the plasma effect on HDP- and TEOS-SiO2 surfaces were rather similar and revealed a slight reduction in surface contamination.No differences in Si 2p spectra were noticed for differently grown SiOlms Fig. . For oxilms Fig. a, c, e.  SI Fig. c. The el SI Fig. e. The obiO2 Fig. f.. In thtra Fig. d. To con3N4/Si surface. In the characteristic C1s spectra, higher C–O peak was observed for as-grown S3N4 films  the substitution of oxygen and depletion of nitrogen at the surface of LP-CVD silicon nitride films after the exposer to oxygen plasma. Like in the SiO2 case, the plasma treatment leads to a slight reduction of C-contamination on the film surface . No change of 2D/G (~ 1.8) ratio was noticed for plasma treated surfaces, indicating no doping or quality reduction after plasma treatment of investigated surfaces before graphene transfer.Additionally, the XPS analysis was performed to determine if Ge cross-contamination and electrolyte residuals (NaOH) appeared after the graphene growth and transfer on the target wafer, respectively. No characteristic Na 1s and Ge 2p peaks were observed in XPS spectra for all investigated transferred graphene samples SI Fig. , or the 2/Si and Si3N4/Si surfaces. The as-grown SiO2 films revealed very low surface roughness (RMS) equal to 0.14 ± 0.02, 0.17 ± 0.02, and 0.22 ± 0.04 nm for HDP-SiO2, thermal-SiO2, TEOS-SiO2 films, respectively. In literature, the RMS values vary from 0.4 to 1.9 nm for as-grown oxide films26, depending on the deposition method. The surface roughness of as-grown Si3N4 films was equal to 0.73 ± 0.06 nm  and confirms the relative surface stability towards oxygen plasma.The measured WCA was equal to 2.4°, 2°, and 2.7° degrees for HDP-, TEOS-SiO2 and Si3N4 surfaces through bombardment with active radicals and particles generated through oxygen plasma . Secondly, in the case of Si3N4, oxygen from the plasma substitutes and exchanges nitrogen in Si3N4 films, leading to the partially oxidized surfaces. As determined by XPS characterization, oxygen plasma treatment leads to surface cleaning and activation, resulting in increased surface reactivity. The surface contamination reduction resulted in the increased number of dangling bonds on the surface and, therefore, higher surface energy in comparison with as-grown surfaces, increasing surface hydrophilicity of investigated SiO2 and Si3N4 surfaces. With increased target surface hydrophilicity the interaction between graphene carbon atoms and the target wafer atoms increases through van der Waals bonding.According to the obtained experimental results, a simple graphical explanation, as possible surface interaction and modification model after interaction with oxygen plasma, is visualized in Fig. 2 surface is presented; however, the same results were obtained for low WCA plasma-treated TEOS-SiO2, PE-Si3N4 surfaces. The ability to transfer graphene on the blanket and structured 200 mm wafers  and plasma-enhanced (PE) CVD processes were developed, allowing depositions also at low temperatures (below 400 °C) suitable for back-end-of-line (BEOL) in integrated circuits (IC) fabrication. Four corresponding target surfaces  were selected for transfer experiments of graphene. HDP-SiO2 layers were deposited at 650 °C deposition temperature using SiH4 as precursor. TEOS-SiO2 films deposited at 400 °C by PE-CVD using tetraethyl(ortho)silicate (TEOS) precursor. Thermal-SiO2 was produced at a temperature of 1000 °C. The Si3N4 films were grown by PE-CVD at 400 °C using silane and NH3/N2 gasses as precursors. The thickness of SiO2 films was measured to be 100 nm, whereas Si3N4 films were 40 nm thick. The wetting contact angle (WCA) measurements to determine hydrophilicity of SiO2/Si, Si3N4/Si surfaces were performed by the “Surftens Automatik” technique at 20 °C temperature. The 49-point measurements were performed pro wafer and 4–6 wafers were investigated for each of investigated as-grown and plasma treated wafer surface. The oxide and nitride surfaces were treated with oxygen plasma for 25 s at 250 °C at 900 W in order to reduce the WCA. The roughness of the target surfaces was investigated by Park Systems NX20 atomic force microscopy (AFM) in the non-contact mode by using the high aspect ratio silicon tips (AR5-NCHR 10 M), operating at 330 kHz resonance frequency and having high operation stability with outstanding sensitivity and fast scanning ability. The surface composition/contamination of SiO2 and Si3N4 films were investigated by XPS  analysis. After deposition or/and transfer processes, the samples for XPS analysis were taken to the XPS measurement chamber within 20 min. Before starting the measurements, samples were annealed in 10–7 vacuum for 20 min. Raman measurements were performed by Renishaw system with a 532 nm Nd:YAG laser, 100X objective and a laser spot size of 1 µm.The SiO28. Afterward, the monolayer graphene was grown on 200 mm Ge/Si wafers by CVD technique (Aixtron Black Magic BM300T CVD) at 885 °C temperature and 700 mbar pressure, using CH4 and Ar/H2 as a source and carrier gasses, respectively29. The poly(methyl methacrylate) (PMMA) polymer was spin-coated on top of the grown graphene to enable and support the graphene during the wet transfer procedure. For the spin coating the E-Beam Resist PMMA 950 K (Allresist) was used and coated with 4000 rpm rotation speed resulting in ca. 400 nm thickness of PMMA on the Gr/Ge/Si stacking.Prior to the deposition of graphene, 2 µm thick Ge(100) films were grown epitaxially on 200 mm Si(100) wafers2 formation on the cathode (PMMA/graphene/Ge/Si) as the results of water electrolysis process. The delaminated PMMA/graphene stack was transferred onto isolating SiO2/Si and Si3N4/Si substrates. Post-transfer processing of the transferred graphene (PMMA/graphene/SiO2(Si3N4)/Si) was performed at 135 °C temperature for about 13 h in an inert atmosphere for water elimination from graphene-target wafer interface. The PMMA was dissolved by immersing PMMA/Gr/Target-Wafer into acetone, followed by thermal annealing at 400 °C in an inert atmosphere for 5 min, in order to remove the resist from the transferred graphene surface.The 200 mm wafers with PMMA/Gr/Ge/Si were cut into different pieces in the sizes from 4 × 8 cm till 13 × 13 cm before the delamination process. The size of transferred graphene flake of 13 × 13 cm is only limited by our equipment, not graphene itself, therefore full 200 mm wafer covered with graphene can be transferred by electrochemical delamination process. PMMA/graphene stack was delaminated from donor Ge/Si wafer by electrochemical delamination process in NaOH electrolyte solution, where the PMMA/graphene/Ge/Si was used as cathode and graphite plate as anode Fig.  in the e2 and Si3N4 surfaces, used as target wafers, were investigated. The exposure of SiO2/Si and Si3N4/Si surfaces to oxygen plasma revealed a decrease of C-contamination on the surface and the decrease of contact wetting angle down to ~ 2° for HDP, TEOS-SiO2, and PE-Si3N4 films on Si wafers. Due to the increase of surface hydrophilicity, the large-scale graphene transfer was improved, resulting in homogenous graphene flakes (from 4 × 8 cm till 13 × 13 cm size) transferred on 200 mm HDP-, TEOS-SiO2/Si and PE-Si3N4/Si target wafers for reliable and reproducible graphene integration in Si-based technology platform.In the present research, the relation between surface morphology, composition, and contact wetting angle of CVD grown SiOSupplementary Information.


Proechimys may represent a strong indication of their role in karyotype diversity of this speciose Neotropical rodent.Signal distribution of L1 and B1 among chromosomes in five species of  Proechimys from different regions of the Amazon. Karyotype comparisons were performed using fluorescent interspecific in situ hybridization. The L1 and B1 retroelements showed a non-random arrangement and a conserved pattern when the genomes of the five species of Proechimys were compared, including the two cytotypes of Proechimys guyannensis. The signal homeology among the chromosomes and the degree of similarity among the formed clusters indicate rearrangements such as fusion/fission, and demonstrates that these retroelements can behave as derived characters shared in Proechimys. The differentiated distribution and organization of these retroelements in the karyotypes and in the chromosomal fiber, respectively, may represent a strong indication of their role as generating sources of karyotypic diversity in the genus Proechimys and provide insights into the evolutionary relationships between taxa.This study aimed to understand the impact of LINE-1 and SINE-B1 retroelements on the architecture and karyotypic diversification of five rodent species of the genus  Transposable elements (TEs) are repetitive nucleotide sequences that are dispersed throughout the genome in euchromatin and heterochromatin regions. This distribution varies according to the taxon and can represent 3–50% of the genome depending on the species .Drosophila melanogaster and Aedes aegypti  and SINE-B1 (B1) are repetitive, interspersed gene sequences that represent a predominant part of the genome of humans and mice . L1 compL1 and B1 comprise about 18% and 2.7% of the mouse genome, respectively. Functional studies in cultured cells of mice revealed 3,000 L1 sequences that are compatible with retrotransposition. However, the number of active B1 sequences remains unknown because of the difficulty in identifying subfamilies given the short length of the sequences .In addition to activity in dynamic, sometimes deleterious processes, such as transcriptional regulation, epigenetic control, cell differentiation, and reprogramming, retrotransposons have also been associated with the structuring and organization of chromosomes . LINEs, Proechimys is a speciose and chromosomally variable Neotropical rodent. The taxonomic identification of Proechimys species is problematic due in part to inter- and intraspecific morphological overlaps, with many species presenting subtle variations in their characteristic morphological traits  allowed us to visualize the distribution of the retrotransposable elements LINE-1 and SINE-B1 in each chromosomal pair and comparison of the pattern of these retroelements between species  PL and PC, (2) PS1 and PS2, (3) PE, and (4) PG (Proechimys ssp. (The cladogram generated from the distribution of L1 and B1 retroelements in the different karyotypes of d (4) PG   of some chromosomes , the L1 signal on chromosome 2a was coincident with CH in a region close to the centromere; in P. echinothrix, blocks of L1 and B1 coincided with the fully heterochromatic short arm of metacentric 10; and in P. longicaudatus, there was no B1 labeling that coincided with heterochromatin. The totally heterochromatic PS1 and PE Y chromosome showed blocks of L1 and B1, and in PS2 and PC there were also strong labeling with L1  PL and PC, (2) PS1 and PS2, (3) PG, and (4) PE. These data reflect some of the taxonomic relationships proposed by Proechimys: PS1 and PS2, corresponding to the two cytotypes of P. guyannensis (Guyenne spiny rat), were grouped together, as were PL and PC, corresponding to P. longicaudatus (Long-tailed spiny rat) and P. cuvieri (Cuvier’s spiny rat), both of which belong to the longicaudatus group according to Proechimys , PG , PS2 , PE , PL (10m) and PC , and B1 in PS1 (Ya), PG (13m), PE , and PC (3a)  . Our resProechimys sex chromosomes, L1 and B1 retroelements seemed to accumulate in the centromeric/pericentromeric regions  and are available in GenBank with the numbers https://www.ncbi.nlm.nih.gov/nuccore/MW027222 and https://www.ncbi.nlm.nih.gov/nuccore/MW027223. Sequenced plasmids were tagged by nick translation with digoxigenin-11-dUTP , according to the manufacturer’s instructions, and used as probes for FISH. The slides containing the target chromosomes were initially treated with pepsin for 10 min at 37°C and washed in distilled water. Chromosomal denaturation was performed in 70% formamide in 2× SSC at 70°C for 2 min followed by dehydration in ethanol. The hybridization mix consisted of 200 ng of digoxigenin-labeled probe and hybridizations in hybridization buffer (50% formamide/20× SSC/50% dextran sulfate) were performed at 42°C over night, with 77% stringency. After post-hybridization washings in 2×SSC/PBST at 37°C and immunodetection with rhodamine-conjugated antidigoxigenin, the metaphases were counterstained with DAPI (0.8 ng/μl) in antifade reagent .The retroelements L1 and B1 were obtained using polymerase chain reaction (PCR). The following primer sets were used: L1-F (5′-AAGAATTCCGCAGGATACAAGATCAACTCA-3′) and L1-R (5′-AAGGATCCCAATTCGATTCCATTGGT-3′) , B1-F 5. The PCRFor fiber-FISH experiments, the protocol by  was usedThe results obtained using FISH for the retroelements L1 and B1 were compared to the CBG-banding patterns and the silver-staining of the nucleolus organizer regions (Ag-NORs) described by Proechimys species from the distribution of L1 and B1 retroelements were coded as binary characters, and used in a cluster analysis using a dendrogram performed with PAST 4.3 software. The comparison of similarity among species was performed using the Jaccard similarity index shown in The probable structural rearrangements in the chromosomes of https://www.ncbi.nlm.nih.gov/genbank) under accession number https://www.ncbi.nlm.nih.gov/nuccore/MW027222 and https://www.ncbi.nlm.nih.gov/nuccore/MW027223.All raw and processed sequencing data demonstrated in this study have been submitted to the NCBI GenBank (


In this study, five mathematical models were fitted in the absence of space charge with experimental data to find a more appropriate model and predict the emission current density of the graphene-based thermionic energy converter accurately. Modified Richardson Dushman model (MRDE) shows that TEC's electron emission depends on temperature, Fermi energy, work function, and coefficient of thermal expansion. Lowest Least square value of  Because of its unmatched prospects in electronics, thermal expansion, optical and mechanical properties, ease of tune-ability of its work function that is seen to operate in the 2D world15 and graphene's tolerance of high temperature16. Recently the mechanical properties were experimentally proven to use also in the 3D world17. In principle, Fig. 20, photo22, secondary23 and field emission27. The thermionically excited electrons work as the working fluid above the potential interface barrier and, after that, is collected at the anode of the TEC. Fundamentally, a TEC setup comprises a cathode (B), an anode (A), a conducting wire, and a load  materials, graphene remains an electrode of choice in the heart of researchers in harvesting electricity via thermionic and photo-enhanced thermionic energy converter due to its unmatched potentials30. This has led to the unnatural death of technology in the 90 s. Nevertheless, a recent revival of interests in thermionic energy converters is attributed to the emerging nanomaterials and ever-growing technology in the twenty-first century8. The technologies that are primarily of interest for applying graphene-based TEC are graphene's growth on solid tungsten and silicon carbide substrates33 and ease of work function engineering of graphene surface36.Despite enormous research in TEC, the TEC technology's potential in generating electricity is still being hindered due to high material work function and space charge related problemsTheoretically, researchers have seen green light about the terrestrial applications of TEC instead of space applications through the usage of nanomaterials as electrodes in TEC. In harnessing a graphene-based TEC's hidden electric power generation potential, specific parameters such as power output, thermal exchange, heat removal rate, and efficiency must be accurately modelled with some other TEC parameters.7 and 8. Once electrons are emitted thermionically at the emitter by overcoming the barrier due to its work function), the electrons find an electric field between the emitter and the anode due to the potential barrier The formation of the potential barrier mentclass2pt{minimmentclass2pt{minims connotes the emitter's surface area.The present study aimed at examining the power output of graphene-based TEC specifically in the absence of space charge:7. Incorrect modelling of efficiency of a TEC energy conservation which was not appropriately modelled except in Refs.37 must be taken fully into account. This is given for a solar TEC by Eq.  can  TEC Eq.  is found49 only in predicting and determining the current density of nano-thermionic energy47 converter. In response to the fitness of the RD equation, some scholars have affirmed the potency of the RD equation in determining the current density of the emitters and collectors in nano-thermionic engines60. At the same time, some researchers opined that using macroscopic RD law to investigate a nano-thermionic energy converter (NTEC) is against the physics of nanoscience and nano-engineering61. To resolve this challenge, the experimental results of Zhu et al.44 for J vs. T in graphene have been adopted to fit the various models and to find the most suitable model for predicting the accuracy of the current density of graphene-based thermionic energy converter.The recent issue being raised in scientific communities is the fitness of the Richardson Dushman (RD) equation that was enacted for metals44. Consequently, the five theoretical models (Eqs. 36–40) were coded in MATLAB to fit the experimental current density of graphene-based TEC.This study has acquired graphene's experimental thermionic emission (J vs. T) data from reliable and standard source49:Nobel Prize-winning Eq.  of RichaTypically, 52 confirmed that Sommerfeld theory could have been a suitable model in predicting electron emission from the edge of two-dimensional (2D) materials like graphene. However, the current density from the edge is too small to be of any practical importance. Hence, the scientific thought of Wei et al.52 and Kim and Lee53 on the electron emission from the graphene sheet with atomic thickness is expressed as:Investigation of Wei et al.Moreover, a complexity in their current density versus temperature model when tunnelling effect is applied at a different energy level and time interval in examining the confinement of electrons in a potential barrier poses a fundamental question on the correctness of their theory. Also, this theory should be conceived in the heart and not in the practical term.20 showed that the RD is a non-valid theory to examine the current emission density of graphene. Their study considered the in-plane mass of graphene electrons as a zero entity. Even though they thought the finite mass of electrons for motion perpendicular to the graphene surface, they got rid of this finite mass in the final equation using some questionable tricks. They assumed a temperature-independent work function. This allows their study to adopt a new Richardson Dushman constant of 54 experiment revealed the non-massless nature of graphene when the estimated dynamical electron mass of graphene is found between 0.01me to 0.024me, which raises doubt on Liang and Ang idea20 in Eq. 208\docum50:Consequently, Khatoon et al. considered how to work function and Fermi energy are a function of temperature in37:That Fermi energy Thermionic emission must be perpendicular to the graphene surface. For in-plane motion, the electrons must possess in-plane momentum components  approach has the potential to model the mechanism of thermionic emission in 2D electrodes(graphene) based on the following conditionsSo far, no model has considered all the above realistic scenarios of dynamics of electron emission from the 2-D graphene, as it is too complicated to handle theoretically.12. They also considered the temperature dependence of work function. With these considerations, the Richardson Dushman equation was modified to model the current density of graphene in terms of Fermi energy and work function dependent on temperature45.0 is the work function of the material, r is the dimensionality of the material, such that 2D graphene, has a r value of 2 while carbon nanotube has a r value of 1, T is the temperature, To avoid the complication associated with a near-perfect model as mentioned above, considering no. (v) De and Olawole assumed a 3D model (due to three momentum of the electrons) considering thermal expansion of the lattice and temperature dependence of work function. The 3D model of graphene is recently found to hold for mechanical properties of grapheneTable Figure r of Eq.  in Fig. 44 of graphene accurately as its electron emission depends on temperature, Fermi energy, work function, and coefficient of expansion. That graphene's Fermi energy is temperature-dependent, precisely at 0 K, which was ignored in Eqs.  show prospect of applying the equation in which the external voltage induced diode current density 57 in Eq. . Moreover, it is not easy to model the tunnelling times because of variation with quantized energies51. Thus, we observe a real challenge in formulating the current density-temperature relation of 2D graphene, considering the energy dispersion in the 2D plane and the discrete energy levels in the direction normal to the 2D plane of graphene.This study also reveals how the Richardson Dushman model