Bilayer graphene consists of two stacked graphene layers bound together by van der Waals interaction. As the molecular analog of bilayer graphene, molecular bilayer graphene (MBLG) can offer useful insights into the structural and functional properties of bilayer graphene. However, synthesis of MBLG, which requires discrete assembly of two graphene fragments, has proved to be challenging. Here, we show the synthesis and characterization of two structurally well-defined MBLGs, both consisting of two π−π stacked nanographene sheets. We find they have excellent stability against variation of concentration, temperature and solvents. The MBLGs show sharp absorption and emission peaks, and further time-resolved spectroscopic studies reveal drastically different lifetimes for the bright and dark Davydov states in these MBLGs. Molecular bilayer graphene — a discrete assembly of two stacked graphene fragments — is challenging to produce in pure form. Here, the authors are able to synthesize and characterize stable molecular bilayer graphenes by the π−π stacking of two identical, well-defined nanographene sheets. SLG sheets display an inherent tendency to stack into multilayered structures due to the inter-planar π−π interaction3. As the simplest manifestation of such structures, bilayer graphene (BLG) exhibits novel characteristics absent in SLG, such as bandgap opening4, bound excitons6, etc., and therefore provides a fertile ground7 for studying two-dimensional materials. Advances in construction and characterization of BLG have also provided theoretical frameworks and instrumental tools in exploring the emerging field of van der Waals heterostructures9. However, current methods, such as chemical vapor deposition11 and layer-by-layer assembly12, often restrict BLG formation on substrate surfaces. Moreover, tendency of solution-dispersed graphene sheets to self-aggregation3 leads to a mixture of multi-layered graphene sheets in solution and merely the dispersions of enriched bilayer or trilayer graphene sheets could be achieved by exfoliation of specific graphite intercalation compounds13.Single-layer graphene (SLG) possesses a variety of unique optical and electronic properties, which stem from its mono-atomic network of sp14. Research on nanographene has provided deep insights into the structure–property relationships of graphene15 and stimulated the field of organic synthesis of graphenic materials with a well-defined architecture16. Although peripheral substitution of nanographene with sterically bulky substituents can preserve its monolayer structure19, stacking nanographenes in a discrete bilayer form is much more challenging. By using surface supramolecular assembly method, bilayer assembly of nanographene can only be found at liquid/solid interfaces in a very limited amount21. This problem is further compounded by the fact that the generated bilayer nanographene is often bound to, and thus difficult to be separated from, the substrate surface. An alternative strategy involves tethering two nanographene sheets together with a covalent linker23. However, the resultant product cannot be considered as a molecular cutout of bilayer graphene because the covalent attachment disrupts the van der Waals interactions between the two layers. On the other hand, although stable bilayer nanographene assembled by π−π interaction can qualify as molecular bilayer graphene (MBLG), to the best of our knowledge, such an MBLG has not been obtained in a pure form so far, because the propensity for multilayer stacking of nanographene often results in a dynamic mixture of columnar superstructures with indefinite numbers of layers28.Nanographene is widely regarded as the molecular model of graphene114H24R6)2, 1 and (C96H24R6)2, 2, R = mesityl], each formed by π−π stacking of two identical nanographene sheets. Mass spectrometric analysis shows that these MBLGs possess a bilayer structure and can dissociate into the corresponding monolayer when exposed to enhanced laser ablation. Nuclear magnetic resonance (NMR) spectroscopy and single-crystal X-ray diffraction (SXRD) clearly validate their bilayer structures. The interlayer H···H proximity in the bilayer structure is revealed by two-dimensional (2D) nuclear overhauser effect spectroscopy (NOESY). The bilayer structure of MBLGs is highly stable against varying temperature, concentration, and solvent. The absorption and emission of the MBLG shows clear vibronic fine structures. The time-resolved fluorescence (TRPL) and transient absorption (TA) spectroscopies indicate that the bright and dark Davydov states of MBLGs have drastically different lifetimes.Herein we report the synthesis and characterization of two stable MBLGs  , the upper level is the allowed state (bright state). On the other hand, if the transition dipoles are in in-line alignment, the upper level is the forbidden state (dark state). Owing to the parallel dipole alignment in π–π stacking configuration of 1 and 2, the optical transition from ground state to the upper and lower Davydov states are allowed and forbidden, respectively46. Taking into account this energy splitting, we tentatively attribute the bright and dark state to the upper and lower states, respectively, and the fast energy relaxation from upper to lower states is responsible for the short lifetime of bright states.Owing to the van der Waals interaction between two aggregated molecules, originally degenerate excited states from the two monomers will form excited states with different energies (Davydov splitting) after forming a dimer. The optical transition to one of these two Davydov states is allowed, while the transition to the other one is forbidden. The energetics of the allowed and forbidden states depend on the transition dipole alignmentWe have demonstrated the chemical synthesis of well-defined MBLGs and validated their structures. The MBLGs are remarkably stable, showing no sign of aggregation or dissociation. The MBLGs here offer the well-defined molecular cutout of bilayer graphene and can help in the understanding of structure and properties of bilayer graphene at the molecular level. Further investigation on rational design of MBLGs with tunable size, edge, and twist angle could be readily envisioned, and perhaps even more ambitious goals, such as hetero-structural MBLG and bilayer graphene nanoribbons, could be aimed. Our current study can also inspire the synthesis of other stable bilayer carbonic nanomolecules, such as geodesic polyarenes, carbon nanobelt, and nanorings.Supplementary InformationPeer Review File


Phenotypic changes in lung fibroblasts are believed to contribute to the development of Idiopathic Pulmonary Fibrosis (IPF), a progressive and fatal lung disease. Long intergenic non-coding RNAs (lincRNAs) have been identified as novel regulators of gene expression and protein activity. In non-stimulated cells, we observed reduced proliferation and inflammation but no difference in the fibrotic response of IPF fibroblasts. These functional changes in non-stimulated cells were associated with changes in the expression of the histone marks, H3K4me1, H3K4me3 and H3K27ac indicating a possible involvement of epigenetics. Following activation with TGF-β1 and IL-1β, we demonstrated an increased fibrotic but reduced inflammatory response in IPF fibroblasts. There was no significant difference in proliferation following PDGF exposure. The lincRNAs, LINC00960 and LINC01140 were upregulated in IPF fibroblasts. Knockdown studies showed that LINC00960 and LINC01140 were positive regulators of proliferation in both control and IPF fibroblasts but had no effect upon the fibrotic response. Knockdown of LINC01140 but not LINC00960 increased the inflammatory response, which was greater in IPF compared to control fibroblasts. Overall, these studies demonstrate for the first time that lincRNAs are important regulators of proliferation and inflammation in human lung fibroblasts and that these might mediate the reduced inflammatory response observed in IPF-derived fibroblasts. The etiology and pathogenesis of the disease are still unclear, although recent research has indicated that persistent epithelial injury and/or exposure to pathogens, leads to the secretion of fibrotic, proliferative and inflammatory mediators such as TGF-β14, PDGF5 and IL-1β6 . These are then thought to act upon surrounding fibroblasts, to induce an exaggerated wound healing response that contributes towards the development and progression of IPF1.Idiopathic pulmonary fibrosis (IPF) is a fatal progressive chronic disease characterised by scar tissue accumulation in the lungs leading to impaired gas exchange and restricted ventilation8 and diminished capacity to synthesis cyclooxygenase 2 and prostaglandin E29. Differences have also been observed in proliferation and release of fibrotic components, although these have resulted in contradictory observations13. Attempts to understand these persistent phenotypic changes at the epigenetic level have shown differences in the pattern of DNA methylation15. However, although there are reports of histone changes associated with individual genes linked to IPF16, there has been no attempt to determine if there are genome wide changes in the profile of histone modifications.Comparison of the phenotype of lung fibroblasts derived from IPF patients with those from non-fibrotic patients has shown that these exhibit multiple differences including reduced apoptosis17. In contrast, little is known about lncRNAs which are commonly divided into three groups: long intergenic non-coding RNAs (lincRNAs) that are located between protein-coding genes, antisense that are transcribed across protein coding genes on the reverse strand and pseudogenes, that are non-translated versions of protein coding genes19. In most cases, it is believed that the actions of lncRNAs are mediated through domains that interact with proteins, acting as scaffolds or to modulate their activity21. At the present time, a number of miRNAs have been implicated in the regulation of fibroblast function and in the development of IPF including let-7d22, miR-17~9223, miR-10124 and miR-15525. In contrast, although there is accumulating evidence to indicate that lncRNAs are important regulators of biological response19, little is known regarding the role of lncRNAs in lung fibroblast function or IPF.High-throughput sequencing indicates that much of the human genome is transcribed into non-coding RNAs (ncRNAs). The majority of ncRNAs (>90%) are involved in house-keeping activities such as translation , splicing (short nuclear RNAs) and post-transcriptional RNA modifications (short nucleolar RNA) whilst the others are broadly classified as either short ncRNAs (<200 nt (nucleotides)) or long ncRNAs (lncRNAs) (>200 nt). The microRNA family of short ncRNAs are the best characterised and are known to induce mRNA degradation or block messenger RNA (mRNA) translation via the RNA interference pathway27 and up-regulation of two lincRNAs, LINC00960 and LINC01140 in IPF fibroblasts. Functional analysis has shown that the both LINC00960 and LINC01140 are required for proliferation and that LINC01140 is a negative regulator of the inflammatory response. Given that LINC01140 is upregulated in both IPF fibroblasts and lung biopsies, our data suggest that LINC01140 mediates the reduced inflammatory response in IPF fibroblasts.In this report, we have demonstrated differences in the functional responses between fibroblasts derived from control and IPF lungs. These are reflected by changes in H3K4me1, a histone epigenetic marker of primed genes and enhancersOur initial aim was to determine whether there were significant differences in the phenotypic responses of lung fibroblasts derived from control lung and IPF patients and to examine the potential role of lincRNAs in these variations. We selected high-throughput approaches to measure the time- and concentration dependency of their TGF-β1-induced fibrotic response, PDGF-induced proliferation and IL-1β-stimulated inflammatory response.28. To assess potential difference in the fibrotic response in control and IPF fibroblasts we examined the time- and concentration-dependent release of PAI-1 release in response to TGF-β1.TGF-β1-induced activation of lung fibroblasts triggers the expression of PAI-1 , a protein known as an important regulator of fibrinolysis and wound healing and therefore implicated in the process of fibrosisInitial studies showed a time-dependent release of PAI-1 from non-stimulated IPF cells, which reached significance at 48 hours (h) and 72 h Fig. . However50 between the control (319 pg/ml) and IPF (139 pg/ml) individuals showed a significant difference . In relation to PAI-1 release, these studies show IPF fibroblasts are more fibrotic since they demonstrate increased sensitivity to TGF-β1 activation.To examine the concentration-dependent response, cells were incubated for 72 h with 0.01 to 10 ng/ml TGF-β1 and compared with time matched non-stimulated cells. As expected, TGF-β1 induced a concentration-dependent increase in PAI-1 release in both control Fig.  and IPF 29. To assess the proliferative response in control and IPF fibroblasts, we investigated the time- and concentration-dependent effect of PDGF-AB on the fibroblast cell number. Initial examination of the proliferation in non-stimulated fibroblasts showed increased proliferation in control versus IPF at 72 h , we observed a concentration-dependent increase in proliferation at 72 h, although we observed wide variation in the response of IPF fibroblasts Fig.  compared31. To assess potential differences in the inflammatory response, we examined the time- and concentration-dependent IL-1β induced IL-6 release from control and IPF lung fibroblasts.The pro-inflammatory cytokine IL-1β has been shown to potently induce IL-6 release from various cell types including fibroblastsInterestingly, we observed a time dependent IL-6 release in non-stimulated fibroblasts, which was significantly increased in control (67.1 ± 19.8 pg/ml) versus IPF (3.7 ± 0.9 pg/ml) fibroblasts at 24 h, 48 h and 72 h Fig. . Exposur50 values showed a significant reduction (p = 0.0234) between control (EC50 = 101 pg/ml) and IPF (EC50 = 550 pg/ml) fibroblasts. Based upon IL-6 release, this data would indicate that IPF fibroblasts are less inflammatory than control cells as they demonstrated both reduced sensitivity to IL-1β and a lower basal release of IL-6.To examine the concentration-dependency, cultured cells were incubated for 72 h with increasing concentrations of IL-1β ranging from 0.03 to 30 ng/ml . Comparison of the logEC31. Exposure to 10 ng/ml TNF-α induced a comparable time-dependent release of IL-6 from control and IPF fibroblasts, which continued to increase over the 72 h period  and IPF (EC50 = 2.2 ng/ml) fibroblasts  despite no history of smoking in either the control or IPF patients. To examine further the potential role of epigenetics changes, we have employed Western blotting to compare the levels of H3K4me1, H3K4me3 and H3K27ac in the five control and IPF patient samples . Amongst this group, PAI-1 (Serpin E1) gave one of the highest fold changes in both the control (44-fold) and IPF fibroblasts (31-fold). Comparison of the lists of genes that were changed following TGF-β1 exposure identified only 77 that were differentially expressed between control and IPF, which includes a single lincRNA (LOC100507516) whose expression was reduced in IPF . To this end, we compared the expression profile in control and IPF lung fibroblasts in non-stimulated cells and those exposed to 3 ng/ml TGF-β1 for 24 h, using the Affymetrix GeneChip™ Human Transcriptome Arrays 2.0  that were increased in IPF  and IPF (n = 20) lungs32  antisense sequences against each lincRNA that produced 50–85% knockdown following overnight transfection into fibroblasts and stimulation with TGF-β1 for 24 h Fig. . FollowiFollowing overnight transfection with LNA antisense to LINC00960, LINC01140 and the 2 negative controls, we observed no effect upon cell count, indicating that this procedure had no immediate action upon cell viability Fig. . FollowiWe once again demonstrated LNA mediated knockdown of LINC00960 and LINC01140 in both control and IPF fibroblasts following overnight transfection and 24 h exposure to IL-1β Fig. . LINC0003. These included TGF-β1-stimuated PAI-1 release as a marker of the fibrotic response34, PDGF-induced changes in cell numbers as a model of proliferation36 and IL-1β-stimulated IL-6 release as a marker of inflammation37.In this report, we have for the first time investigated the role of lincRNAs in the regulation of lung fibroblast function and whether changes in their expression might be involved in the development of IPF. In our initial studies, we employed high throughput assays to undertake detailed examination of the time and concentration responses of control and IPF fibroblasts, examining phenotypes associated with IPF13. In contrast, the concentration response curves showed a significant leftward shift in IPF fibroblasts, indicating that these had increased sensitivity to TGF-β1.As previously reported when using collagen release as a measure of fibrosis, we observed no difference in the magnitude of PAI-1 release from control and IPF fibroblast in the absence and presence of a maximally effective TGF-β1 concentrationet al.10.In the absence of PDGF, we observed a reduction in the proliferation of IPF fibroblasts. Following exposure to PDGF, the small increases and the variability of the response between individual fibroblasts samples, meant there was no significant difference in the magnitude or concentration dependency of the proliferation. This variability in the PDGF-induced proliferative response had previously been observed by Jordana 38. Unlike TGF-β1 and PDGF, the role of IL-1β in the development of IPF is yet to be established although there is a report of showing increased levels in bronchoalveolar lavage39. In addition, there are conflicting reports as to whether IL-1β elicits profibrotic or antifibrotic activities41. IL-1β was previously shown to drive IL-6 expression in orbital and synovial fibroblasts in vitro42, although nothing is known regarding its effect on lung fibroblasts. Significantly, measurement of IL-6 indicated that non-stimulated IPF fibroblasts were less inflammatory and showed a reduced sensitivity to IL-1β stimulation. In contrast, examination of the IL-6 release in response to TNF-α showed no difference between control and IPF fibroblasts indicating that reduced sensitivity might be specific to IL-1β. In support of these phenotypic observations, a recent meta-analysis of four previous microarrays studies in control and IPF fibroblasts which showed repression of inflammation and immune pathways43. Interestingly, this reduced inflammation in non-stimulated and IL-1β-stimulated IPF fibroblasts may explain the lack of efficacy observed when corticosteroids and immunosuppressants have been examined as therapeutic options for the treatment of IPF3.In general, IL-1β is considered to be a potent pro-inflammatory cytokine that can induce the release of multiple pro-inflammatory mediators, including IL-615, with none having examined histone modifications. We therefore measured the distribution of H3K4me1, a marker for priming at promoter and enhancer regions that is thought to work through the recruitment of multiple chromatin-remodelling complexes44. Significantly, we identified multiple regions demonstrating differential expression of H3K4me1, with unsupervised hierarchical clustering showing a clear separation between control and IPF fibroblasts. In addition, although the patients variation meant that we failed to reach significance, we have shown a trend towards reduced expression of H3K4me1, H3K4me3 and H3K27ac in IPF fibroblasts and speculate that this might to linked to phenotypic changes such as the reduced inflammatory response. Although there have been a number of reports showing changes is histone marks localised to specific genes implicated in IPF16, this is the first evidence to show genome-wide changes in histone modifications and that targeting the acetylation of H3K4 and other histones might provide a novel therapeutic strategy. Indeed, this latter contention is supported by studies into Brd4, a bromodomain containing protein that can bind to acetylated histones and act as a scaffold to attract components of the transcriptional machinery. Thus, inhibitors of Brd4 have been shown to attenuate migration, proliferation and IL6 release in isolated fibroblasts and inhibit fibrosis in a bleomycin-induced model of flung fibrosis45.Having demonstrated significant differences in fibrotic and inflammatory responses between control and IPF fibroblasts, we then proceeded to show that this was also reflected at the epigenetic level. To date, the only genome wide epigenetic studies in IPF have examined the changes in the profile of DNA methylation47 and pulmonary fibrosis associated lncRNA (PFAL)48 contribute to lung fibrosis by acting as sponges (and therefore inhibitors) for miRNAs. Using microarray analysis and qRT-PCR, we identified 2 lincRNAs, LINC00960 and LINC01140 that were up-regulated in IPF compared to control fibroblasts. Given that isolated and cultured fibroblasts might not reflect the situation in the whole lung, we were able to analyse historical RNA sequencing data obtained from lung biopsies32 and confirm up-regulation of LINC01140 but not LINC00960. Knockdown studies indicated that neither appeared to regulate TGF-β1-induced PAI-1, although it is not possible to eliminate the possibility that these lincRNAs might regulate another aspect of the fibrotic response. In contrast, knockdown studies indicated that both LINC00960 and LINC01140 were required for proliferation. Interestingly, these knockdown studies appeared to contradict the results in non-stimulated cells which showed increased proliferation in control fibroblasts despite reduced levels of LINC00960 and LINC01140. The reason for this discrepancy is unknown, although we speculated that other pathways, rather than LINC00960 and LINC00960, might play a dominant role in regulating the increased proliferation in non-stimulated control fibroblasts. It might also be argued that measurement of cell number is not the optimal approach and that lincRNA knockdown may have impacted upon cell viability rather than decreased proliferation. However, we believe that this is unlikely since we observed no significant effect of lincRNA knockdown upon TGF-β1-induced PAI-1 release and increased IL6 release in response to IL-1β exposure, when decreased cell viability might have been expected to also reduce these responses. Examination of the inflammatory response showed that LINC01140 was a negative regulator of IL-1β-induced IL-6 release. In the latter case, there was a greater elevation in IL-6 release from IPF fibroblasts following LINC001140 knockdown, possibly reflecting the increased expression of LINC01140 in IPF versus control fibroblasts. This increased LINC01140 levels in IPF fibroblast might also explain the absence of IL-6 release in non-stimulated IPF fibroblasts compared with controls and the shift in the concentration response curve in response to IL-1β exposure. Although the mechanism of action is yet to be determined, negative regulation of the inflammatory response is commonly observed with both lncRNAs and miRNAs including interleukin-7-antisense (IL7-AS)49 and miR-146a50. Of relevance, we have recently shown that increased inflammatory response is IPF fibroblast might also be related to reduced expression of MIR3142HG/miR-146a51.At the present time, little is known regarding the role of lincRNA either in the regulation of fibroblast function or whether changes in their expression are associated with the phenotypic changes associated with IPF. The exception is studies showing that H1953. With regards to inflammation, immune-modulatory lncRNAs have been shown to bind and regulate the activity of (i) transcription factors such as nuclear factor-κB (NF-κB), signal transducer and activator of transcription 3 (STAT3) and the glucocorticoid receptor (GR), and (ii) chromatin associated proteins including the heterogeneous nuclear ribonucleoproteins (hnRNPs) family and components of the polycomb repressor complex 2 (PRC2)55. However, the identification of these functional domains (conserved sequences) has been hindered by their poor evolutionary conservation, which in contrast to protein coding genes, does not require the maintenance of a conserved open reading frame for optimal translation. Instead, it is thought that lncRNAs conservation is geared towards the maintenance of genomic position (synteny) and short conserved domains (microdomains)53. In addition, it has been suggested that the action of lncRNAs might be mediated through acting as sponges for miRNAs, although this remains a controversial issue,57. Future studies will examine the mechanism of action of LINC00960 and LINC01140 in fibroblasts responses.Overall, this report is the first to demonstrate that phenotypic differences between control and IPF fibroblasts are associated with genome-wide changes in histone modifications and increased expression of the lincRNAs, LINC00960 and LINC01140. Significantly, we also demonstrate that these lincRNAs can regulate fibroblast proliferation and inflammation whilst changes in LINC01140 expression might mediate the reduced inflammatory in IPF fibroblasts. Currently, the mechanisms by which these lincRNAs regulate these responses is unknown. By analogy with proteins and the non-coding RNAs involved in mRNA translation (tRNAs/rRNAs) and splicing (snRNA), it is speculated that the biological actions of lncRNAs are mediated through domains containing conserved sequences, that interact with proteins and/or base pair with RNA/DNA2 humidified incubator. Upon reaching approximately 80–90% confluency cells were washed in sterile 1x PBS   followed by treatment with StemPro® Accutase® cell detachment solution . All experiments were performed using cells plated at passage 6 to 7.Control  and IPF fibroblasts  were obtained from Professor Carol Ferghali-Bostwick  and the Coriell Institute of Medical Research . Approval was obtained from the Medical Board of the Medical University of South Carolina and the Coriell Institute of Medical Research with patients proving material with informed consent. All methods were performed in accordance with the relevant guidelines and regulations. Neither the control or IPF patients had a history of smoking. Isolation of lung fibroblasts was initiated using explants of minced lung tissue. Fibroblasts were cultured in DMEM  growth media  supplemented with 10% (v/v) FBS  , 1% (v/v) Penicillin-Streptomycin  and 0.1% (v/v) Fungizone . All cultures were maintained in a 37 °C, 5% (v/v) CO4 cells were plated in 96 cell culture wells on day 1 and allowed to adhere overnight. On day 2, cells were serum-deprived by reducing FBS to 0.1% in 200 μl of starvation media. Fibroblasts were then treated with the required concentration of TGF–β1 , IL-1β , TNF-α  and PDGF-ΑΒ  and incubated for the indicated time before supernatants were collected for protein quantification assessment or cell viability assays.1 × 105 cells of control and IPF lung fibroblasts were seeded in 25 cm2 cell culture flasks  and medium was replaced until the cells reach near-confluency state. Their growth medium was then replaced with 3 ml starvation medium (0.1% FBS) and left overnight. The following day, the cells were treated with and without 3 ng/ml TGF-β1 for 24 h before all supernatants were collected and cells were lysed.5 × 10Lung fibroblast proliferation was evaluated by measuring cell viability with the Cell Counting Kit-8 . Cells (5000) were seeded in a 96-well plate and incubated overnight in 100 μl growth media (10% FBS). The following day, the cells were serum-deprived and treated with the indicated concentration of PDGF-ΑΒ and then incubated for the indicated times. Before the end of each assay, 10 μl of the CCK-8 solution was added into a final volume of 100 μl cultured media and incubated for 2 h before absorbance was measured using a microplate reader . The absorbance wavelength was measured at 450 nm and 600 nm which was then subtracted during data analysis.5 cells per well in 200 µL of growth media and incubated with the transfection mixes at 37 °C, 5% (v/v) CO2 overnight. The next day, 800 µL of media (0.1% FBS) was added to the wells to dilute out the lipid-LNA complexes and reduce the toxicity of the reaction. The cells were stimulated with either 3 ng/ml of TGF-β1 or 3 ng/ml of IL-1β and incubated for 24 h before harvesting for RNA extraction and ELISA analysis. The same transfection protocol was followed for the cell viability assays (CCK-8 kit) with or without 100 ng/ml of PDGF-AB stimulation for 72 h, however 96 well plates were used and the reaction volume and reagents were reduced accordingly. LNA Gapmer sequences: LNA1 –TCATACTATATGACAG; Negative Control LNA2 – GACGGTAAGTAGGCGA; LINC00960 LNA1 – GGCGTGAGAGTAAAGC; LINC00960 LNA2 – GTGCTTAGGCTTAGAG; LINC01140 LNA1 – TTTAATTGGGCCGTCT; LINC01140 LNA2 – TTGACACGGCTGACTT.On the day of transfection, 5 µL of HiPerFect (Qiagen) was mixed with 200 µL of growth media without antibiotics, serum or antifungals to prepare the transfection mix. LNA Gapmers were added to 200 µL of the transfection mix at a final concentration of 30 nM, placed in 12-well plates and incubated for minimum 10 mins at room temperature. Fibroblasts were then seeded at a density of 5 × 10Supernatants of cultured lung fibroblasts were collected and used to assess secretion of IL-6 and PAI-1, using the DuoSet ELISA  Development System Kits  following the manufacturer’s instructions. Samples and standard curve samples were diluted as appropriate in reagent diluent. Absorbance was measured at 450 nm with wavelength correction at 570 nm using a microplate reader .For all samples, total RNA was extracted using the RNeasy kit (Qiagen), included an on-column DNase treatment (Qiagen), according to the manufacturer’s guideline. RNA concentration was determined using the Qubit 2.0 (Life Technologies). RNA quality was measured using the Agilent Bioanalyzer and produced RIN values of >8.0.−(ΔΔCt) method was used to determine relative-quantities of individual mRNAs and lncRNAs which were normalized to 18S ribosomal RNA. qRT-PCR primer sequences: 18S – AAACGGCTACCACATCCAAG (Forward), CCTCCAATGGATCCTCGTTA (Reverse); IL-6 – ACTCACCTCTTCAGAACGAATG (Forward), CCATCTTTGGAAGGTTCAGGTTG (Reverse); LINC00960 – TCCAGGCGTCATAACCAACC (Forward), CGGTGCTTAGGCTTAGAGGG (Reverse); LINC01140 – CATCTCATCGGCATGGACCT (Forward), CAAACTGGACTGACTTTCACCA (Reverse).For quantitative PCR (qPCR), cDNA libraries were prepared from total RNA using the High capacity cDNA RT kit . Expression of mRNAs and lncRNAs were determined by qPCR using the SYBR® Green PCR mix  supplemented with a protease inhibitor cocktail . SDS-PAGE electrophoresis was used to separate proteins using the Mini-PROTEAN® TGX™ Precast Gels  with Precision Plus Protein™ Dual Xtra Prestained Protein Standards . Samples were transferred to PVDF membranes using the Trans-Blot® Turbo™ Transfer System (Bio-Rad) and the membranes were blocked for 1 h at room temperature (RT) in blocking buffer (3% BSA in PBS) before they were incubated overnight at 4 °C in a 3% BSA-TTBS  blocking buffer with the following antibodies: β-actin , H3K4me1 , H3K4me3  and H3K27ac . The membranes were washed 3 times in TTBS for 5 mins and incubated with IRDye® 800CW Goat anti-Mouse  and IRDye® 680RD Donkey anti-Rabbit  for 1 h at RT before they were washed and imaged on the LI-COR Odyssey CLx imaging system.The Affymetrix GeneChip™ Command Console Software was used to summarise probe intensity data and to generate a CEL file for each sample. The CEL files were then processed by the Affymetrix Expression Console™ using Robust Multi-chip Analysis (RMA) to generate CHP files (Probe-level summarisation) following the manufacturers manual. The CHP files were then used for Quality Control analysis which generated a full report with the array QC metrics and appropriate algorithm parameters. Using the data of the CHP files, a 3-dimensional Principle Component Analysis (PCA) graph was generated accounting for the majority of variance based on a set of variables PCA1, PCA2 and PCA3 in the original data set. CHP files generated by the Expression Console Software were used in the Transcriptome Analysis Console (TAC) Software to perform statistical analysis and obtain a list of differentially expressed genes. To run the TAC software, a library folder containing the annotation files was required (HTA-2_0.na36.hg19.probeset.csv) and installed. CHP files were imported and separated into different condition groups for analysis using the Gene Level Differential Expression Analysis tool. A master table was generated containing signal expression levels of all 67528 transcript clusters (genes) covered in the arrays, 44699 protein coding and 22829 non-protein coding. Signal intensity for each transcript cluster was presented as a Bi-weight average signal (log2) value and depending on which two conditions were compared the values of ANOVA p-value, FDR p-value (based on Benjamini-Hochberg Step-Up FDR-controlling Procedure) and gene fold change (linear) were adjusted accordingly.32 were downloaded from Sequence Read Archive (SRA) (https://www.ncbi.nlm.nih.gov/sra) using the following command in SRA tools: fastq-dump −I–split-files <file_name>. The paired end reads were aligned to the human reference genome (hg38) using Hisat2 (version 2.0.4)59 using the following command line options: hisat2 −q–dta–rna-strandness FR −x < reference-genone.gtf > −1 < forward_strand.fa > −2 < reverse-strand file.fa > −S < output.sam > . Output SAM files were then sorted and converted to BAM files (samtools sort −@ 8 −o output.bam output.sam) and indexed (samtools index −b output.bam) in Samtools60. The profile of gene expression (using the Gencode v27 database) in the BAM files for each samples were determined using Stringtie61: stringtie < sample.BAM > −G < GenCodev26.gtf > −1o < samples.gtf > −e −A < sample.txt > . Following feature counting: featureCounts -a < reference-genome.gtf > −g gene_name -o counts.txt Control_*.bam IPF_*.bam the differential gene expression was assessed using DeSeq2 and the following R script: curl −s -O http://data.biostarhandbook.com/rnaseq/code/deseq 2.r cat simple_counts.txt | Rscript deseq2.r 19 × 20 > results_deseq2.txt.Previously reported sequencing data obtained from the biopsies of 19 control and 20 IPF lungs (GSE92592)62: bowtie2 −q–very-fast < reference_genome.gtf > −U < file_name.fastq > −S < output_file.sam > . Output SAM files were then sorted and converted to BAM files (samtools sort −@ 8 −o output.bam output.sam), indexed (samtools index −b output.bam) in Samtools60 and then converted to BigWig format using BamCoverage (which is part of the deepTools suite63) using the following command line: bamCoverage −b < input_bam.bam > –normalizeUsingRPKM–binSize 30–smoothLength 300 -p 10–extendReads 200 −o < output_file.bw > . Significant ChIPseq peaks (q =< 0.1) in each sample were called with MACS264 using the broadpeak options: macs2 callpeak –t < sample > −c < backgrounf_igG > −broad − < output_files > −g hs. The differential expression of H3K4me1 peaks was determined by inputing the individual Bam files (Bowtie2) and BED files (Broadpeaks output - MACS2) for control and IPF samples into Diffbind (version 2.2.1) on Galaxy 65. Control 1 was omitted as an outlier following PCA analysis.Chromatin immunoprecipitation (ChIP) using a H3K4me1 antibody  and the iDeal ChIP-seq kit for Histones kit  was performed on the 5 control and 5 IPF fibroblast samples. In addition, we performed ChIP on a single control and IPF using an IgG antibody to provide a background control. Paired-end 75 bp sequencing data was obtained using the Illumina HiSeq. 4000 at the Oxford Genomics Centre at the Wellcome Centre for Human Genetics (funded by Wellcome Trust grant reference 203141/Z/16/Z). FASTQ sequencing data from control (n = 5) and IPF (n = 5) fibroblasts was aligned to hg38 using Bowtie 266. Data was log2 transformed following the addition of 1 FPKM. The threshold for reporting gene expression at FPKM > 1 is based upon the ability to validate sequencing data using qRT-PCR.The abundance of Gencode v27 defined genes in individual samples was defined as the fragments per kilobase exon per million reads mapped (FPKM) and determined using Stringtie (RNA). PCA and hierarchical clustering on Gencode v27 genes demonstrating an expression > 1 FPKM was performed using Genesis (v1.7.7)All statistical analysis and graphs were generated using GraphPad Prism 7 software.Dataset 1 Table showing ChIPseq H3K4me1 binding in control and IPF fibroblasts (MOESM1)Dataset 2 Images of original Western blot gels showing expression of H3K4me, H3K4me 3 and H3K27ac in control and IPF fibroblasts (MOESM2)Dataset 3 Overall microarray data showing gene expression in non-stimulated and TGFß-stimulated control and IPF fibroblasts (MOESM3)Dataset 4 Differential gene expression in TGFß-stimulated control fibroblasts (MOESM4)Dataset 5 Differential gene expression in TGFß-stimulated IPF fibroblasts (MOESM5)Dataset 6 Differential gene expression between TGFß-stimulated control and IPF fibroblasts (MOESM6)Dataset 7 Differential gene expression between non-stimulated control and IPF fibroblasts (MOESM7)Dataset 8 Differential gene expression between control and IPF biopsies (MOESM8)


New architectures of transparent conductive electrodes (TCEs) incorporating graphene monolayers in different configurations have been explored with the aim to improve the performance of silicon-heterojunction (SHJ) cell front transparent contacts. In SHJ technology, front electrodes play an important additional role as anti-reflectance (AR) coatings. In this work, different transparent-conductive-oxide (TCO) thin films have been combined with graphene monolayers in different configurations, yielding advanced transparent electrodes specifically designed to minimize surface reflection over a wide range of wavelengths and angles of incidence and to improve electrical performance. A preliminary analysis reveals a strong dependence of the optoelectronic properties of the TCEs on (i) the order in which the different thin films are deposited or the graphene is transferred and (ii) the specific TCO material used. The results shows a clear electrical improvement when three graphene monolayers are placed on top on 80-nm-thick ITO thin film. This optimum TCE presents sheet resistances as low as 55 Ω/sq and an average conductance as high as 13.12 mS. In addition, the spectral reflectance of this TCE also shows an important reduction in its weighted reflectance value of 2–3%. Hence, the work undergone so far clearly suggests the possibility to noticeably improve transparent electrodes with this approach and therefore to further enhance silicon-heterojunction cell performance. These results achieved so far clearly open the possibility to noticeably improve TCEs and therefore to further enhance SHJ contact-technology performance. Graphene has been regarded as a promising candidate for the new emerging generation of transparent electrodes in several applications such as displays, touch screens and/or solar cells ,2. Its uSignificant effort has been devoted to improve the overall performance of PV devices using graphene. Thus, this material has been reported to play diverse positive roles acting as electrode, active layer, interfacial layer or electron acceptor in photovoltaic cells ,10,11,12PV market is dominated by crystalline-silicon technology that requires exhaustive technological solutions to achieve thinner and cheaper wafers. In this sense, silicon-heterojunction (SHJ) technology is emerging as a reliable low-temperature and high-efficiency solution, where new architectures of transparent conductive electrodes (TCEs) to generate and extract the current in a more efficient way are being required . In thatRegarding the prominent and unique properties of graphene, several approaches incorporating it have proven to be especially interesting. In particular, hybrid concepts combining graphene with metal grids  or nanowIn the present work, new architectures of TCOs and graphene electrodes incorporating one, two and three atomic graphene layers in different configurations have been fabricated in order to evaluate the optoelectronic properties of the resulting structures. The key parameters for choosing the most appropriated and reliable combination of layers for the electrode have been determined.A commercial copper foil was used as a catalyst for the chemical vapor deposition (CVD) of the graphene . This foil was cut into 5 cm × 6 cm rectangles and subsequently dipped in 55 wt.% phosphoric acid and then electro-polished for 10 min. After that, a hot peroxide treatment at 80 °C for 10 min was carried out.2), and methane (CH4)) connected to the CVD chamber were flushed before the growth. After that, the temperature was raised to the synthesis temperature (1050 °C) under Ar atmosphere. Once the temperature was stabilized, a mixture of 0.7% CH4/31% H2/68.3% Ar (in volume) was introduced for 15 min at 50 mbar pressure. A cold-wall CVD reactor  for 4-inch-diameter samples was used for the synthesis of graphene monolayer. The system has two heaters, a bottom one under the sample and one at the top heating the gases from the showerhead and also capable of radiatively heating non-flat or thick samples . All the4)2S2O8) 0.3 M), which is gradually changed into deionized water (DIW) for the final steps. The synthesized graphene on the Cu foil was transferred to the different substrates used throughout the study using a home-built system designed to automatically transfer graphene to arbitrary substrates . This sy−5 Pa. The purity of Ar gas used for the sputtering processes is 99.999%, and its flux is controlled with a mass-flow controller. In this work, aluminum-doped zinc oxide (AZO) and indium tin oxide (ITO) were chosen as TCO materials. The 4-inch commercial ceramic targets of ZnO:Al2O3  and In2O3:SnO2  for AZO and ITO deposition respectively were sputtered by radio-frequency (RF) and direct-current (DC) power for AZO and ITO, respectively.A commercial UNIVEX 450B sputtering system from Leybold, Cologne, Germany equipped with four magnetron sources was used for the fabrication of TCO thin films. This system has a confocal geometry where the guns are placed at around 0.15 m from the substrate centre. The base pressure of the process chamber is 1 × 104 Ω·cm). The native silicon oxide (SiOx) was removed from the silicon-wafer surface by chemically etching with a dilute (2%) hydrofluoric acid. After that, the wafer was rinsed in DIW and finally, dried by blowing nitrogen over it. On the other hand, glass substrates were also used to determine the TCE optical transparency. The glass substrates were ultrasonically cleaned using an special detergent that is a phosphate free surface active cleaning agent radioactive decontaminant from DECON laboratories , rinsed in DIW, and finally, immersed for 2 min in isopropyl alcohol and dried by blowing nitrogen over it.The TCE structures were deposited on 4-inch polished resistive float zone <100> silicon wafers (resistivity > 10λ/4) in order to lead to the destructive interference of the beams reflected from the silicon and TCO surfaces, thus acting as an anti-reflectance (AR) coating. The number of GML was chosen in order to obtain a TCE with a good conductivity and low optical losses.Two different configurations were studied for the graphene-based TCE. In In the particular case of the configuration 1, an Ar plasma bias etching on the substrate surface was carried out at 120 W under 0.5 Pa for 5 min without intentional heating prior to the sputtering deposition. This process was used to improve TCO thin-film adherence. After that, the substrate was rotated at 20 rpm, and heated at 190 °C in the holder system. Regardless the TCO material deposited, the gas flow rate and working pressure were maintained constant at 5 sccm and 0.18 Pa, respectively. The samples were sputtered at the constant values of 75 W DC power and 250 W RF power applied to ITO and AZO targets, respectively. In the case of configuration 2, no bias was applied and softer sputtering conditions were used to avoid the possible damage to GML structure, that is, at room temperature and 25 W DC power and 150 W RF applied to ITO and AZO targets, respectively.The structural quality of the graphene in the TCE was evaluated from Raman measurements. A WITec Alpha 300AR, Ulm, Germany, confocal microscope was used for the Raman spectroscopy of the samples. Raman spectra were obtained in backscattering geometry using a ×100 objective lens  in ambient conditions. A 532 nm wavelength laser set to a power of 3 mW was used as excitation source. 2 quartz substrates. Four metal coplanar parallel electrodes unequally spaced, pictured in TCER) and to compare them to the theoretical values expected corresponding to an electric circuit with two parallel electric resistances, as described by the following Equation: TCOR is the sheet resistance of the TCO film and GGR, the corresponding to GML.The metal combination tested to have a low contact resistance was Ti (50 nm)/Ag (500 nm). Transmission-line-model (TLM) measurements were done by testing 4-point electrical resistance between all the possible electrode pairs with two contacts on each electrode. Contacts were made by means of 4 micro-positioners, a power supply was used to bias the samples, an electrometer measured currents and a voltmeter provided voltages. These TLM measurements were used to determine the actual sheet resistance of the hybrid TCEs developed  is the global spectral AM1.5G irradiance [The total optical reflectance spectra were measured in the wavelength range from 300 to 1500 nm by using a UV/Visible/NIR Perkin-Elmer Lambda 1050 spectrophotometer equipped with a 6 mm integrating sphere accessory. From these measurements the weighted reflectance radiance .T) of the fabricated TCEs deposited on glass were measured by using a three-detector module in the Perkin Elmer spectrophotometer. This measurement serves to determine the fraction of light that reaches the absorber of the solar device. Finally, optical transmission maps were obtained from the TCEs fabricated on glass by means of a home-made system combining a focused white-light lamp, an X-Y linear-positioner set, a pair of current preamplifiers, a reference photodiode and a pair of digital voltmeters. Specular transmittance spectra (BT), and the other one is directly related to the combined transmittance of the sample plus the background (SAT + BT). SAT comprises the transmittance of both the substrate and the different TCEs (TCO and/or GML). Thus, knowing the transmittance of the substrate (SUBT), it is possible to calculate the amount of light transmitted by the different layers (GGT for the GML and TCOT for the TCO) as follows:The system is controlled by specific software that returns an image where each point registered is associated with a specific transmittance value. Once the data is collected, it is represented in the form of a histogram where two peaks appear: one of them corresponds to the background transmittance  , where tTCO of 120 ± 10 Ω/sq, and GGR of 390 ± 10, 230 ± 30 and 120 ± 5 Ω/sq for one, two and three GML, respectively, were used. The evaluation of the compatibility between the TCO and the GML was carried out in terms of the electrical and the optical properties of the whole structure. 2. As it can be appreciated, the measured TCER values differed considerably from the theoretical ones, especially in the case of configuration 2 where the TCO is sputtered on top of the transferred graphene. This suggests that GML might get damaged by the bombardment of the high-energy sputtered atoms during the sputtering process of the TCO thin film. This process could induce significant disorder, affecting the electrical performance of the GML [The contact resistivity of the Ti/Ag metallization was in the range of 3 to 10 mΩ·cm the GML ,22. TCER in configuration 1 (graphene transferred onto sputtered TCO), summarized also on On the other hand, the results of the TCOR is 85 ± 5 Ω/sq was used. Therefore, another TCO material free of aluminum in its composition, such as ITO, was tested. This material was preferred because of its good conductivity and its optical transparency. The electrical performance of the ITO-based TCEs are summarized in The results from With regard to optical performance of hybrid TCEs developed, the optical characteristics were also addressed in both total reflection and transmission geometries. Thus, Finally, the white-light optical-transmission maps of the samples on glass substrates can reveal the number of GML transferred and the homogeneity of the transfer process. This technique is very useful because of its high sensitivity to small variations in thickness (either produced during the graphene growth or due to folds and wrinkles arising from the transfer process). Optical-transmission maps yielded white-light transmissions of 95% that corresponds to On the other hand, regardless the TCE configuration, the white-light transmission values for the AZO-based TCEs are slightly lower than the bare AZO, as it was expected. In addition, no re-cover effect during thermal evaporation process was observed in 12 cm−2.The structural quality of the optimum hybrid ITO-based TCE, with configuration 1, was assessed. 2/min. The laboratory relative humidity was 50% and its temperature was 23 °C. All the electrical values were extracted using mathematical models detailed in [Finally, THz-TDS conductance and resistance maps acquired in reflection-mode for optimum hybrid ITO-based TCE deposited on Si substrate are pictures in ailed in . From Fiailed in . In compRegarding the resistance b, a centIn this work, the compatibility of the graphene and the TCO deposition processes was analyzed with the aim of developing hybrid TCEs for silicon-based solar cell technology. The evaluation of TCEs made by coating the GMLs with a sputtered TCO layer indicated that this process severely affected the structure of graphene, leading to a significant detriment of sheet-resistance . In contrast, the GML placed on top of a TCO led to TCEs with relatively good sheet resistances, particularly when the TCO material was ITO. This hybrid TCE showed sheet resistance values as low as 55 Ω/sq. These results suggest the convenience of avoiding aluminum in the TCO material, and hence, the choice of ITO is considered as suitable to achieve an optimum TCE structure. The in-depth electrical analysis using the THz-TDS technique led to average conductance values as high as 13.12 mS and a sheet resistance of 76.2 Ω/sq, improving the values of a common TCO. Optically, it was also verified that graphene transferred on TCO caused a total reflectance decrease, reaching values of weighted reflectance 2–3% lower than those obtained without graphene. All these results suggest the possibility of a noticeable improvement for TCEs by developing structures combining graphene with TCOs. Such hybrid TCEs would have optical and electrical properties favoring the absorption of more photons, hence leading to positive effects on the short circuit current, as well as lowering the cell series resistances.


In this study, indentation tests of graphene-based polymer nanocomposites were carried out to determine the local elastic mechanical properties. The samples consist of epoxy matrix with graphene additives. Additives were added at levels of 0% as a control, 0.5%, 1%, 2.5%, 5% and 10% by weight. The local elastic properties such as moduli and hardness were calculated. After each indentation, the prints were characterized using scanning electron microscopy (SEM). It seems that the local mechanical properties of nanocomposite samples were improved as the amount of nano-additives increased. Based on the curve displacement and surface imaging, we can conclude that the nano-additives influenced the overall plastic mechanical behavior of the samples. For simulating micro-indentation test, a finite element analysis model was developed using ABAQUS software and compared to experimental tests. Good correlation was observed. To enhance the mechanical performance of monolithic epoxy, many researchers have used nanofillers such as graphene, carbon black, carbone nanotubes, and other carbonaceous materials to reinforce it, due to their high modulus of elasticity and capacity to stop crack propagation ,2,3. IndIndentation has been intensively used to characterize local mechanical properties of different materials such as polymer, composites, etc. The aim of this study was to quantitatively understand the effects of neat epoxy and NGP/epoxy on its indentation measurement with experimental and numerical approaches. Micro-indentation tests of graphene-based polymer nanocomposites were carried out to determine the local elastic mechanical properties. The samples consisted of epoxy matrix with graphene additives. Additives were added at levels of 0% as a control, 0.5%, 1%, 2.5%, 5% and 10% by weight. Our results clearly demonstrate that the influence of the presence of NGP in epoxy matrix on its indentation measurement can be significant.The nanocomposite samples were produced using the conventional dry mixing method and adding 0.5, 1, 2.5, 5 and 10 wt % of graphene to epoxy. To maintain uniformity and produce sound samples, the unfilled polymer and the composites were processed under identical conditions. Graphene used in this study was produced by exfoliation and reduction of graphite in our own laboratory . The polGraphene tends to agglomerate in chunks because of its small particle size, large surface area and superior particle activity. To improve the dispersion of graphene in epoxy resin, the graphene bundles were dry-blended in a blender to obtain a uniform density graphene. It was then weighed to obtain five concentrations ranging from 0 to 10 wt % with the epoxy resin system. The lowest graphene concentration selected was 0.5 wt %, an intermediate value of 5 wt %, and the maximum concentration used was 10 wt %. It was found that, for concentrations higher than 10 wt %, the mixing process became challenging and a well dispersed compound was very difficult to obtain.The nanofiller was added, based on the desired filler concentration, to the epoxy resin, which was previously mixed with the curing agent in a plastic container by hand for 5 min in a weight ratio of 100/26.4 (epoxy/curing agent). This blend was subsequently loaded in a three-roll mill mixer to obtain a homogeneous dispersion of graphene in the epoxy. The procedure was the same for all compositions, with the exception of 10 wt % of graphene. As the amount of nanofiller increased, the viscosity of the compound increased. Therefore, the 10 wt % compound was prepared in several steps. Here, a small amount of graphene was added to the resin, then it was mixed in the three-roll mill until the viscosity of the compound was reduced. Subsequently, more graphene was added. This step was repeated until all the graphene was incorporated in the resin. A three-roll mixing mill, from Lehmann Mills a, was emThe mixing of the compound was started by slowly pouring the pre-mixed compound  between feed and center rolls. The mixture then automatically passed between center and apron rolls by sticking to them, and a knife edge blade scraped the processed material off the apron roll and stored in the pan . When thAfterwards, both sides of the composite plates were polished by hand for 1.5 min each with a 180 grit emery paper, and then they were washed in water and dried.The mechanical properties of each constituent are listed in The mechanical properties of epoxy matrix with grapheme additives specimens were evaluated for each volume fraction using the indentation test. A CSM Micro-Hardness Tester  using ViFor this type of material, and to control the dispersion of graphene in the matrix, micro-indentation tests were carried out in the specimen . MinimumThe mechanical and physical properties of nanocomposites depend on the dispersion of filler agent into the matrix. Here, the dispersion of graphene in epoxy was analyzed using optical and scanning electron microscopes. The optical microscope specimens were produced by mounting a 5 mm × 5 mm × 5 mm composite cube in epoxy holder and polishing its surface to optical flatness through several steps. The SEM samples were obtained by breaking a thin strip of the composite in tension. The graphene layers in epoxy can be identified via color contrast caused by the polarized light. This method is qualitative because the color contrast often varies from one laboratory to another . HoweverFirst step: Vickers indenter approaches the surface.Second step: Loading phase up to the maximum load. The maximum load applied was 1000 mN.Third step: Holding the load. It was done to avoid the creep effect on the unloading characteristics.Fourth step: Unloading phase.The mechanical properties of epoxy matrix with graphene additives specimens were evaluated for each weight fraction using an indentation test. In this study, a CSM Micro-Hardness Tester with a Vickers diamond indenter was used. A minimum of eight indentation tests were carried out for every sample. Minimum distances of 0.5 mm between indentation marks were used to avoid any hardening effect from previous tests. For all the micro-indentation tests carried out, the maximum effort was set 1000 mN. The micro-indentation parameters used for the tests are: approach speed of 50 μm/min, contact load of 10 mN, load rate of 2000 mN/min, unload rate of 2000 mN/min, maximum load of 1000 mN and 20 s of pause. The elastic moduli, indentation depth, stiffness and hardness were determined as an average value of all specimen tests. For a rigorous approach and a realistic comparison, the testing conditions were the same for all specimens. Micro-indentation tests were performed using Vickers indenter, which monitors and records the load vs. displacement of the indenter. this the total depth under a load, Pt;ehis the elastic rebound depth during unloading;fhis the residual impression depth;ahis the surface displacement at the perimeter; andphis the contact indentation depth.The indentation depths:S, is defined as the slope at the beginning of the unloading curve.The contact stiffness, For a qualitative and quantitative study, it is necessary to ensure test reproducibility. The load–displacement curves for each graphene-based nanocomposite are shown in Pmax.As shown in 2, which increases to 28.90 (8.83%), 31.59 (19%), 33.37 (26%), 35.02 (32%) and 39.89 (50%) with the addition of 0.5 1, 2.5, 5 and 10 wt % NGP, respectively. This increment in hardness is due to a good dispersion and interfacial bonding between NGP and the epoxy matrix.E, δ, S and H) are listed in The load–displacement curves for the neat epoxy resin and various NGP/epoxy-based nanocomposites were analyzed. These curves were obtained by micro-indentation testing under a maximal load of 1000 mN. As a result, adding the graphene nanoparticles (NGP) to neat epoxy, the mechanical properties were altered. Indeed, the elastic modulus and the stiffness (slope for the unloading) were improved. The elastic modulus of the epoxy increased from approximately 2.7 GPa to 3.6 GPa upon the addition of 10 wt % of NGP to the epoxy resin. Our results also showed that the stiffness and hardness of NGP/epoxy samples increased by 42% and 40%, respectively. The values of mechanical properties  . A homogThe total mesh of indentation geometry was composed of 45,310 linear hexahedral elements of C3D8R for specimen, 4986 linear quadrilateral elements of R3D4 and 2 linear triangular elements of R3D3 for rigid indenter. We developed a numerical model of micro-indentation of the composites based on the implicit scheme using Abaqus FEA software. The force at each increment time was examined and correlated to the experimental variations to validate our numerical model and enhance the convergence of results. The boundary condition applied to the indenter is the displacement resulting from the experimental results . The corE), Stiffness (S) and hardness (H). Micro-indentation tools have provided a new method to evaluate the degree of dispersion of nano-additives and the local mechanical properties at the micrometric scale. The local mechanical properties can be considered representative of those of the bulk. This study shows that the indentation test could be an excellent technique in characterizing the mechanical properties of composites laminate reinforced with graphene nano-additives. It seems that the local mechanical properties of nanocomposite samples were improved as the amount of nano-additives increased. Based on the curve displacement and surface imaging, the influence of nano-additives on the overall plastic mechanical behavior is noticeable. A proper numerical model was developed for validating the experimental results. A small difference between two approaches was remarked and defined by the projected contact area and the graphene nano-additive distribution. It seems that graphene tends to improve the fracture toughness of composite and interfacial resistance.Local mechanical properties of an epoxy matrix reinforced with graphene nanoparticles (NGP) were investigated using depth-sensing micro-indentation. The addition of low percentage weight of NGP was considered as part of a strategy to enhance the mechanical properties of the nanocomposites. The graphene additives were uniformly distributed into the epoxy matrix, forming a nanocomposite. The study focused on the effect of the concentration of NGPs on the mechanical performance. It was found that the addition of NGP to epoxy matrix reduces the indentation depth (δ) and increases the Young modulus (


The incoming 5G technology requires antennas with a greater capacity, wider wireless spectrum utilisation, high gain, and steer-ability. This is due to the cramped spectrum utilisation in the previous generation. As a matter of fact, conventional antennas are unable to serve the new frequency due to the limitations in fabrication and installation mainly for smaller sizes. The use of graphene material promises antennas with smaller sizes and thinner dimensions, yet capable of emitting higher frequencies. Hence, graphene antennas were studied at a frequency of 15 GHz in both single and array elements. The high-frequency antenna contributed to a large bandwidth and was excited by coplanar waveguide for easy fabrication on one surface via screen printing. The defected ground structure was applied in an array element to improve the radiation and increase the gain. The results showed that the printed, single element graphene antenna produced an impedance bandwidth, gain, and efficiency of 48.64%, 2.87 dBi, and 67.44%, respectively. Meanwhile, the array element produced slightly better efficiency (72.98%), approximately the same impedance bandwidth as the single element (48.98%), but higher gain (8.41 dBi). Moreover, it provided a beam width of 21.2° with scanning beam capability from 0° up to 39.05°. Thus, it was proved that graphene materials can be applied in 5G. Over the next few years, researchers and scientists anticipate that the next communication technology—fifth generation cellular network technology 5G)—will have greater overall capacity and implement a new spectrum . The sysG—will haMuch effort has been put into achieving the requirements of 5G such as broadband dual-band antennas , multi-iPrior to this study, previous works on 5G antennas have been conducted, particularly on bandwidth, gain, and beam steering in terms of the projection of higher data rates, further distance, and reliable communications, respectively. Bandwidth improvement can be shown in many ways such as balun integration , coupled2 V−1 s−1 compared to other materials. It becomes an attractive material for the manufacturing of ultra-high speed electronics [In conjunction with the emergence of 5G, the properties of graphene have been considered in this work. Graphene is the thinnest two-dimensional layer of carbon atoms  bound inctronics  besides ctronics  or tunabctronics . The swictronics  or Diracctronics . This prctronics . Based octronics ,39,40,41ctronics , transpactronics , switchectronics ,44,45, ectronics , reconfictronics , tunablectronics , and beactronics , the opeBased on the 5G requirements and limitations aforementioned, this paper proposes the development of graphene antennas in single and array which operates at a frequency of 15 GHz for 5G applications. The 15 GHz frequency was selected since the range is at high frequency. Thus, it can produce large bandwidth to support higher speed . Note thThe structure of this paper is as follows: rε = 3.5, and tan δ = 0.002 at 1 kHz. The graphene used was from graphene dispersion with ethyl cellulose in terpineol, or commonly known as graphene ink, which has a sheet resistivity between 0.003 Ω.cm and 0.008 Ω.cm with a thickness near 100 nm. The antenna was printed by a screen-printing method and fed by a 50-Ω CPW. The top substrate was coated with the patch and ground plane. Then, the antenna was excited through CPW to achieve a large bandwidth [andwidth ,56, wherWp antenna size was estimated by Equation (1):c is the velocity of light, 3 × 108 m/s, οf is centre frequency, and rε is the dielectric constant of a substrate. Then, the distance of the separation between the two ground planes of the waveguide (CPW), Dcpw, was calculated by Equation (2) [s1 is the slot width between the ground plane and the feedline, and Wf is the centre strip conductor width or feedline width.Before optimisation, the patch width, tion (2) ,58:(2)DcThe antenna was simulated in Computer Simulation Technology (CST) Microwave Studio. The printed graphene antenna had a width and length of 12.2 mm and 11.8 mm, respectively, and a thickness of 76 μm. The details of the antenna parameters are summarised in Screen printing is a technique of printing design patterns from an ink solution onto a substrate surface through a mask using a squeegee rubber movement. The method has been widely used in electronic manufacturing ,64,65,66Related to the graphene ink which needs a curing process, a Kapton film was adopted as a substrate. This was due to the film’s unique combination of properties which make it ideal for variety of applications in many different industries . It alsoSeveral parametric studies were executed to observe the antenna’s performance and to identify problems. There are many factors that could disturb the accuracy of the antenna’s results which happen during the fabrication process and other technical works. During fabrication, the graphene ink was handled manually; thus, the antenna produced may have an inaccurate size for small parts. During the curing process, the temperature and time set may have affected the condition of graphene and substrate. Then, after the curing process was completed, the graphene antenna was cut using penknife where small defects may have been created.The parametric studies were the dielectric constant of the substrate, conductivity of the graphene, and cut length. These parameters were selected due to the fact of their significant influence on the frequency resonant. It was conducted by a single parameter which was varied while others were kept constant. The simulation results of the parametric studies are shown in 2 S/m to 3.33 × 106 S/m shifted the resonance frequency to the right by approximately 1.53 GHz, from 13.65 GHz to 15.18 GHz. Conductivity variation can also be caused by the heat treatment value or length of the curing process. The parametric study on the cut length is depicted in Lc, is a critical part of the fabrication process for the proposed screen-printed antenna.The curing process was needed to break the binder contained in the graphene ink, so that the conductivity would increase. The binder contained in graphene functioned to change the graphene flakes into ink solution and as an insulator . The conDisturbance was observed on the other type of graphene antenna with the same design and operating frequency. The two minor lobes that appeared in the radiation pattern at the E-plane for the antenna made with graphene ink also appeared in the antenna made by graphene sheet but to a smaller degree due to the higher conductivity, while, the radiation pattern at the H-plane for the graphene sheet was similar to the simulation. When graphene has a higher conductivity, it will not be disturbed by the adaptor and connector during radiation pattern measurement compared to graphene ink which does not have enough conductivity as stated in the manufacturer’s data sheet. Also, the graphene sheet does not go through a curing process which could affect the results. However, the radiation pattern of the graphene sheet was not included in this work.oe, was estimated through the calculation of the multiplication between the reflection (mismatch) efficiency, re, and the antenna radiation efficiency, cde [With the respect to the resonance frequency in graphene ink, the simulated gain value was 2.39 dBi, while the measured gain was 2.87 dBi. The total efficiencies obtained for the simulation and measurement were 65.25% and 67.44%, respectively. The total efficiency, ncy, ecd . The radS11 of smaller than −10 dB from 14.32 GHz to 15.87 GHz which covered the proposed frequency bands of 5G communication, while S12, S23, and S34 were −17.47 dB at 15 GHz. However, when each element of the printed graphene antenna array was combined with the adjacent element to form a 4 element and terminate in the same ground plane, the radiation pattern at the E-plane presented an omnidirectional pattern, while the H-plane deteriorated with higher side lobe level (SLL) at −9.6 dB, as shown in Wd, of 2 mm and length, Ld, of 10.8 mm in between adjacent elements were then introduced, as shown in S11 showed that the printed graphene antenna array had a frequency range of 14.30–15.71 GHz, where the impedance bandwidth was reduced to 140 MHz compared to the antenna array before introducing the rectangular slot, but it was still larger than 1 GHz which is enough for the 5G high data rate, while the mutual coupling was reduced by approximately −4.66 dB. Even though a small reduction occurred in the mutual coupling, a large effect showed up in the radiation pattern and gain, where a bidirectional pattern was exhibited in the E-plane and directive beam in H-plane, as shown in According to the radiation pattern deterioration, the mutual coupling of the adjacent elements should be reduced through a technique of defected ground structure (DGS) for the improvement of the radiation pattern. Rectangular slots with a width, d, and the progressive phase shift, β, can control the characteristics of the array factor and the total field of the array [AF) is given by Equation (3):n is number of elements, and θ is the angle observed.The variation of the inter-element spacing or separation, he array . The arrBased on Equation (3), the inter-element spacing was observed for the effect of the gain obtained on the printed graphene antenna array after introducing the rectangular slot. The variation of inter-element spacing was between 0.6λ and λ. The gain value increased from 0.6λ to 0.8λ, then decreased until λ. The gain versus inter-element spacing is plotted in S11 measurement via a performance network analyser and then for comparison with the simulated S11 as displayed in S11 were less than −10 dB in the frequency bands of 14.30 GHz to 15.70 GHz and 11.10 GHz to 18.30 GHz, respectively, which are included in the proposed frequency for 5G. The resonance frequency for the measured result was approximately the same as the simulation’s result, 14.78 GHz. However, some ripples appeared in the measured S11 due to the cable loss from the Re-Formable Semi-Rigid Cable Assemblies that were connected between the printed graphene antenna array and the external 1-to-4 power divider as depicted in S11 also had broad bandwidth compared to the simulation because of the unpredicted dielectric, conductivity or composition formed by the graphene ink after the curing process. For validation of the proposed designed, the printed graphene antenna array with a rectangular slot was fabricated as presented in Re-Formable Semi-Rigid Cable Assemblies were connected to the antenna array which were located at 90° during the rotation of the measurement as shown in The performance of the printed graphene antenna array with the rectangular slot was validated by measuring the radiation pattern. The simulated and measured normalised radiation patterns were plotted in the E-plane and H-plane at a respective frequency as shown in Theoretically, scanning phased array can be evaluated by Equation (4):The performances of printed graphene antennas were studied for single and array elements at 15 GHz. The 4 element antenna array was successfully fabricated by introducing a rectangular slot for reducing mutual coupling. From the results of the impedance bandwidth, antenna gain, and antenna efficiency, the printed graphene antenna at a single element fulfils the 5G requirement, on par with the array element which has high gain, a narrow beam, and is steerable. This tiny antenna is comparable with other thin film metals and offers another selection for alternative methods to produce smaller-sized antennas. The challenge in this work was to control the dielectric substrate and conductivity of graphene during the curing process which caused a slightly different result. In the future, the graphene antenna can be improved by using a higher conductivity graphene ink or the choice of graphene ink must not involve a curing process for maintaining conductivity and dielectric stability.


The metal-free synthesis of graphene on single-crystal silicon substrates, the most common commercial semiconductor, is of paramount significance for many technological applications. In this work, we report the growth of graphene directly on an upside-down placed, single-crystal silicon substrate using metal-free, ambient-pressure chemical vapor deposition. By controlling the growth temperature, in-plane propagation, edge-propagation, and core-propagation, the process of graphene growth on silicon can be identified. This process produces atomically flat monolayer or bilayer graphene domains, concave bilayer graphene domains, and bulging few-layer graphene domains. This work would be a significant step toward the synthesis of large-area and layer-controlled, high-quality graphene on single-crystal silicon substrates.The online version of this article (10.1007/s40820-017-0173-1) contains supplementary material, which is available to authorized users. Graphene was successfully grown on single-crystal silicon substrates using metal-free, ambient-pressure chemical vapor deposition.Atomically flat monolayer or bilayer graphene domains, concave bilayer graphene domains, and bulging few-layer graphene domains can be produced by controlling the growth temperature.In-plane propagation, edge-propagation, and core-propagation processes are proposed to evaluate the sequentially changing graphene domains.Graphene is a novel two-dimensional material with remarkable mechanical and electrical properties, such as high carrier-mobility and outstanding mechanical flexibility . Since t2 . D. D42]. DAt a higher temperature, both the hydrocarbon radicals and the silicon surface should become more active, and an additional layer of graphene forms on the active edge of the monolayer graphene domain. The growth mechanism of graphene changes to edge-propagation instead. Therefore, concave bilayer domains with larger size are formed, as depicted in Fig. n-type and p-type silicon). We found that the growth regimes of graphene are independent of the Si surface structure, further indicating that the direct growth of graphene on silicon is a self-propagating process and follows the van der Waals growth model. The growth of graphene on Si is mainly controlled by the temperature and the confined, local environment of the precursors.The growth of graphene was further explored using different orientations and doping types of single-crystal silicon substrates Below is the link to the electronic supplementary material.


In this paper, we propose a metamaterial structure for realizing the electromagnetically induced transparency effect in the MIR region, which consists of a gold split-ring and a graphene split-ring. The simulated results indicate that a single tunable transparency window can be realized in the structure due to the hybridization between the two rings. The transparency window can be tuned individually by the coupling distance and/or the Fermi level of the graphene split-ring via electrostatic gating. These results could find significant applications in nanoscale light control and functional devices operating such as sensors and modulators. Electromagnetically-induced transparency (EIT) is a concept originally observed in atomic physics and arises due to quantum interference, resulting in a narrowband transparency window for light propagating through an originally opaque medium ,2. The EGraphene is a two-dimensional material which is composed of single layer of carbon atoms, which has been widely used in optoelectronic devices, such as optical modulators ,29,30 duRecently, L.S. Yan et al. have investigated that a plasmonic waveguide system based on side coupled complementary split-ring resonators and the electromagnetic responses of CSRR can be flexibly handled by changing the asymmetry degree of the structure and the width of the metallic baffles . W. Yu e2O3 and the graphene split-ring is composed of 5 layers of graphene in our following simulations so as to enhance the modulation depth [1 whose material is gold as shown in 1 is negligible because of the relatively small size with respect to the structure. The incident light propagates in the z direction and its polarization is along x direction. Both the gold split-ring and graphene split-ring are symmetric in y direction and their symmetry axis are coinciding. The (complex) refractive index of Al2O3 is obtained from Table I, page 662 in Ref. [18cm−3 and its refractive index can be obtained from Ref. [2O3 and doped Si have been considered having the dispersion and absorption effects in our simulations.As shown in on depth . The bot in Ref.  and the rom Ref. . Both thσ(ω) is dominated by interband and intraband transitions within the random-phase approximation, which can be calculated as [ω is the optical angular frequency, T is the ambient temperature (300 K in this paper), fE is the Fermi level of graphene, ħ = h/2π is the reduced Plank’s constant, Bk is the Boltzmann constant, τ is the carrier relaxation time and τ = ħ/2Γ [Γ is the scattering rate and it is set 0.00099 eV in this paper (corresponding to the carrier relaxation time τ ≈ 0.33 ps). It can be seen from the above formula that the surface conductivity is greatly affected by the Fermi level and the Fermi level is determined by the carrier concentration gn and Fermi velocity fV. The formula fE = f(πng)ħV1/2 with the Fermi velocity of fV = 106 m/s can be obtained from Ref. [gn = ε0dVgε/eH1, where gV is the applied voltage and dε is the permittivity of Al2O3 and H1 is the thickness of Al2O3. In the following simulation, graphene is modeled as a thin anisotropic material film and the in-plane permittivity is approximated to be [gt is the thickness of graphene which is estimated as 1 nm [0ε is the permittivity in vacuum. In addition, the conductivity of the N-layer graphene is Nσ [The optical properties of the gold material are obtained from Palik’s experimental data. The surface complex conductivity of graphene lated as ,45:(1)σεg=1+ as 1 nm  and ε0 ine is Nσ ,50.In this paper, we use the finite-difference time-domain (FDTD) simulations to numerically investigate the properties of the structure. The perfectly matched layer (PML) absorbing boundary conditions are set on the z direction while the periodic boundary conditions are set on the x and y directions.fE of the graphene split-ring is set as 0.9 eV and the coupling distance d is 20 nm. From the transmission spectra in To demonstrate the EIT effect, we numerically calculate the simulated transmission spectra of the unit cell with the graphene split-ring and the gold split-ring are shown in d is 20 nm. It can be observed that dip II is obviously blue-shifted from 7.97 μm to 4.35 μm while dip I blue-shifts slightly and its transmittance increases with the layers of the graphene split-ring N increasing from 1 to 5. In addition, it is clear shown that the transmission peak is blue-shifted and the line width of the transmission peak becomes smaller and narrower. This is because the hybridized of the gold split-ring and graphene split-ring become stronger with increasing the layer number N. It is also found that 4 layers or 5 layers of the graphene split-ring are suitable. Here we choose the graphene split-ring of 5 layers because the quality factor of transmission peak is larger than that of the 4 layers. In Then, we study the response of EIT effect resulting from the variation of the layers of graphene split-ring a and thed between the two rings. As shown in d. When d comes to 400 nm, the peak will disappear. It reveals that the interaction of gold split-ring and graphene split-ring becomes weaker and their interaction eventually disappears when the coupling distance is large enough. It can also find that dip I red-shifts slightly while the dip II blue-shifts with the increase of d and both their transmittance decrease. Therefore, the coupling strength of the metamaterial structure greatly depends on the distance of the gold split-ring and graphene split-ring.Furthermore, we also investigate the dependence of the EIT spectra on the distance λ = 3.83 μm) and wavelength II (λ = 4.35 μm) are shown in d is 20 nm, and the Fermi level of graphene split-ring is 0.9 eV.To get further insight into the physics of the EIT phenomenon of our structure, the electric-field distributions of the z-component at wavelength I (d is small enough, their local surface plasmons will couple to each other, which is analogous to σ hybridization of p orbital electron in the atomic outer layer. From λ = 3.83 μm. In contrast, the positive and negative induced charges are distributed on the left and right sides of the graphene split-ring. The different charge distributions of the two rings are similar to the anti-bonding mode of the hybrid molecular system. On the other hand, one can find that the distribution of induced change in the gold split-ring remains unchanged at the wavelength λ = 4.35 μm, while the distribution of induced change in the graphene split-ring is opposite to the situation at λ = 3.83 μm from The placement of graphene split-ring and gold split-ring can be treated as asymmetric nanoparticle dimer which has been investigated in Ref. . When d f1/2λ~1/E [As mentioned earlier, the complex surface conductivity of graphene can be changed by controlling its Fermi level, which can be realized by applying different gate-voltages to the graphene split-ring. Compared with the reported bright-bright mode coupling EIT structure fabricated by metallic material, we can realize the dynamically control of the EIT window in our metamaterial structure without reconstructing the geometries or imbedding other actively controlled materials. ~1/Ef1/2 . At the In this paper, we have successfully proposed that the EIT effect can be realized through bright-bright modes coupling in the MIR regions. The EIT window appears due to the hybridization between the gold split-ring and graphene split-ring and it is sensitive to the coupling distance. Moreover, we investigate the response of EIT effect resulting from the variation of the layers of graphene split-ring and periods in the x direction. Particularly, the EIT window can be dynamically tuned by adjusting the Fermi level of the graphene split-ring instead of reconstructing the structure. These properties could lead to new opportunities for many important applications, such as sensors and slow light modulators in MIR regions.


In this paper, we report the molecular docking study of graphene oxide and glucose oxidase (GOx) enzyme for a potential glucose biosensing application. The large surface area and good electrical properties have made graphene oxide as one of the best candidates for an enzyme immobilizer and transducer in the biosensing system. Our molecular docking results revealed that graphene oxide plays a role as a GOx enzyme immobilizer in the glucose biosensor system since it can spontaneously bind with GOx at specific regions separated from the active sites of glucose and not interfering or blocking the glucose sensing by GOx in an enzyme-assisted biosensor system. The strongest binding affinity of GOx-graphene oxide interaction is −11.6 kCal/mol and dominated by hydrophobic interaction. Other modes of interactions with a lower binding affinity have shown the existence of some hydrogen bonds (H-bonds). A possibility of direct sensing (interaction) model of glucose by graphene oxide (non-enzymatic sensing mechanism) was also studied in this paper, and showed a possible direct glucose sensing by graphene oxide through the H-bond interaction, even though with a much lower binding affinity of −4.2 kCal/mol. It was also found that in a direct glucose sensing mechanism, the sensing interaction can take place anywhere on the graphene oxide surface with almost similar binding affinity. T network ,7,8,9,10 network , gas ads network , and opt network . The larThe large surface area combined with a very good electrical conductivity has made graphene-based biosensors powerful in terms of sensing accuracy and selectivity. The excellent biocompatibility and non-toxic properties also favor graphene for biosensor applications. Utilizing graphene in biosensing system requires incorporation of other materials to build a graphene composite or thin film electrodes. Some materials such as Nafion, polyaniline, and gold nanoparticles have been combined with graphene to immobilize the enzyme and to facilitate the electrochemical sensing mechanism. Some applications of graphene in biosensors include cholesterol detection ,15, DNA The use of graphene in glucose biosensing systems has been utilized intensively for more than a decade. There are two methods of glucose biosensing widely known, first the enzyme-assisted, and second, the non-enzymatic (direct) mechanism. For the enzyme-assisted method, researchers have focused on synthesizing the graphene nanocomposite system using various combinations of materials  to immobilize the glucose oxidase enzyme and to facilitate the electrochemical process in the biosensors . Fu et aThe molecular docking method is a computational approach to analyze the receptor-ligand interaction by utilizing a rigorous physical concept combined with the optimization and statistical method. Through a docking method, we can have a good prediction on the likelihood of a particular molecule in making a complex with another molecule. There are two types of docking, rigid docking, and flexible docking. In this paper, we use a rigid docking where the target structure is rigid, while the ligand structure is flexible. The docking simulation of graphene oxide with glucose oxidase in this paper was inspired by the experimental work by Wang et al. , where td-glucose. Secondly, we assume that the interaction between GOx with graphene oxide will not change the conformation of GOx in its native state and do not interfere with the glucose sensing process in the active sites of GOx. Although in reality the binding of graphene oxide with GOx enzyme in an aqueous condition most likely will change the enzyme’s conformation, but for the simplicity we assumed here that the adsorption of enzyme on graphene oxide surface is solely based on rigid docking calculation, without considering the dynamics of the complex in a particular environment (solvent). Further evaluations and more precise simulation of GOx-graphene oxide complex’s structural integrity through a molecular dynamics simulation [d glucose) molecule directly interacts with graphene oxide. The binding affinities and binding sites of this mechanism are our interest.The aim of this research is to elucidate the molecular interaction between graphene oxide with GOx enzyme and glucose with graphene oxide to explore the graphene oxide’s potential in a biosensing system, whether as an enzyme immobilizer or as an active sensing material. There are some assumptions made at the beginning of this research. Firstly, we focus on the molecular interactions between graphene and GOx enzyme and not delving too much into the interaction between graphene and other materials, such as Nafion, polyaniline, gold nanoparticles, and etc. The blind docking method was chosen to enclose the whole surface of GOx enzyme and to identify the binding sites of graphene oxide as compared to the binding (active) sites of β-mulation  and molewww.rcsb.org with PDB code of 1CF3 [Aspergillus Niger bacteria. The enzyme structure was cleaned from water molecules, natural ligand, and other residual substrates using the Chimera program [*.pdb format. The docking preparations for the enzyme receptor were done using Autodock Tools ADT 1.5.6 [*.pdbqt format. The structure of the enzyme receptor and the graphene oxide are shown in The structure of glucose oxidase for the enzyme receptor was downloaded from  of 1CF3 . This en program , then itDT 1.5.6 . In this*.sdf format from https://pubchem.ncbi.nlm.nih.gov. The 2D structure then converted into a 3D structure using an online server at https://www.mn-am.com/online_demos/corina_demo. As we know, there are three kinds of graphene, pristine graphene, graphene oxide, and reduced graphene oxide [*.pdb format using Chimera. The polar hydrogen bonds and the Gasteiger charges were added to the ligand before it was saved in the *.pdbqt format by using Autodock tools ADT 1.56. The two-dimensional structure of graphene oxide (PubChem CID:124202900) was downloaded in the Since there is only limited information regarding the binding of graphene oxide on GOx, we had to perform a blind docking method to identify the possible binding modes. The blind docking was performed using Autodock Vina software  to find The sensing mechanism of glucose by GOx can be expressed asd-glucose into d-glucono-1,5-lactone, which is then hydrolyzed to gluconic acid and hydrogen peroxide (H2O2). The hydrogen peroxide then dissociates into 2H+, O2, and two free electrons, which can be captured by the electrode and measured by the electronic instrument [The glucose oxidase enzyme catalyzes the oxidation of β-strument . The send-glucose, the sensing mechanism as explained by Equation (1) was not disturbed and still took place. In this mechanism, graphene oxide was acting as an enzyme immobilizer which facilitates, and not interferes with the glucose sensing activity by GOx enzyme.In this proposed biosensing system, the graphene layers were attached to the cathode (by coating technique) and expected to bind with GOx enzymes with some kind of non-covalent interaction at particular binding sites. By assuming the GOx-graphene oxide interaction occurred at a different pocket as compared to the active sites of β-d glucose are GLU 412, HIS 516, HIS 559 [d glucose and negates the possibility of using graphene in biosensing applications. On the other hand, if graphene does not interact with the active sites of glucose, then graphene acts as a non-competitive ligand to glucose and can be used as a GOx enzyme immobilizer in an enzyme-assisted biosensor, such as in [The catalytic sites of GOx enzyme which interacts with β- HIS 559 . Throughch as in . The AutIt was found that mode 1 to 3 have almost similar binding sites see , while mUnlike mode 1, the binding affinity of mode 3 is slightly weaker . There are 18 residues involved in the hydrophobic interaction in addition to two residues involved in H-bond interaction. Two H-bonds (double bonds) present in mode 3, with one H-bond, occurred in GLY 169 (H-bond distance of 3.149 Å), and one in ASN 168 (H-bond distance of 2.859 Å) as seen in d glucose) sensing by the graphene oxide through a simulation approach. We performed a blind-docking method to explore any possible interaction between the glucose molecule with the surface of the graphene oxide layer. The docking simulation was set by the grid box size of x = 30 Å, y = 24 Å, and z = 26 Å, and the origin point at x = 0.135, y = 0.041, and z = 0.018. The number of modes and exhaustiveness were set to 20 and 16 respectively. The docking results of this non-enzymatic sensing model are shown in The large surface area has made graphene oxide very promising for biosensor applications due to its superior electronic and electrochemical properties . Some red-glucose and graphene oxide. The strongest interaction (mode 1) has the binding affinity of ΔG = −4.2 kCal/mol, while the weakest (mode 20) has ΔG = −3.7 kCal/mol . From 20 binding modes, only mode 1 and 8 which have H-bond interactions. In mode 1, one H-bond is identified with a distance of 2.262 Å. Mode 8 has two H-bonds, each with a distance of 2.023 and 2.236 Å  interaction with a small portion of a strong non-covalent (H-bond) interaction presents. Since graphene oxide binds in the different pocket than β-d-glucose, the binding of GOx and graphene oxide will not interfere the glucose sensing process in the active sites of GOx . At this stage of research, we only consider the binding between graphene oxide and GOx enzyme. The point mutation effect on the particular amino acid of GOx enzyme will definitely change the enzyme adsorption at the graphene surface, since the adsorption free energy of each amino acid is different. A molecular dynamics simulation will be conducted in the future to reveal the conformational ensemble of the adsorbed enzyme as shown by [d-glucose and graphene oxide shows a promising application of graphene oxide layer as an active biosensor material through a non-enzymatic mechanism. The relatively the same binding affinity of β-d-glucose on graphene oxide , regardless of the position of interaction on the graphene oxide surface, offers an almost uniform glucose-probing sensitivity of the graphene oxide layer. Despite a much weaker binding affinity of β-d-glucose with graphene oxide as compared to the GOx-graphene oxide binding, the large surface area of graphene oxide with relatively the same sensitivity makes graphene oxide also promising to be developed into a non-enzymatic biosensor. The direct glucose sensing simulation here is only a preliminary study of a possible direct biosensing mechanism, where glucose (β-d glucose) molecules directly interact with graphene oxide. Further study must be done on this subject in the future. Unlike the drug discovery research where a high throughput virtual screening with multiple docking is common, the multiple docking of various analytes with graphene (or graphene oxide) to study the graphene’s selectivity, to the best of authors knowledge, has never been found in the literature and would be interesting to do in future.In this work, we have performed a molecular interaction analysis based on the molecular docking results to explore the potential of graphene oxide in glucose biosensing applications. Two mechanisms were simulated in this paper, first the enzyme-assisted mechanism and second the non-enzymatic (direct) mechanism. Based on the simplified docking parameter assumptions used here, which in some cases might be favorable compared to experimentally achievable results, molecular docking analysis has revealed that graphene oxide can be used as an enzyme immobilizer as shown by its strong binding affinity  and its binding sites which do not overlap with the active sites of β-shown by . The mol