The amplitude and sign of the potential can be modified without altering its periodicity by employing photoreactive molecules or their reaction products. In this regard, the supramolecular lattice/graphene bilayer represents the hybrid analogue of fully inorganic van der Waals heterostructures, highlighting the rich prospects that molecular design offers to create ad hoc materials.The rise of 2D materials made it possible to form heterostructures held together by weak interplanar van der Waals interactions. Within such van der Waals heterostructures, the occurrence of 2D periodic potentials significantly modifies the electronic structure of single sheets within the stack, therefore modulating the material properties. However, these periodic potentials are determined by the mechanical alignment of adjacent 2D materials, which is cumbersome and time-consuming. Here we show that programmable 1D periodic potentials extending over areas exceeding 10 et al. use a monolayer supramolecular lattice with a tunable one-dimensional periodic potential to modify the electronic structure of graphene.Two-dimensional material heterostructures enable unique electronic features by introducing periodic potentials. Here, Gobbi  Van der Waals (vdW) heterostructures, in which different two-dimensional 2D) materials are superimposed in a stacked configuration, represent a versatile experimental platform to study fundamental properties for device physics and materials science1D materia5From a more fundamental point of view, the mutual interaction between vertically-stacked 2D materials generates electronic properties that are different from those of the isolated materials, as experimentally demonstrated for graphene on boron nitride9101112In this context, the use of molecules offers two attractive features. First, organic molecules can prompt doping effects in 2D materials161718202123242526272829303132333426363738402. The amplitude of the periodic potential is mediated by the specific interaction between graphene and molecular dipoles, while the nanoscale periodicity is determined by the geometry of the self-assembly. In particular, by using a photoreactive organic molecule, we show how a subtle photo-induced change in the chemical structure of the starting molecule leads to a different amplitude of the potential, while leaving the periodicity unaltered.In this work, we demonstrate that a tunable periodic potential with 1D geometry on graphene can be realized at ambient conditions in a hybrid vdW heterostructure composed of a 2D supramolecular lattice (SL) self-assembled on graphene, with single domains extending over areas exceeding 250 × 250 nmThe analogy between hybrid and fully inorganic vdW heterostructures goes beyond their layered nature; in analogy to stacks of inorganic 2D materials, vdW forces drive the interaction between graphene and the self-assembled molecular layer, and similar inter-layer interactions can be envisaged. However, the paradigm leading to the formation of such heterostructure is substantially different. While in inorganic vdW heterostructures the different monolayers are mechanically superimposed with an empirical  alignmentin situ generated carbene binds to the solvent molecules in its proximity, generating modified MBBs in a mixture which we call MBB-2. As detailed in the ad hoc synthesized molecules . Absorption spectra allow to follow the change in the molecular head of MBB-1, as shown in λ=365 nm , the characteristic absorption at λ=365 nm of MBB-1 disappears, revealing that MBB-1 was efficiently photolyzed.Our approach is schematically illustrated in 432 as substrate  imaging. We performed STM imaging in ambient conditions on dry SLs, formed by simply spin-coating a solution of either MBB-1 or MBB-2  nm, b=(0.9±0.1)  nm and α=(84±2)° therefore leading to an area A=(3.4±0.1) nm2, with each unit cell containing two molecules (a=(3.8±0.2) nm, b=(0.9±0.1) nm, α=(84±2)° giving rise to an area A=(3.5±0.2) nm2. The nearly identical unit cells are justified if one considers that on ultraviolet irradiation only a few atoms in the molecular head are replaced. Instead, the long alkyl chains are unaltered in both cases. Hence, MBB-1 and MBB-2 form SLs that possess the same lattice parameters but differ in the head groups, resulting in different electronic interactions with graphene.In the case of MBB-1, the functional heads lie close to each other, with the alkyl chains forming an interdigitated structure, exhibiting a unit cell where olecules . Similarolecules . Again, 2 interfaces2 to consider a situation closer to that of the actual devices, as shown in the 2 substrate. However, even on CVD graphene, it is possible to clearly observe the existence of ordered lamellar structures whose size is in good agreement with that monitored on HOPG. This finding confirms that the MBBs assemble in the same way on both HOPG and graphene/SiO2. Large-size STM images show that the crystalline order is maintained over wide areas even on CVD graphene on SiO2, as displayed in the For both MBB-1 and MBB-2, crystalline domains with different orientations of the lamellae may form on the graphene surface. This evidence reflects the three-fold symmetry of the substrate and may give rise to misorientation in the direction of the 1D potential. Therefore the size of single-crystalline domains is a key parameter for the relevance of this study on the device physics. To gain detailed insight into this issue, we recorded survey STM images mapping the surface over a scale of a few hundred nanometres. IDS on the gate voltage VGS is displayed in 2 V−1 s−1 for the device in 3, and the mobility decreased to its initial value . The devices were measured before and after the formation of the MBB-1 and MBB-2 SLs, to evaluate their effect on the electrical characteristics of graphene. The measurements were reproduced on three different devices for each MBB, and the results were in qualitative and quantitative agreement (see the ‘Methods' section for details). Representative dependence of the drain current  see the .V>25 V), as shown in p>5 × 1012 cm−2, according to the widely used parallel-plane capacitor modelIn the case of MBB-2, after the formation of the SL the charge neutrality point was shifted towards positive voltages  measurements on a contacted graphene flake, acting as the device active layer, before and after formation of a MBB-1-SL. After formation of the SL, some inhomogeneities in the conductance are introduced with a length scale of a few hundred nanometres, which is the typical size of the single domains imaged by STM. Although the lamellas cannot be visualized by C-AFM, this finding supports the presence of anisotropic conductance within graphene covered by a single-domain SL, as detailed in 3 group is pointing parallel to graphene, whereas the axis of the N=N double bond is normal to the graphene plane , while in the case of MBB-2 it is significantly higher (μz=−0.82 D mol−1). The doping effect in the MBB-2 case, therefore, results from the higher vertical dipole moment of the SLs, as expected from the geometry of the assembly. By accounting for molecular motion at room temperature from MD simulations, we predict a negligible and a significant work function shift for MBB-1 and MBB-2, respectively, as experimentally verified by photoemission spectroscopy in air (see p=5 × 1012 cm−2) and a minor p-type doping for MBB-1 (Δp<5 × 1011 cm−2), in excellent agreement with the device experiments. Moreover, our theoretical analysis confirms that the origin of the measured effects can be entirely ascribed to the geometry of the head groups, while the assembly of the alkyl chains, similar for MBB-1 and MBB-2, is not a source of doping.To gain more insight into the origin of the doping effects, the interactions of the molecular assembly with graphene were elucidated through molecular mechanics/molecular dynamics (MM/MD) simulations. For the case of MBB-2, we assumed that all the molecules at the surface possess the structure shown in ne plane . Convers surface . Interes surface . Insteadne layer . Based o air see . The worProceeding with the analysis, the spatially resolved electrostatic potential at the graphene/molecule interface was calculated by means of classical microelectrostatic calculations (see V (z=8 Å)–V (z=0 Å) for both MBB-1 and MBB-2, where V (z=8 Å) is the potential calculated on the parallel plane just above the SLs, and V (z=0 Å) is the potential at the graphene surface. Such differential potential probes the dipolar field associated with the out-of-plane component of the molecular dipoles and has an effect similar to that of a fixed externally applied gate voltage. In both assemblies, such induced gating potential is characterized by a 1D-modulation, with higher negative values localized at the molecular heads separated by inert alkyl chains. The amplitude of the induced gating effect increases by a factor of 4 from MBB-1 to MBB-2, as expected on the basis of the different vertical dipoles. Note that image charge and depolarization effects associated with intermolecular interactions have been estimated using DFT electronic structure calculations implementing periodic boundary conditions. These were found to reduce the electric dipole per molecule by ∼30% for MMB-1 and 20% for MBB-2. Thus, image effects are found to be fairly limited in our case and the graphene layer acts primarily as a template breaking up symmetry in picking selected dipolar conformations for the MBBs at the surface, which then translates into the observed electrostatic potential shown in In This different amplitude induced by the MBB-1 and MBB-2 SL demonstrates the ability of introducing significant variations in the SL-induced periodic grating with a subtle light-induced change in the molecular structure. In both cases, the in-plane distribution of the potential can be considered as a nanoscopic experimental realization of a Kronig–Penney potential. We highlight that not only its geometry, but also its amplitude is within the same order of magnitude of that considered in the initial prediction of anisotropic behaviour of charge carriers in graphene superlatticesa=(3.8±0.2) nm, b=(0.9±0.1) nm and α=(84±2)° which lead to an area A=(3.5±0.2) nm2, analogous to that of MBB-1 and MBB-2. Again, the atoms incorporated through photolysis do not perturb the assembly at the nanoscale, which is determined and imposed by the alkyl chains. Instead, the intensity of the induced potential is determined by the interaction with the head groups. The overall effect can be measured in a three-terminal device, by covering a graphene device with a MBB-3 SL. In this case  might introduce a 1D periodic potential capable of locally varying the position of the Fermi level, resulting in alternating hole- or electron- rich regions. In this way, one would obtain a series of consecutive nanoscale p–n junctions with distinctive charge transport properties and optical response.The concept that interplanar interactions are capable of modifying fundamental electronic properties of pristine materials lies at the heart of the research field of vdW heterostructures. In this context, the possibility of introducing tunable 1D potentials through organic SLs is particularly appealing. For instance, one could combine graphene covered by a SL with other inorganic 2D materials to fabricate more complex multi-layered hybrid vdW heterostructures. In such systems, an ordered lamellar assembly modifying the momentum dispersion of graphene would induce anisotropy not only on the in-plane (graphene) but also on the inter-layer charge transport, for which momentum conservation plays an important role2 ref.  and blac2 ref.  to demonIn conclusion, our study shows that organic SLs represent an ideal system for generating periodic potentials whose periodicity, amplitude and sign can be pre-programmed by careful molecular design. In particular, here we have shown that a periodic potential with 1D geometry can be generated at the graphene surface and manipulated by making use of molecular photo-reactivity. In perspective, a 1D potential will offer the opportunity to introduce anisotropy in otherwise isotropic materials, hence paving the way for design and implementation of novel functional vdW heterostructures, for example, for 1D charge transport. The control over periodic potentials can hardly be achieved in vdW heterostructures based solely on inorganic 2D materials, while their combination with SLs allows expanding substantially the 2D material library. Chemistry can offer an almost unlimited choice of SL that can form hybrid vdW heterostructures with controllable structural and electronic properties, exerting periodic potentials with tunable amplitude and periodicity. In perspective, a great deal of novel electrical, magnetic, piezoelectric and optical functionalities arising from such hybrid vdW heterostructures are expected by taking full advantage of the infinite degrees of freedom offered by the design of the MBBs.Full details regarding the characterization and synthesis of the MBBs are given in –3 M. Photochemical reaction of MBB-1 was performed in air-equilibrated CHCl3 (Uvasol Merck-Millipore) or in degassed diethyalmine (Sigma-Aldrich) by using an Ultraviolet lamp (UV-6 L/M Herolab) with λirr=365 (±5) nm and a power density=1.7 mW cm−2.Absorption spectra were recorded at room temperature (∼25 °C) with a JASCO V-670 spectrophotometer and all solutions were examined in quartz cells with 2-mm pathlength (HELLMA) with a concentration of 2.2 × 102 (90 nm) substrates with a Microtech laser writer, exposing with a λ=405 nm laser a standard photoresist . Gold (without adhesion layer) was thermally evaporated onto the patterned photoresist and lift-off was carried out in warm acetone (40 °C). After fabrication, the devices were immersed in warm NMP (40 °C) overnight, rinsed with chloroform, acetone and isopropanol. The devices were kept in a nitrogen-filled glove box in which they could be measured in a probe station connected to a Keithley 2636. By employing this procedure, the graphene device showed almost-ideal, symmetric and stable current–voltage IDS−VGS characteristics, with the charge neutrality point close to a gate voltage VGS=0 V, and hole mobility reproducibly above 1,800 cm2 V−1 s−1. The carrier mobility was determined by the conventional parallel-plane capacitor model:Back gated devices were fabricated on Si/SiOL and W are the graphene channel length and width, t is the thickness of gate oxide, ɛr is the relative dielectric permittivity of SiO2. The mobility was extracted at the carrier concentration value of |n|=1 × 1012 cm−2, by measuring dIDS/dVGS at 4.5 V away from the charge neutrality point. In all the electrical measurements, the potential applied between the graphene electrodes was V=10 mV. The reproducibility of the effect of each SL on the electrical characteristics of devices was tested by repeating the measurement on three different devices for each MBB. For every test, a fresh solution of MBB was employed. The doping effect is reproducible: MBB-1 was found to induce small p-type doping (ΔV<5 V in the three tested devices); MBB-2 significantly higher p-type doping (ΔV>25 V in the three tested devices); and MBB-3 n-type doping (ΔV in the range between −7 and −12 V in the three tested devices). Moreover, the experiments were repeated several times on the same device, by washing the MBBs away and re-forming the SL. In the cases of MBB-1 and MBB-3, the increase in mobility after the formation of the SL was also found in the three tested devices.where 2 and onto devices from chloroform solutions (1 mg ml−1). For the sake of consistency, the solutions were spin-coated onto the graphene devices by employing the same parameters used for the STM imaging. In the case of irradiation in diethylamine, the molecules were dried after irradiation and re-solubilized in chloroform before spin-coating. In the case of devices, the solution were spin-coated in situ in the nitrogen-filled glovebox. The SLs could be washed away by rinsing the substrates or the devices with CHCl3.To form the SLs, molecules were spin-coated onto either HOPG or CVD graphene on SiO2 (300 nm) purchased from Graphenea. The graphene sample has been used as received, without any additional cleaning step, and has been stored in air for 4 months before the STM experiments. The substrates were glued onto a magnetic disk and an electric contact was made with (conductive) silver paint .STM measurements were carried out by using a Veeco scanning tunneling microscope  operating with an A piezoelectric scanner which allowed the mapping of a maximum area of 1 μm × 1 μm. As substrates, we used highly oriented pyrolytic graphite and commercial CVD graphene supported on Si/SiOVt to 10 mV and setting the average tunneling current It=60 pA. Tip height and current were measured for all STM images.The STM tips were mechanically cut from a Pt/Ir wire . The images were obtained in air at room temperature. The raw STM data were processed through the application of background flattening, and in the case of the HOPG substrates in Full details regarding the MD/MM and DFT calculations are given in The data that support the findings of this study are available from the corresponding authors on request.How to cite this article: Gobbi, M. et al. Periodic potentials in hybrid van der Waals heterostructures formed by supramolecular lattices on graphene. Nat. Commun.8, 14767 doi: 10.1038/ncomms14767 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Methods, Supplementary Note and Supplementary References.


The primary objective was to assess OS according to PD-1, PD-L1, PD-L2 expression status on PBMCs and lymphocyte subsets. Our results indicate that the percentage of PD-L1+CD3+, PD-L1+CD3+CD8+ PD-L2+PBMCs, PD-L2+CD3+, PD-L2+CD3+CD4+ cells was higher in patients than in healthy donors. Survival was decreased among patients with a high percentage of either PD-1+PBMCs, PD-1+CD3+, PD-L1+CD3+, PD-L1+CD3+CD8+, PD-L2+CD3+, PD-L2+CD3+CD4+, or PD-L2+CD3+CD8+ cells. IL-2 and TNF-α showed the strongest association with PD-L1 and PD-L2 expression on specific subsets of T-lymphocytes. Our findings suggest that increased PD-1/PD-L1/PDL-2 expression in PBMCs, particularly in T-cells, may be an additional mechanism leading to tumor escape from immune control. This study is registered with ClinicalTrials.gov, number NCT02758314.Binding of programmed death-1 (PD-1) with its ligands (PD-L1/2) transmits a co-inhibitory signal in activated T-cells that promotes T-cell exhaustion, leading to tumor immune evasion. The efficacy of antibodies targeting PD-1 and PD-L1 has led to a paradigm shift in lung cancer treatment but the prognostic and predictive value of tumor PD-L1 expression remains controversial. Evaluating PD-1, PD-L1/2 expression in peripheral blood cells may serve as a potential biomarker for prognosis and response to therapy. In this prospective observational study, plasma cytokine levels and PD-1, PD-L1 and PD-L2 expression was evaluated in circulating CD3 Lung cancer remains the leading cause of cancer-related deaths in men, and had, in 2015, the second highest absolute incidence globally. It has been estimated that in 2017 lung cancer will account for 13% of all new cancer cases and for 26% of cancer-related deaths .Transformed cells can evade immune system elimination by decreasing the expression of antigen presentation molecules and co-stimulatory molecules or, by increasing the expression of co-inhibitory molecules –8 such aAnti-PD-1 ICIs have achieved higher therapeutic responses than standard-of-care chemotherapy in patients with advanced NSCLC, both in the second-line –19 and f+, CD3+CD8+, PD-1+PBMCs, PD-1+CD3+, PD-1+CD3+CD4+, PD-1+CD3+CD8+, PD-L1+CD3+CD4+ and PD-L2+CD3+CD8+ cells .No differences were found between patients and controls with regards to the percentage of CD3+CD4+ cells than controls  as well as a lower percentage of PD-L1+PBMCs .However, patients had a lower percentage of CD3+CD3+ cells , PD-L1+CD3+CD8+ cells , PD-L2+PBMCs , PD-L2+CD3+ cells  and PD-L2+CD3+CD4+ cells , . A negative correlation was also found between IL-6 levels and the percentage of CD3+CD4+PD-L1+, CD3+PD-L2+ and CD3+CD8+PD-L2+ cells . Similarly, the levels of IL-8 negatively correlated with the percentage of CD3+PD-L2+ and CD3+CD4+PD-L2+ cells . Finally, the plasma concentration of IL-17A negatively correlated with the percentage of CD3+CD8+PD-L1+, CD3+CD8+PD-L2+ and CD3+CD4+PD-L2+ cells . In contrast, there was a positive correlation between the concentration of TNF-α and the percentage of CD3+PD-L1+, CD3+PD-L2+, CD3+CD4+PD-L1+, CD3+CD4+PD-L2+, CD3+CD8+PD-L1+ and CD3+CD8+PD-L2+ cells . Similarly, the concentration of IL-31 positively correlated with the percentage of CD3+PD-L2+ and CD3+CD4+PD-L2+ , , .+CD3+CD4+ and PD-1+CD3+CD8+ cells positively correlated with age . Tobacco exposure was associated with higher percentage of PD-1+PBMCs [P=0.010]. The percentage of PD-1+, PD-L1+ and PD-L2+ cells was not associated with gender, histology pattern, or clinical stage. EGFR mutation status was associated with a lower percentage of PD-L2+CD3+ and PD-L2+CD3+CD8+ (P=0.051 and P=0.008). Decline in functional status (ECOG ≥ 2) was associated with an increase in the percentage of PD-1+CD8+ cells , (Table The percentage of PD-L1P=0.08), The mean follow-up of patients was 22.9 months, with a range of 18.6 to 27.2 months. The median OS was 17.5 months (95%CI 9.7 - 25.4). Clinical and demographic characteristics  did not affect median OS when assessed in a multivariate analysis. Patients with an ECOG ≤1 also had a better OS . Patients with >1.25% of PD-1+PBMCs had an OS of 12.6 months (95% CI 5.8-19.3) vs patients with ≤1.25% of PD-1+PBMCs, whose OS was 23.3 months (95% CI 16.3-30.2). Patients with >1.1% of CD3+PD-1+ had an OS of 9.9 months (95% CI 3.9-15.9) vs. patients with ≤1.1% of CD3+PD-1+, whose OS was 23.3 months (95% CI 18.7-27.8), . Patients with >3.6% of CD3+PD-L1+ or with > 1.5% of CD3+CD8+PD-L1+ cells had an inferior survival rate (6.8 months and 6.9 months) compared with the patients with ≤ these cutoff values (P=0.012), , PD-L2+CD4+ (>0.5%) and PD-L2+CD3+CD8+ (>0.625%) cells showed approximately a 15 month decrease in OS , , PD-L1+CD3+ , PD-L1+CD3+CD8+  and PD-L2+CD3+ cells . In contrast to the univariate analysis, in the multivariate analysis a high percentage of PD-1+CD3+, PD-1+PBMCs, PD-L2+CD3+CD4+ and PD-L2+CD3+CD8+ cells was no longer significantly associated with reduced OS.Calculation of the correlation coefficients for every combination of the predictors selected from the univariate analysis  revealedThe expression of PD-1, PD-L1 & PD-L2 is modulated by the inflammatory milieu, through the action of cytokines . Our resIt has previously been reported that IL-2 increases PD-L1 expression on human T cells , which wThe efficacy of PD-1/PD-L1 ICIs has been demonstrated in numerous preclinical models , 32 as wIt is likely that some of the reliability and reproducibility issues surrounding the use of PD-L1 as a biomarker derive from variable assay methodology, heterogeneity of tumor sampling and surgical inaccessibility . Novel s+, CD3+CD4+ and CD3+CD8+ cells, NSCLC patients had a higher percentage of circulating PD-L1+CD3+ and PD-L1+CD3+CD8+ cells (but not PD-L1+CD3+CD4+), as well as a higher percentage of PD-L2+PBMCs, PD-L2+CD3+ and PD-L2+CD3+CD4+ cells (but not PD-L2+CD3+CD8+).Our results concur that there are mechanisms independent of tumoral PD-L1 expression by which tumor evasion occurs. Although no differences were found between patients and controls with regards to the percentage of peripheral PBMCs, CD3+PBMCs, PD-1+CD3+, PD-L1+CD3+, PD-L1+CD3+CD8+, PD-L2+CD3+, PD-L2+CD3+CD4+ and PD-L2+CD3+CD8+ cells. However, in the multivariate analysis only a high percentage of PD-1+CD3+CD4+, PD-L1+CD3+, PD-L1+CD3+CD8+ and PD-L2+CD3+ cells were significantly associated with reduced OS in NSCLC patients. This is likely due to the tight interrelationship governing the expression of receptor and ligands from the same signaling pathway, which causes multicollinearity problems that reduce the power of a multivariate analysis. It is possible that a larger scale study or a different regression model would confirm that, in addition to the predictors previously described, a high percentage of PD-1+CD3+, PD-1+PBMCs, PD-L2+CD3+CD4+ and PD-L2+CD3+CD8+ cells would also be associated with reduced OS in NSCLC patients.Furthermore, the survival analysis revealed a dramatic reduction in the OS of patients with a high percentage of PD-1Nevertheless, our results indicate that an additional source of PD-1 inhibitory ligands are T-cells themselves. This could represent an additional and important mechanism by which tumor immune evasion occurs in advanced NSCLC patients. However, future studies are necessary to provide a mechanistic insight into how the expression of these proteins leads to tumor immune escape.et al. reported a significant difference between the mean levels of PD-L1 in the blood serum of advanced NSCLC patients and healthy controls, being 0.723 (±0.081) ng/ml and 0.565 (±0.048ng/ml), respectively. By establishing a cut-off point of 0.636 ng/ml, the authors distinguished a clear correlation with survival and found an area under the curve of 0.956 (95%CI 0.927–0.985)  [CB/683]) of the INCan. This study is registered with Blood samples were prospectively collected (pretreatment) and handled as follows: a) Eight milliliters of blood were drawn into a plastic EDTA tube . Upon collection, the plasma was separated by centrifugation and stored at -80°C for subsequent cytokine analysis; b) Eight milliliters of blood were drawn into 3.2% citrate tubes (BD Biosciences). Upon collections samples were diluted with PBS  and separated by density gradient centrifugation . The interphase, containing peripheral blood mononuclear cells (PBMCs), was stored in liquid nitrogen for subsequent immunophenotyping.+, CD4+ and CD8+ T lymphocytes): anti-CD3-FITC, anti-PD-L1-PE, anti-CD8-APC Cy7, anti-PD-1-PE Cy7, anti-PD-L2-APC and anti-CD4-PerCp . The PBMC fraction was blocked for 5 minutes with a FAB anti-IgG. Samples were incubated for 45 minutes with the appropriate antibodies (2.5 μl) at room temperature and protected from light exposure. After incubation, 2 ml of a 1:1 solution of PBS-Fetal bovine serum (FBS) were added to each sample. Samples were then centrifuged at 1100 rpm for 5 minutes. The supernatant was decanted and cells were fixed in paraformaldehyde (1%).Expression of PD-1/PD-L1/PD-L2 was assessed retrospectively in prospectively collected blood samples. The following combination of human monoclonal antibodies were used according to the manufacturers’ instructions to identify different immune cell populations (CD3Gating strategy were set using fluorescence minus one (FMO) for PD-1 /PD-L1 &PD-L2. The samples were acquired in a FACS Aria II Flow Cytometer  and analyzed with FlowJo software 10.1 . The leukocyte population was gated based on morphological parameters on a forward vs side scatter (FSC/SSC) plot.Plasma levels of 14 cytokines  were quantified as previously described . Briefly2) test. Immune parameters associated with clinical variables were determined by bivariate analysis. Correlations were computed by linear regression and analyzed using the Spearman rank correlation test. The Receiver Operating Characteristic (ROC) method was performed to find the best cut-off point value for PD-1, PD-L1 and PD-L2 to be used for the survival analysis. OS curves were estimated by the Kaplan–Meier method while comparisons among groups were analyzed with log-rank or Breslow tests. Statistically significant and borderline variables  were included in the multivariate analyses. Statistical significance was determined as P ≤0.05 with a two-sided test. All data were analyzed using the SPSS software package version 20 .Continuous data were summarized as arithmetic means with standard deviations (SDs) or medians with ranges according to data distribution. Two group comparisons were tested using Student's t test or Mann-Whitney U (according to data distribution determined by the Kolmogorov Smirnov test). Nominal data was analyzed using the chi square (X


The world of dentistry is approaching graphene-based nanomaterials as substitutes for tissue engineering. Apart from its exceptional mechanical strength, electrical conductivity and thermal stability, graphene and its derivatives can be functionalized with several bioactive molecules. They can also be incorporated into different scaffolds used in regenerative dentistry, generating nanocomposites with improved characteristics. This review presents the state of the art of graphene-based nanomaterial applications in the dental field. We first discuss the interactions between cells and graphene, summarizing the available in vitro and in vivo studies concerning graphene biocompatibility and cytotoxicity. We then highlight the role of graphene-based nanomaterials in stem cell control, in terms of adhesion, proliferation and differentiation. Particular attention will be given to stem cells of dental origin, such as those isolated from dental pulp, periodontal ligament or dental follicle. The review then discusses the interactions between graphene-based nanomaterials with cells of the immune system; we also focus on the antibacterial activity of graphene nanomaterials. In the last section, we offer our perspectives on the various opportunities facing the use of graphene and its derivatives in associations with titanium dental implants, membranes for bone regeneration, resins, cements and adhesives as well as for tooth-whitening procedures. Tissue engineering is an interdisciplinary science which aims at developing biological substitutes to restore, maintain, or improve tissue function by using a combination of cells, scaffolds and suitable biochemical factors . ScaffolOver the years, several natural and synthetic three-dimensional 3D) scaffolds have been successfully developed and employed for various tissues, such as skin D scaffol, muscle In the last decade, many advances have been made in the dental tissue engineering field aimed at the regeneration of dental pulp , periodoIn the last few years, graphene and its derivatives have emerged as a new class of nanomaterials . GraphenSince its discovery in 2004 , many apThe present review intends to provide the reader an overview of the current state of the art of the graphene-based nanomaterials in tissue engineering in dentistry. Different aspects of the graphene-based nanomaterials will be discussed in detail. First, we highlight the role of stem cells and their interactions with graphene nanomaterials. We consider the influence of graphene-based nanomaterials both on biocompatibility, cytotoxicity and differentiation properties of stem cells. In this context, particular attention will be given to stem cells of dental origin. The interactions with immune cells and antibacterial properties of graphene, which are critical aspects to consider in the hostile environment of the oral cavity, are subsequently discussed. Finally, applications of graphene-based nanomaterials on dental implants, membranes, resins, cements and adhesives as well as on teeth-whitening are extensively presented.The first aspect to consider when developing a new nanomaterial for biomedical applications is its biocompatibility. Excellent biocompatibility is indeed essential in order to avoid any adverse effect of a material in living tissues . Apart fGraphene-based nanomaterials are rapidly spreading as scaffolds in tissue engineering applications, also in the dental field; consequently, assessments of their biocompatibility and cytotoxicity have to be done. Dosages, viability assays, type of nanomaterials, type of cells and uses need to be evaluated before developing a new reliable graphene-based nanomaterial.Over the years, biocompatibility of graphene and its derivatives has been largely discussed among authors. The comprehension of the toxicological potential of graphene-based nanomaterials, indeed, has to be carefully evaluated in order not to interrupt future in vivo studies and translational efforts . AccumulThe 3--2, 5-Diphenyltetrazolium bromide (MTT) assay, which measures the mitochondria function of the cells, is one of the preferred method for evaluating the effect of nanomaterials in cell culture . In one Shortly after, another work evaluated the toxicity and biocompatibility of GO to human lung A549 cells, a widely used cell line for toxicity studies . CompareAnother work that stresses this concept is the one by Sasidharan and colleagues . They fo2O2 decomposition and ⋅OH formation. Among various types of ROS, ⋅OH is an extremely reactive free radical and indiscriminately induces oxidative damages on various biomolecules, including DNA, lipids and proteins [The physicochemical mechanism of the oxidation-state dependent cytotoxicity of GO was proposed recently by the group of Zhang . They syproteins .Soon after, another distinct process contributing to the molecular basis of graphene cytotoxicity was unraveled by Duan and colleagues . They deWith regard to in vivo experiments, there are few studies concerning the biocompatibility of graphene-based nanomaterials since most of the investigations have only focused on the in vitro aspect of research. Nevertheless, the in vitro cell culture environment is definitely different from the in vivo complex 3D physiological condition. In addition, typical cytotoxicity assays measure the effect only during the first 12–24 h after exposure to a toxic substance although many biological reactions in vivo continue beyond 24 h.As for in vitro experiments, the in vivo effects of graphene-based nanomaterials were observed to be dependent on their concentration, physicochemical properties, exposure time, but also on administration route and on the characteristics of the animals used in the assays .Wang and colleagues observed that intravenous injection of GO at 0.1 mg and 0.25 mg showed no obvious toxicity to mice, whereas almost half animal population died after administration of 0.4 mg GO . All deaIn another study, GO or pluronic-dispersed graphene was administered by intratracheal instillation to mice . The autLater, the group of Yang tested several Polyethylene Glycol (PEG) functionalized GO materials with varied sizes and surface chemistry after oral and intraperitoneal injection to mice . The autConsidering certain inconsistencies and contradictory results in several studies, generalizations about graphene toxicity should be avoided. Indeed, it is generally accepted that the in vitro and in vivo behaviors of graphene-based nanomaterials are closely associated with their physicochemical properties, such as surface functionalization, coating, size and importantly, the administration routes. However, based on the studies presented in this section, we may summarize that the hydrophobic forms of graphene-based nanomaterials, which accumulate on the surface of cell membranes, are more toxic compared to the most hydrophilic ones. These, in fact, are more prone to infiltrate the cell membrane and to be internalized, as well as being removed from the application site. At the same time, when comparing GO with its reduced counterpart, rGO, the latter results less toxic than the hydrophilic form. Anyway, by controlling the GO reduction and maintaining the solubility, it is possible to minimize the GO toxicity and unravel the wide range of biomedical applications.As mentioned before, one of the key characteristics of the ideal scaffold is an effective and controlled guidance of the proliferation and differentiation of stem cells into specific tissue lineages, either in the absence or presence of chemical inducers and growth factors. In the past few years, graphene-based nanomaterials have been increasingly used as scaffold materials for stem cell growth and differentiation. In the specific field of dental tissue engineering, most of the studies focused on evaluating the role of graphene in driving the osteogenic differentiation of stem cells. Indeed, bone regeneration plays a fundamental role in regenerative dentistry and how incorporation of graphene-based nanomaterials can enhance osteoconductivity through stimulating both cellular osteogenic differentiation and biomineralization have been extensively investigated .In one of the first in vitro studies using stem cells, graphene produced by the Chemical Vapor Deposition (CVD) method and combined with different substrates was found to increase human Mesenchymal Stem Cells (MSCs) proliferation with no signs of cytotoxicity . MSCs arStarting from these results, another group investigated whether the graphene-induced osteogenesis of human MSCs was correlated to the chemical properties of graphene . In the Other studies have further tested the effects of GO on MSCs growth and osteogenic differentiation and the results varied. In the interesting work of Wei and colleagues, the authors compared the cellular behaviors of BM-MSCs towards pristine GO nanosheets using two in vitro biomimetic culture methods, that imitate similar situations in vivo . The seqIn recent years, also 3D graphene-based scaffolds have been actively explored and developed for the growth and differentiation of stem cells. The group of Crowder was the first to employ 3D graphene foams as substrates for human BM-MSCs culture . When seBesides being used in their pristine form, graphene nanomaterials have also been combined with different polymers, nanofibers, or nanoparticles to create novel composites for controlling the growth and differentiation of stem cells. Hydroxyapatite (HAp), for example, is a calcium phosphate ceramic commonly used for bone repair or regeneration due to its chemical similarity to that of natural apatite in bones . The recNatural bone is a composite material in which inorganic HAp nanocrystals are orderly embedded in an organic matrix made of collagen . CollageAmong others available materials, peptides containing the repeating arginine-glycine-aspartic acid (RGD) sequence, which is often found in Extracellular Matrix (ECM) proteins, have been widely applied to enhance cell adhesion to synthetic polymers or metallic surfaces ,59. In oOne last work worth mentioning is the one of Lim and colleagues . The autAll the works presented in this section demonstrate that, over the last few years, graphene has been found as one of the most promising biocompatible scaffolds for human MSCs adhesion, proliferation and differentiation, particularly towards the osteogenic lineage. These recent investigations clearly indicate a potential or active ability of graphene and its derivatives to induce the osteogenic differentiation of MSCs. Graphene alone seems to drive the spontaneous osteogenesis of stem cells; however, the presence of osteogenic inducers or growth factors in culture medium contributes in promoting this effect. Further summarizing the studies presented here, we may conclude that the association of graphene with stiff substrates, such as HAp, or with natural bone components, such as collagen, enhances the intrinsic properties of graphene in guiding the osteogenic differentiation of stem cells. Also mimicking the natural bone ECM, for example through the production of 3D graphene-based scaffolds, has proved to be a novel strategy in directing the stem cell fate towards the osteogenic lineage. Nevertheless, a deeper understanding of the ability of graphene to improve the biological properties of different scaffold materials will be essential to engineer graphene-based substrates for targeted biomedical applications.Regarding stem cells of dental origin, few data are available to date. Dental tissues are a very rich source of stem cells, which are collectively called Dental Stem Cells (DSCs) . In the Dental pulp is the soft connective tissue of the tooth and Dental Pulp Stem Cells (DPSCs) are a unique MSCs population that is present in the cell rich zone and core of the pulp. These cells were firstly discovered in 2000 by Gronthos and colleagues, thanks to their ability to differentiate into odontoblasts/osteoblasts, adipocytes and neural-like cells .Previous studies established that DPSCs and BM-MSCs are analogous cell populations, since they share expression profile for thousands of genes, among which those associated with the initiation of mineralization and bone homeostasis . Other cIn the study of Rosa and coworkers, the effect of GO-based scaffolds on DPSCs proliferation and differentiation was evaluated and compared to that of glass substrates . Cell moIn another study, the authors aimed at evaluating the potential of graphene to induce odontoblastic or osteogenic differentiation of DPSCs without the use of any chemical inducers . Dentin Therefore, authors seem to agree with the fact that graphene is a biocompatible material for DPSCs and it might be used as a valid substrate for dental tissue engineering since it is able to promote either osteogenic or odontogenic differentiation of DPSCs depending on the surface characteristics and functionalization of graphene, as also emerged in the previous section.The periodontium refers to those structures which surround and support teeth. It is composed by four parts: gingiva, alveolar bone, cementum and periodontal ligament. The function of the periodontal ligament is to literally attach the teeth to the bone and when a chronic process, called periodontitis, affects these structures, the teeth stability and health are compromised. All the periodontium is constantly maintained by Periodontal Ligament Stem Cells (PDLSCs), which have the ability to differentiate into cementoblasts, odontoblasts and fibroblasts .Thanks to their potential to differentiate into several types of specialized cells, PDLSCs can be used as substitute to BM-MSCs in order to determine the osteogenic potential and the viability onto different substrates . Even ifIn one of the few studies, the effects of GO, Silk Fibroin (SF) and GO combined with SF were investigated on PDLSCs adhesion, proliferation, viability and expression of MSCs markers . SF is wLater, the same group investigated the potential of SF and GO composites to promote human PDLSCs spontaneous differentiation into osteo/cementoblast-like cells . In the The dental follicle is the loose connective tissue surrounding the enamel organ and the dental papilla of the developing tooth germ. Its principal role is the coordination of tooth eruption through the regulation of the osteoclastogenesis and osteogenesis processes . The DenTo date, only one study evaluating the behavior of human DFSCs on graphene-based nanomaterials has been reported . In the A critical step for the application of nanotechnology in medicine is the interaction of a material with the immune system because, once administrated, it immediately enters in contact with the immune cells. Graphene and its composites are not exempt from the interaction with cells of the immune system and, as in the last few years the biological applications of graphene-based nanomaterials have grown, it results necessary to investigate the immune-related impact of these nanomaterials. Many papers about this issue underlined stimulation or suppression effects of graphene-based nanomaterials on immune system cells, depending mainly to graphene purity, oxidation level, shape dimension, functionalization and to the type of immune cells analyzed.The first extended investigation on graphene and immune cells was made by Sasidharan and coworkers . After cTo best characterize the impact of GO on immune cells, Orecchioni and colleagues studied and compared the effects of small-size (100–500 nm) and large-size (1–10 µm) GO flakes on a pool of PBMCs . The autLymphocytes, which represent about the 20–40% of the white blood cells and the 70–80% of PBMCs, are responsible for the antigen-specific and innate immune response. The most of research have been made analyzing the effects of graphene-based nanomaterials on T lymphocytes, concluding that the cytotoxicity of graphene depends on its dosage, time of exposure and type of material with which these are combined. For example, Ding and coworkers demonstrated that low doses of GO have no cytotoxic effects on T lymphocytes, while a dose over 100 µg/mL causes oxidative stress related apoptosis after 24 h of incubation . In partHalf of the studies about graphene and the immune system have been carried out on macrophages, not only because these represent the first phase of body immune reaction, but also because macrophages are the easiest long-term culturing primary immune cell population .The phagocytic activity of macrophages makes them the most exposed cells to the negative influence of graphene and its derivatives . It has Regarding the activation of macrophages, Chen and colleagues showed that pristine graphene and GO promoted the production of several cytokines, such as IL-1α, IL-6, IL-10 and Tumor Necrosis Factor alpha (TNF-α), via NF-κB pathway, that is activated via Toll-Like Receptors (TLRs) . GraphenDendritc cells (DCs), representing the 1–2% of PBMCs, play a fundamental role in the activation of antigen-specific T lymphocytes . Few datInfection is a frequent process during biomaterial implantation procedures. Since antibiotics often impact negatively on bacterial flora and pathogens are able to acquire resistance against different antibiotics, in the last few years various nanotechnologies with antimicrobial properties have been developed and studied ,104, tryIt has been reported that some graphene-based nanomaterials possess antibacterial properties; nevertheless, the effects of graphene on bacteria structure, metabolism and viability depend on the materials’ concentration, time of exposure, physical-chemical properties, as well as on the characteristics of bacteria used in the tests ,106.A lot of studies indicated that graphene and several graphene nanocomposites have a remarkable antibacterial ability against both Gram-negative and Gram-positive bacteria ,109,110.Another way for graphene to exert antibacterial activity seems to be through the separation of microorganisms from the microenvironment. Aggregated graphene sheets in suspension can isolate bacteria from the surrounding environment hindering nutrient consumption, reducing their ability to proliferate and favoring their inactivation .Graphene antibacterial activity is also probably related to oxidative stress induction. This is mainly mediated by ROS generation, especially when GO is used, nevertheless oxidative stress can be triggered without ROS generation. Li and coworkers showed that graphene is able to act as an electron pump pulling out electrons from bacterial membranes and inducing ROS-independent oxidative stress that affects microorganisms .Obviously, these mechanisms are not specific against bacteria but can also affect other cells, although with less effectiveness. Pang and colleagues tried to clarify the ratio between biosafety and antibacterial activity of graphene and GO . They obEscherichia coli compared to smaller one, probably due to the capacity of larger GO sheets to completely cover bacteria inhibiting their proliferation and colony formation  and GO-chitosan against ectively . The resAll these studies would suggest that the antibacterial activity of graphene-based nanomaterials is variable and dependent on the conditions of the tests; in particular, results change depending on the type of material and the size of particles, on its concentration and its state (in solution or adsorbed on a support), on the type of bacteria used in the study and on the medium used for their growth.Graphene and its derivatives display many potential applications in the dental field, as can be seen from the literature. Thanks to their potentiality, particularly in driving the osteogenic differentiation of stem cells and antibacterial abilities, the application of graphene-based nanomaterials to already existing dental technologies are being studied. In addition, graphene seems to be interesting as a platform able to release therapeutic molecules to improve implants osseointegration and bone formation. In the following paragraphs, we will present several studies discussing the association of graphene with dental implants, membranes, resins, cements and adhesives, as well as strategies for teeth-whitening.Nowadays, Ti dental implants are considered as the best substitutes for missing teeth thanks to their reliability and predictability, besides to their mechanical strength and favorable biocompatibility ,130. SinTi inertness represents a disadvantage because it may induce the development of fibrous tissue which could lead to the failure of the implant . For thiAs evidenced earlier, GO-coating was found to be a successful substrate for anchoring and growing of DPSCs on its rough surface and for promoting their osteogenic differentiation through up-regulation of MSX-1, PAX-9, RUNX2, COLI, DMP-1 and DPSS in particular after 14 days of culture . To imprAnother approach to improve the osseointegration of Ti implants could be its coating with bioactive proteins. For bone regeneration, BMP are the most potent osteoinductive proteins and among these, BMP-2 has been reported to induce osteogenic differentiation of stem cells, which can enhance the osseointegration of implants by forming bone at the space between the implants and the implantation site ,134. La Staphylococcus aureus), facultative anaerobic bacteria (E. coli) and anaerobic bacteria (S. mutans) thanks to the synergic effect of GO contact-killing and minocycline release-killing [S. mutans and P. gingivalis [Implant GO-coating represents an advantage also due to its antibacterial properties. Despite the fixation of graphene on Ti implant makes it to lose a bit of antibacterial activity, there is the possibility to functionalize the coating with antibacterial substances, such as antibiotics or silver nanoparticles. As demonstrated by Quian and colleagues, minocycline hydrochloride added to GO-coating improved the antibacterial activity against aerobic or facultative anaerobic bacteria  substrates . The resTo increase efficiency of bone repair, especially in periodontal and periimplant bone defects, the use of Guided Bone Regeneration (GBR) membranes has been important for years in oral surgery. These membranes are placed on the bone defect regeneration site and their principal function is to not allow infiltration of soft tissue cells into the growing bone. Thus, these act as physical barriers that separate connective tissue and the regenerating bone providing the slow osteogenic cells migration . In the All membranes need to satisfy the five main criteria underlined by Scantlebury: biocompatibility, space-making, cell-occlusiveness, tissue integration and clinical manageability . In ordeThe same GO-coating applied on collagen membranes was tested using DPSCs to analyze osteoblastic differentiation and inflammatory response . The resS. mutans, without altering the standard adhesion properties of the dental adhesive.Resins, cements and adhesives are the most used materials for dental restoration. Nevertheless, their porosity and adhesiveness make them receptacles for bacteria in the oral cavity and in particular in proximity of dental restoration. These polymeric materials, indeed, facilitate the adhesion of bacteria and the formation of biofilms that are the main cause of dental restoration failure . An examS. mutans. The results showed that up to the 95% S. mutans cell viability reduction was obtained using ZNGs even at a very low concentration (5 μg/mL). Moreover, the biomass and exopolysaccharide production, which are necessary for the biofilm formation, were evaluated: the ZNGs showed to behave as an obstacle for the biofilm growth. The authors concluded that the use of ZNGs is a viable instrument to control caries disease as it decreases S. mutans growth.One of the main limitation of GNPs in dental application is its grey color. In order to ameliorate this aspect, Zanni and colleagues tested a hybrid material composed by Zinc Oxide Nanorods (ZnO-NRs) grown on GNPs, thus combining the antimicrobial effect of GNPs with the light color and biocidal properties of ZnO-NRs . The autx/MgO catalyst [Biris and colleagues successfully prepared graphene sheets embedded with various amounts of gold nanoparticles (Gr-Au-x) using the radiofrequency catalytic chemical vapor deposition technique over a Aucatalyst . Sarosi catalyst . The autAs a last example, Li and collaborators demonstrated that the functionalization of glass ionomer cements with fluorinated graphene is not only useful to inhibit bacterial growth but also to ameliorate mechanical properties of the cements, increasing microhardness and compressive strength and decreasing friction coefficient, all important parameters for cements .Nowadays, a lot of people would change the color of their teeth, which often is ruined by food, beverage and smoke; consequently, the level of demand for tooth-whitening is increasing. Tooth color is determined by intrinsic factors, associated with the light scattering and absorption properties of the enamel and dentine and extrinsic factors, associated with the absorption of materials  on the surface of the enamel.2O2) chemistry, because of its bleach-properties and the techniques constantly evolve to ameliorate the rate of whitening, the ease of the treatment and to decrease the side effects. The most common risks of teeth-whitening include increase dental sensitivity and gingival irritation, besides changes in tooth microstructure. These effects are directly related to the concentration of H2O2, the duration of the treatment and the non-bleach component of the products [The products used for tooth-whitening are extensively-based upon Hydrogen Peroxide  phthalocyanine, together with H purpose . Based o purpose . They thent time b. This rWhile graphene is one of the most promising materials in nanotechnology, the synthesis on large scale represents an important aspect to be evaluated. Most of the studies described so far have been carried out using small amount of uncontaminated and controlled defect-free samples , but in Another limitation on graphene biomedical application is the scarce existing information on in vivo toxicity mechanisms and more studies are needed to support safe biological applications of these materials. However, some reports showed that graphene-based nanomaterials mainly accumulate in liver, spleen and lung after intravenous administration . Few stuTherefore, the scarce synthetic control, the high variability in production and the multiple parameters that modulate activity and toxicity of graphene and graphene-based nanomaterials, together with the scarce available studies, are big limitations for large scale application of graphene and its derivatives.The research on biomedical applications of graphene-based nanomaterials has seen dramatic progress in the last few years. In this review, we focused on the recent advances in dental tissue engineering using graphene and its related nanomaterials. Graphene exhibits numerous outstanding properties. In addition to its exceptional mechanical strength, electrical conductivity and thermal stability, what makes graphene extremely interesting is the possibility to functionalize and combine it with different biomaterials and biomolecules. This confers new properties to existing materials and allows the generation of nanocomposites with enhanced characteristics.In this review, we discussed recent studies concerning the biocompatibility, cytotoxicity and antibacterial activity of graphene-based nanomaterials both in vitro and in vivo. Then, we analyzed the effect of graphene-based nanomaterials on the adhesion, proliferation and differentiation of stem cells, focusing in particular on stem cells of dental origin. We examined the potential of graphene to promote osteogenic differentiation of stem cells, which is a key point for its future application in dental field. In this context, we presented recent studies employing graphene and its related nanomaterials for surface modification of dental implants or other scaffolds used in dentistry, such as membranes, resins and adhesives. As clearly highlighted in this review, graphene-based nanomaterials have emerged as promising scaffolds for a wide range of biomedical applications and all the presented works seem to agree with the fact that existing dental materials show improved characteristics following the addition of graphene; nevertheless, their behavior is closely dependent on graphene’s physicochemical properties, such as surface functionalization, coating and size.The increased interest in graphene and its derivatives have led to concerns about the risk of exposure not only to humans but also to the environment. Therefore, evaluation of the safety and potential risks of these nanomaterials is mandatory to ensure the safe use of graphene materials in biomedical applications. However, there is still much investigation to perform in order to assess the potential long-term toxicity of graphene and its composites and their effects on different cells, tissues and organs, including those of oral cavity. Moreover, in-depth studies are necessary to understand cell-signaling, metabolic pathways and osteogenic effects triggered by graphene-based nanomaterials. Overall, we believe that the use of graphene-based nanomaterials in the dental field deserves to be deeply explored as it can lead to even more reliable dental treatments in the near future.


L1 regularization. In order to better reconstruct the piecewise constant and sparse coefficient distributions, TV and L1 norms are combined as the regularization. The forward problem is discretized with the discontinuous Galerkin method on the spatial space and the finite element method on the angular space. The minimization problem is solved by a Jacobian-based Levenberg-Marquardt type method which is equipped with a split Bregman algorithms for the L1 regularization. We use the adjoint method to compute the Jacobian matrix which dramatically improves the computation efficiency. By comparing with the other imaging reconstruction methods based on TV and L1 regularizations, the simulation results show the validity and efficiency of the proposed method.Optical tomography is an emerging and important molecular imaging modality. The aim of optical tomography is to reconstruct optical properties of human tissues. In this paper, we focus on reconstructing the absorption coefficient based on the radiative transfer equation (RTE). It is an ill-posed parameter identification problem. Regularization methods have been broadly applied to reconstruct the optical coefficients, such as the total variation (TV) regularization and the  Optical tomography with near-infrared light is a promising technique for noninvasive studying of the functional characters of human tissues. One can find many applications of this technique, for example, the early detection of breast cancer, cervical cancer screening, monitoring of infant brain tissue oxygenation level and functional brain activation, and the study of dosimetry in photon dynamic therapy; see –4. UnlikL1 norm has been used in optical tomography  ∈ ℝ𝔡×N is defined as follows:Jμai] can be computed by direct method which differentiates Mζi(ξj) with respect to the perturbation of μa on each element. In order to compute [Jμai], for each i, we need to solve forward problems 𝔡 × N times. For a total of s sources, we need to compute s × 𝔡 × N times, so the direct method is very time consuming. Therefore, we use the adjoint formulation to compute [Jμai] instead of direct method.Since we adopt the Jacobian-based minimization method to solve  in this Fi′(μa)∗ denotes the adjoint operator of Fi(μa) and φ is the solution of the adjoint RTEx in direction ω due to a source at x0 in direction ω0 is the same as the angular density at x0 in direction −ω0 due to a source at x in direction −ω. Here, G presents the Green function of Fi′μjoint RTE−ω·∇φx,ω+conditionφx,ω=ω·νx4Fi′μjoinjoint RTE−ω·∇φx,ω+conditionφx,ω=ω·νxd solver:ω·∇φ^x,ω+conditionφ^x,ω=ω·νζi and one detector position ξj, then the kth column of the Jacobi matrix [Jμai] can be computed as follow:φj is the solution of the following boundary value problem:Jμai], for each 1 ≤ i ≤ s, we only need to solve forward problems 𝔡 times. For s sources, we need to solve forward problems s × 𝔡 times, which dramatically reduces the computation burden and improves the efficiency of the algorithm.If we consider one source position μa can be estimated through the regularization method. The quality of reconstructed image strongly depends on the choice of the penalty term R(μa). If we choose the L2 norm as penalty, that is, R(μa) =  ‖μa‖22, the reconstructed image usually blurs with a low resolution. If we choose L1 norm as the penalty, that is, R(μa) =  ‖μa‖1, the reconstructed image tends to find a sparse solution  denotes the Jacobi matrix of the forward operator Fi with respect to μan. For solving Step  1∗, in order to avoid the nondifferentiability of the total variation term ∫X|∇μa| at a zero point, we approximate it with a smooth functional ‖μa‖εTV, defined asϵ is a constant, and it cannot be too large or too small. The Euler-Lagrange equation for Step  1∗ isT denotes the transpose, Ri(μan) is the short notion of Fi(μan) − Mi, the term L(μan)μa is defined asL1 norm regularization problem and it can be solved efficiently through the shrinkage operator, that is,L1 regularization is described in L1 regularization with the split Bregman formulation is applied to 2D test problems. We assume the distribution of the scattering coefficient is known. Reconstructions of spatially dependent distributions of absorption coefficient inside the medium are performed and discussed. In our simulation, a circular domain X which contains different inclusions is investigated. The radius of the circle is 10 mm. 12 sources and 12 detectors are located on the boundary of the domain with equal space. This yields totally 144 source-detector pairs to be used in the inversion.In this section, the TV-π].Noise-free synthetic data are generated by solving the forward problem on triangular meshes with the method we mentioned in −1 and absorption coefficient of 0.01 mm−1, respectively, and these values keep the same throughout this paper. The initial guess of reconstruction is set to be identical to the properties of the background medium. That is, the iterative procedure started with the background value of the absorption coefficient.The background medium has scattering coefficient of 10 mmBi,ωi is a piecewise linear function whose spatial support is Si and achieves the value 1 at the center node of Si, where Si is the element through which the ith incident impulse passes. The direction of uiin, points approximately from the center of Si to the center of X.The incident impulse on the inflow boundary is settled asL1 regularization. Our purpose is to show the following results.Based on above settings, we use various simulations to validate the proposed split Bregman algorithm for our TV-g, the proposed algorithm works better when g is bigger.First, by comparing the reconstruction with different anisotropic factors L1 regularization can reconstruct the location and the quality of the coefficient more accurately than the TV regularization and the L1 regularization.Second, for small sparse inclusions, the TV-L1 needs less computation time than the Levenberg-Marquardt algorithm for TV regularization. Even though our proposed algorithm needs more computation time than the split Bregman algorithm for L1 regularization [L1 regularization.Third, the proposed split algorithm for TV-rization , the recμa = 0.02 mm−1 and μs = 20 mm−1. Our goal is to reconstruct μa. For solving the inverse problem, we use a mesh of 772 nodes and 1484 triangular elements. To avoid the inverse crime, we use a mesh of 1296 nodes and 2440 triangular elements for the synthetic data. The simulated absorption distributions and the meshes can be seen in J(μa) by using the split Bregman method for TV-L1 method with exact data, that is, δ = 0, in this example. Then we compare the reconstruction results for different anisotropic factors g. For the comparison purpose, we use the same parameters α, β, η, and ε for different g. Noticing that the so-called exact data in fact contains noise, so the regularization parameter α,  β should not be too small. Since the inclusion is very small in this example, we enhance more weight of L1 penalty than the weight of TV penalty. Here we take α = 10−4 and β = 10−3. The other two parameters are taken as η = 10−5 and ϵ = 10−6, respectively.In this simulation, we reconstruct one circle inclusion with the radius 0.5 mm centered at . In this circle, the absorption coefficient g = 0.1 and g = 0.4 from the left to the right. The second row of g = 0.7 and g = 0.9 from the left to the right. As it can be seen, the reconstructions for the bigger g are more clear than that for smaller g. There are some apparent blurred parts in the left side of the inclusion in Figures The reconstruction results can be seen in g. The following simulations in this paper are all performed with g = 0.9.From this example, we can find that the proposed algorithm works better for the bigger anisotropic factor L1 regularization and the TV regularization with noise-free synthetic data. In the first case, a small inclusion with radius 0.5 mm centered at  is designed for μa = 0.02 mm−1 and μs = 20.0 mm−1, respectively. In the second case, a middle inclusion with radius 2 mm centered at  is designed for μa = 0.02 mm−1 and μs = 20 mm−1, respectively. In the third case, a middle inclusion with radius 4 mm centered at  is designed for μa = 0.02 mm−1 and μs = 20 mm−1, respectively. The simulated true absorption coefficient can be seen on the first column of L1 regularization and TV regularization are shown in L1 regularization and TV regularization are shown in L1 regularization and TV regularization are shown in ε = 10−6 as the last section, we will not present it repeatedly in There are two groups of experiments in this section. In the first group of experiment, we design three types of inclusions to compare the performance of the TV-L1 regularization performs better than the TV regularization in both localizing the location and quantifying the values; see Figures L1 regularization and Figures L1 regularization imposes both TV penalty and L1 penalty on μa. The TV penalty tends to find edges and the L1 penalty tends to find the sparse details of the inclusion. As can be seen from L1 penalty.Observing from the results, we can see that, for small and middle inclusions, the TV-L1 regularization and TV regularization perform no big differences, but we still can see that the blurred parts in L1 regularization are smaller than that in For the big inclusion, TV-L1 regularization, TV regularization, and L1 regularization.In the second group of experiments of this section, three types of complicated and multi-inclusions are designed for comparing the reconstruction results by TV-μa = 0.02 mm−1 and μs = 20 mm−1. We use a mesh of 1277 nodes and 2488 triangular elements for the inverse problem and a mesh of 1861 nodes and 3616 triangular elements for the forward problem. The true distributions of μa and reconstruction results with various regularizations can be seen in In the first case, we reconstruct two small circle inclusions centered at  and  with the same radius 0.5 mm. In both of the two inclusions, μa = 0.02 mm−1 and μs = 20 mm−1. We use a mesh of 1671 nodes and 3264 triangular elements for the inverse problem and a mesh of 2141 nodes and 4176 triangulations for the forward problem. The true distributions of μa and reconstruction results with various regularizations can be seen in In the second case, we reconstruct three small circles centered at , , and  with the same radius, 0.5 mm. In all the three inclusions, μa = 0.02 mm−1 and μs = 20 mm−1. We use a mesh of 1323 nodes and 2568 triangulations for the inverse problem and a mesh of 1809 nodes and 3512 triangulations for the forward problem. The true distributions of μa and reconstruction results with various regularizations can be seen in In the third case, we design three inclusions. One big circle centered at  with radius 2 mm. Two small circles centered at  and  with the same radius 0.5 mm. In all the three cases, we take the same coefficient values α, β, η), the steps of the iterations Iters, the relative residual error Eresi, and the relative solution error Eμa in the sense of L2 norm corresponding to each regularization method. Eresi and Eμa are defined asIn L1 regularization can preferably identify the location and the value of the inclusions. In other words, the advantages with TV-L1 regularization are indeed apparent over TV regularization and L1 regularization; see Figures L1 regularization. The LM type algorithm for the TV regularization converges slowly especially when the inclusions are very small; see Figures L1 regularization is an efficient algorithm with high convergent rate; we can see this from the number of iterations in L1 regularization.We say that in the first and the second case, compared with the inclusions near the boundary, the inclusion deep in the domain is difficult to identify. In the third case, it is more difficult to reconstruct the small inclusion than the big inclusion. From the reconstruction results, we can see that the TV-X. Moreover, we investigated the reconstruction under different noise level to show the robustness of the proposed algorithm. The noises added to the exact data Mitrue are by the following rule: Mi = Mitrue(1 + δ∗random), where δ is the signal-to-noise ratio; random is a Gaussian random variable with zero mean and unity variation. The background values of the absorption and the scattering are 0.05 mm−1 and 5 mm−1, respectively. The absorbing inclusion for which we set the absorption coefficient is μa = 0.1 mm−1 which is centered at  with radius 2 mm. The scattering inclusion for which we set the scattering coefficient is μs = 10 mm−1 which is centered at  with radius 2 mm. For the discretization, we use a mesh of 463 nodes and 856 triangulations for the inverse problem and a mesh of 681 nodes and 1288 triangulations for the forward problem. See the first row of the μa. In μa with exact data is presented. The parameter value in this case is chosen as α = 0.0005, β = 0.0005, and η = 10−6. α = 0.0005, β = 0.0005, and η = 10−6. In α = 0.005, β = 0.0005, and η = 10−6. From the four reconstructions under four different noise levels, we can find that as the noise level increased, the quality of the reconstruction gets worse. Here, we take the noise level under 1%, since the iteration will not converge if the noise level is bigger than 1% which reflects the severe ill-posedness of the problem. We also find that in order to obtain the reconstruction results with the relative error in L2 norm no more than 20% the noise level on the synthetic data should be less than 1%.As the last simulation, our purpose is to show the validity of our algorithm in the situation that high absorbing inclusion and low scattering inclusion are contained in the domain L1 regularization is proposed. The forward problem is discretized with the discontinuous Galerkin method on the spatial space and the finite element method on the angular space which both are implemented on the piecewise linear basis. We discretize the absorption and scattering coefficients on the piecewise constant basis. The minimization problem is solved by a Levenberg-Marquardt type method which is equipped with a split Bregman algorithm for the L1 penalty. The adjoint method is used to compute the Jacobian matrix which is the discretized Fréchet derivative of the forward operator. We numerically compare the proposed reconstruction method with the other imaging reconstruction methods based on TV and L1 regularizations. The simulation results show the validity and robustness of the proposed method.In this paper, forward and inverse problems of the radiative transfer equation are considered. An image reconstruction method based on the TV-


Graphene has been proposed as the current controlling element of vertical transport in heterojunction transistors, as it could potentially achieve high operation frequencies due to its metallic character and 2D nature. Simulations of graphene acting as a thermionic barrier between the transport of two semiconductor layers have shown cut-off frequencies larger than 1 THz. Furthermore, the use of n-doped amorphous silicon, (n)-a-Si:H, as the semiconductor for this approach could enable flexible electronics with high cutoff frequencies. In this work, we fabricated a vertical structure on a rigid substrate where graphene is embedded between two differently doped (n)-a-Si:H layers deposited by very high frequency (140 MHz) plasma-enhanced chemical vapor deposition. The operation of this heterojunction structure is investigated by the two diode-like interfaces by means of temperature dependent current-voltage characterization, followed by the electrical characterization in a three-terminal configuration. We demonstrate that the vertical current between the (n)-a-Si:H layers is successfully controlled by the ultra-thin graphene base voltage. While current saturation is yet to be achieved, a transconductance of ~230  Additionally, the advent of 2D materials and their physics could play a decisive role for operation levels not yet achieved by traditional semiconductors. Specifically, graphene has been proposed by Mehr et al.  in 2012 s (<0.1) . To alles (<0.1)  proposeds (<0.1) .5. Here we present the electrical characterization of a graphene layer embedded between two (n)-a-Si:H layers deposited by VHF-PECVD and the ability of graphene to modulate the vertical current in the structure up to 40%.Along with high operation frequencies, mechanical flexibility is another desired feature in the new generation of bendable electronic devices. In this field, graphene has positioned itself as a promising candidate . HydrogeThis manuscript is organized as follows. In +)-a-Si:H deposited over a ZnO:Al substrate by VHF-PECVD (140 MHz) at a constant temperature of 180 °C using a gas mixture of silane, hydrogen and 0.1% phosphine (PH3) as n-type dopant. The deposition system is described in detail elsewhere [2 single layer of CVD graphene grown on Cu foil (by Graphenea S.A.) was transferred onto the surface of the (n+)-a-Si:H (collector) layer by the polymer-assisted (PMMA) method. To remove any oxide from the surface of the (n+)-a-Si:H layer, the sample was dipped in 1% HF for 30 s. It was also observed that the HF treatment improved the quality and integrity of the graphene transfer. After the removal of the sacrificial PMMA layer with acetone, another 100 nm (n)-a-Si:H layer was deposited on the surface of graphene using a low power VHF-PECVD regime with 0.0225% PH3 as dopant. A section of the graphene layer was protected from the a-Si:H deposition to place the metal electrode. Due to restrictions in the fabrication process, the deposited emitter layer partially exceeds the surface of the graphene layer. The conductivities of the emitter and collector layers are 5 × 10−5 S/cm and 3 × 10−3 S/cm, respectively. Although heavily doped emitter is the usual configuration to achieve high gain, the highly doped collector layer was chosen since an improvement of the cut off frequency has been reported [In HF-PECVD 0 MHz at reported . AluminuThe base-collector (BC) and base-emitter (BE) IV characteristics were analyzed independently in the same structure by applying a voltage to the graphene contact while leaving the other contact grounded. Assuming an undoped or p-doped graphene channel, we expected a diode-like behavior between graphene and both (n)-a-Si:H layers. The assumption of a p-doped graphene layer is in agreement with reports of transferred graphene grown on Cu foil by CVD ,17,18 asA non-ideal diode behavior with a dominating thermionic transport mechanism is expected for the (n)-a-Si:H/graphene diodes . Hence, e diodes ,20. Large diodes . More thagnitude , it is kThe devices were further studied by temperature dependent IV measurements to determine the Schottky barrier heights, The values of 0/T plot b.To corroborate the obtained results, the Cheung and Cheung method  was useds J plot a the ide. J plot b. From tson plot b a qΦb~0Although in principle both BC and BE diodes are based on (n)-a-Si:H/graphene interfaces, the results of their electrical characterization diverge largely, which could be related to inadvertent interfacial oxide layers, surface defects and/or contaminants . It mustIn summary, the Schottky barrier of the BC diode could be largely affected by the presence of residual elements and/or interfacial states induced during the transfer of graphene onto the a-Si:H layer. At the BE interface, the (n)-a-Si:H layer was deposited on the upper surface of graphene as in previous experiments. Nevertheless, limited by the aluminum- and temperature-induced crystallization of the a-Si:H layers at temperatures larger than 150 °C ,29,30, nFollowing the individual characterization of the BC and BE diodes, the test device was also analyzed in a three-terminal configuration. All currents were measured as the collector-emitter voltage First, the behavior of the device will be discussed at As -barrier c and thepacities ,35. TherIn an ideal GBHT device, both BE and BC junctions exhibit significant thermionic barrier heights. The barrier heights, and thus the collector-emitter current, are controlled by the base voltage applied to the graphene layer. Since ilibrium a illustrThe electrical behavior of the device will now be discussed at At 2 junctions and found a transition of the main transport mechanism from thermionic emission to FN tunneling around 1 V bias. Similarly, FN tunneling could occur in our vertical a-Si:H/graphene structure. However, it must be noted that differently from the gated Ti/MoSe2 of ref. [In unneling , could bunneling  investig of ref. , where t of ref. ). Therefexpected . The lar+)-a-Si:H heterostructures in the vertical GBHT configuration. We found that the collector-base diode is characterized by a small Schottky barrier and low rectifying ratios, which could be caused by atomic residues and/or humidity-induced interfacial states. The base-emitter diode is characterized by a rectifying ratio of more than one order of magnitude and a small Schottky barrier In conclusion, we presented the electrical characterization of the interfacial diodes in (n)-a-Si:H/graphene/(ndry-transfer of graphene layers grown on germanium substrates [2, MoSe2 or WSe2, have been extensively used in gate-tunable heterojunctions or in field effect transistor reaching high on/off ratios of up to 107 [Enhancing the current control in vertical transistor structures could be achieved by further optimizing the diode behavior of the interfaces, specifically the lack of rectification at the base-collector junction. A possibility to increase the barrier at the BC interface is by reducing the doping of the collector layer. Along with this, the quality of the BC interface could be improved by avoiding atomic Cu residues and humidity-related issues. This could be achieved, e.g., by the bstrates . Anotherbstrates . Since sp to 107 ,41. Howep to 107 , compare


B. This finding opens the possibility for nanomechanical control of graphene magnetism and has potential applications in spintronics and magnetic sensing.The effect of shear distortion on the vacancy induced magnetism in graphene is investigated using extensive first-principles calculations. It is found that shear distortion can lead to magnetic transition between two states with high and low magnetic moments. Such a transition is reversible and results from the breaking of the in-plane symmetry of the local atoms, which reverses spin polarization of the π bands of the vacancy states near the Fermi level and leads to the change of magnetic transition by 1 µ Magnetism of graphene can be created in several different ways by for example atom vacancies12, adsorption of magnetic atoms14 and recently by hydrogen modifications16. Among them, single atom vacancies break the local bonding symmetry of the sp2 states in the pristine lattice and introduce dangling bonds at the vacancy site. It generates spin polarization in the valence bands near the Fermi level, resulting in local magnetism of the graphene lattice. It has been known that the ground state of the monovacancy graphene is a ferromagnetic state with a magnetic moment of 1~1.7 µB depending on the vacancy concentration23. Local spin polarization of the graphite24 and graphene25 vacancy can be probed by spin-polarized scanning tunneling microscope.Introducing magnetism into graphene, which is intrinsically a nonmagnetic 2D material, has been the endeavor of many ongoing researches26 applied. It has recently been proposed that the spin channels of graphene nanoribbons can be modified using single tetragon molecules27. Nano-mechanical and vibrational coupling26 would also be interesting to explore. So far, the control and manipulation of graphene magnetism has not yet been well understood.In light of any potential application in spintronic devices and quantum information, it would be highly desirable to achieve dynamical and reversible control of the local magnetization of the graphene. In essence, this depends on how the local spin polarization of the graphene lattice couple and respond to external perturbationsHere we propose and demonstrate a robust way to control the graphene magnetism using nanomechanical deformation, based on extensive and rigorous density functional theory (DFT) calculations. It is found that shear deformation of graphene with single-atom vacancy can lead to an electronic transition between two magnetic states with high and low magnetic moments Fig. . Such a 28, and lead to antiferromagnetic states. In the small concentration limit, however, it is well known that the ground state of monovacancy graphene is a ferromagnetic state, and its relaxed geometry is nearly flat as shown in the insert of Fig. 3) of the pristine lattice is broken due to Jahn-Teller distortion30 and re-bonding between two of the nearest neighbor carbon atoms , the spin up density (the red surface) is dominantly distributed at the vacancy site and decays slowly away from the vacancy. Consistent with the magnetic moment, there is a change of color map at Δθ = 1° on most atomic sites, as the lattice is slightly deformed. It then remains almost the same at larger angles (1–5° as shown in panels (b)–(f))). Figure It is interesting to find out how this magnetic jump happens in space across the lattice. Figure θ. These structures are obtained by fully relaxing all atomic positions under the given distortion. Panel (a) is the structure of the monovacancy graphene in the 12 × 12 supercell without distortion. The lattice shows overall flat. Within the plane, atoms 2 and 3 contract slightly and rebond via the Jahn-Teller distortion with a distance between atoms 2 and 3 of 1.93 Å30. The lattice geometries at Δθ = 1° and 2° have the same shape and the lattice forms a wrinkled structure  shift in opposite directions. More specifically, the spin up state of the π bands shifts upwards from −0.131 to −0.008 eV at the M point and from −0.135 to −0.012 eV at K, and from 0.182 to 0.170 eV at Γ. In contrast, the spin down band shifts oppositely from 0.210 to 0.152 eV at Γ and from −0.017 (−0.014) to −0.225 (−0.219) eV at M (K) when θ goes from 0° to 1°. Simultaneously, one σ band located at −0.61 eV of the vacancy states, which is associated with the in-plane dangling bond of atom 1, disappears from the valence band gap at Δθ = 1° and is shifted downward to the valence band. Such a downward shift indicates a large interaction yet does not contribute to the change of magnetism. Instead the change of magnetic moment results mostly from the reversal of spin polarization of the π bands of vacancy states near the Fermi level. For the planar structure at undeformed state, the total magnetic moment of 1.53 µB is the sum of the σ band of 1 µB and the extended π orbitals of 0.53 µB. In contrast, the transformed σ band still provides the magnetic moment of 1 µB, reversal of π band spin polarization gives a negative value of −0.5 µB, leading to a low magnetic state ~0.45 µB. The reversal of spin polarization of the π band is thus mainly responsible for the jump of the total magnetic moment by 1 µB. From the analysis of local density of states (LDOS), it can be confirmed that this σ band is dominantly localized on atom 1 at Δθ = 0° as it shows no energy dispersion. It becomes delocalized under distortion, but does not contribute to the change of the total magnetic moment because it is occupied before and after the transition. Instead, the opposite shift of the π bands is responsible for the long-range spin transition across the lattice, as shown by the long-range spin density distribution shown in Fig. θ further increases. In particular, there is no change of spin polarization when the geometry transforms at Δθ = 3°. It suggests local bonding symmetry of the atoms around the vacancy is mainly responsible for the magnetic transition. This also suggests that magnetic transition could also be induced by other external deformations.To find out the origin of the magnetic transition, we analyzed in detail the electronic structure of the defect graphene upon distortion. Figure 28. These issues will be pursued in future investigations. This finding has thus general implications to the nanoscale control of graphene magnetism using defects and other related materials.We have investigated the effect of shear distortion on the vacancy induced magnetism of graphene, based on extensive first-principles calculations, and found a reversible transition between two distinct magnetic states with high and low magnetic moments. This transition results from a mechanical response to shear distortion and is associated with the local symmetry breaking of the vacancy. It opens the possibility of dynamical control of graphene magnetism via manipulation of lattice deformation. We believe that such a reversible control is versatile and could find potential applications in spintronics and magnetic sensing. In light of practical application, it would be desirable to investigate possible long-range ordering, in particular the competition and stability of possible antiferromagnetic ordering as implied by the Lieb’s theoremAb initio Simulation Package (VASP)31. The projector augmented-wave (PAW)33 pseudopotentials and the generalized gradient approximation (GGA)34 in Perdew-Burke-Ernzerholf form35 for the exchange-correlation energy are employed. Spin polarization is invoked in all calculations, and spin-orbital coupling can be neglected in graphene. Most calculations are carried out with an energy cutoff of 600 eV in the plane wave basis set, and the main results are also double checked with 800 eV cutoff. The supercells contain a vacuum layer ~20 Å and all atoms are relaxed until the forces on each atom are less than 0.01 eV/Å. The C-C bond length of pristine graphene is found to be 1.424 Å, which is consistent with the experimental value of 1.42 Å. The structural and electronic properties of the monovacancy defect are calculated using the 8 × 8 and 10 × 10, and 12 × 12 supercells. The Brillouin zone is integrated using the Monkhorst-Pack scheme with a 3 × 3 × 1 mesh sampling, and checked with larger (6 × 6 × 1) k-mesh to ensure convergence.Our DFT calculations are performed using Vienna Controlling magnetic transition of monovacancy graphene by shear distortion