The films show quality, mechanical and electrical properties comparable to the mechanically exfoliated samples. Growth mechanism studies show that the second layer is grown underneath the first layer by precipitation of carbon atoms from the solid Pt, and the small energy requirements for the movements of graphene nucleus on the liquid Pt3Si enables the interlayer epitaxy to form energy-favorable AB stacking. This interlayer epitaxy also allows the growth of ABA-stacked trilayer graphene and is applicable to other liquid/solid substrates.Large-area high-quality AB-stacked bilayer graphene films are highly desired for the applications in electronics, photonics and spintronics. However, the existing growth methods can only produce discontinuous bilayer graphene with variable stacking orders because of the non-uniform surface and strong potential field of the solid substrates used. Here we report the growth of wafer-scale continuous uniform AB-stacked bilayer graphene films on a liquid Pt 3Si/solid Pt substrates showing high quality and improved mechanical properties comparable to the mechanically exfoliated flakes.Specific stacking sequence of graphene can enable observation of unusual properties however it has been difficult to obtain this over wider areas. Here, the authors report wafer-scale growth of 100% AB-stacked bilayer graphene films by CVD on liquid Pt For these applications, it is critical to develop a scalable approach to synthesize large-area high-quality uniform AB-BLG films that can be manipulated to complex devices and integrated in silicon device flow. Mechanical exfoliation of highly ordered graphite provides a direct way to obtain high-quality AB-BLG, but suffers from low yield, small size , and poor controllability on the thickness8. In recent years, chemical vapor deposition (CVD) has emerged as a promising scalable method for producing AB-BLG by using solid metal foils as substrates such as Cu and Cu–Ni alloy15. However, the weak van der Waals potential between graphene layers cannot completely screen the strong potential field of solid substrates16. In this case, the nucleation, growth and orientation of the second layer in BLG are more strongly influenced by the solid substrates than the first layer. On the other hand, solid metal substrates usually have variable crystallographic lattices, defects, big surface roughness and abundant grain boundaries13. Such non-uniform surface structures and strong potential field of solid substrates result in only discontinuous BLG films with variable stacking orders and small grain size15 (Supplementary Table 13.The widely tunable bandgap of AB-stacked bilayer graphene (AB-BLG) leads to many interesting physical phenomena and makes it a promising material for a number of electronic, photonic and spintronic devices3Si/solid Pt substrate, we grow high-quality wafer-scale 100% AB-BLG films with 100% coverage and millimeter-sized grains by CVD. The Young’s modulus and breaking strength of the films are as high as 1.02 TPa and 122 GPa, and the mobility based on large-size field effect transistors (FETs) can reach 2100 cm2 V−1 s−1 at room temperature with a tunable bandgap >26 meV at displacement field of 1.0 V nm−1. The interlayer epitaxy mechanism is analyzed based on isotope experiments and density functional theory (DFT) calculations. It is found that the 2nd layer is grown underneath the first layer by precipitation of carbon atoms from the solid Pt core with high carbon solubility through the liquid Pt3Si shell with low carbon solubility, and the small energy requirement for the movement of graphene nucleus on liquid Pt3Si enables the interlayer epitaxy to form energy favorable AB stacking. We also demonstrate the growth of ABA-stacked trilayer graphene by using the same substrate and AB-BLG films by using a similarly structured liquid Pd silicide/solid Pd substrate.Here, we show that a liquid top layer can screen the strong potential of the solid substrate underneath to enable interlayer epitaxy of AB-BLG, in which the second layer grows epitaxially with the first layer graphene as the seed layer. Using a shell-core structured liquid Pt3Si/Pt substrate was synthesized by coating a 500-nm-thick Si film on a 250-μm-thick polycrystalline Pt foil by magnetron sputtering, followed by annealing in H2 atmosphere at 1095 °C for 12 h. According to the Pt-Si phase diagram17, Si element can form stoichiometric intermetallic compound, platinum silicide, with Pt element. As shown in the energy dispersive X-ray spectroscopy (EDS) maps in Fig. 3Si  and hydrogen (H2) at 1100 °C for a short time, and then slowly decreased to 1025 °C with a constant cooling rate (12.5 °C h−1), followed by rapid cooling to room temperature. Since the melting point of Pt3Si is around 830 °C17, the Pt3Si regions in the substrate turned into a liquid phase during the growth process, forming a liquid Pt3Si/solid Pt shell-core structure. In this structure, the solid Pt core with high carbon solubility (0.86 wt.% at 1100 °C17) acts as a reservoir of carbon atoms, while the liquid Pt3Si shell with low carbon solubility provides a diffusion channel of carbon atoms  continuous BLG film was formed , suggesting the robustness of our method for BLG growth. In contrast, under the same experimental conditions, only non-uniform multilayers and uniform MLG were synthesized on Pt and Pt3Si, respectively  of ~54 cm−1, about twice that of MLG, can be fitted into four Lorentzian bands with different frequencies  and selective area electron diffraction (SAED). The following four features were observed. First, the film shows a hexagonal SAED pattern with ots Fig. , both ofern Fig. , which i13. This interlayer epitaxy allows us to tune the grain size and GBs of the AB-BLG film by controlling the grain structure of the first layer graphene. For instance, we synthesized MLG with millimeter-sized grains by using a small flow rate ratio of CH4 to H2 in the first step . The identical loading and unloading curves demonstrate the elastic behavior of the film and no slippage at the periphery of the hole. Due to the slip between the two layers of BLG25, the curves show a softening for displacement larger than ~50 nm. Therefore, we fitted the data up to 50 nm of displacement to obtain the second-order elastic modulus of the membranes25. Fitting the results of 23 individual tests yield a mean 2D Young’s modulus (E2D) of 684 N m−1 (±50 N m−1)  used in our experiments, the mean 2D strength (σ2Dm) of AB-BLG was estimated to be 82 N m−1  of exfoliated AB-BLG25, further confirming the high crystalline quality of our CVD-grown AB-BLG film. Because the indentation area (1 μm) is significantly smaller than the grain size  of AB-BLG film, the influence of GBs on the mechanical properties of AB-BLG film can be ignored in our experiments, but it is a very interesting topic deserving further studies in the future.We measured the mechanical properties of the AB-BLG films by using nanoindentation as reported previously−1) Fig. . This vam−1 Fig.  using th2. As shown in Fig. 2 layer and 60 nm Al2O3 layer, which act as back- and top-gate dielectrics, respectively. The channel size is 80 μm (width) × 20 μm (length), which is much larger than those reported, typically <10 μm × 10 μm. Compared to small-size device, large-size device has much higher requirements on the quality and uniformity of the channel material since the probability of defects and cracks dramatically increases with the channel size, which can easily lead to device performance degradation27. The resistance R versus the back-gate voltage (VBG) plot shows typical ambipolar characteristics expected for graphene devices  of exfoliated AB-BLG with similar device structures but much smaller channel size1 (Supplementary Table VTG) at different fixed VBG, the maximum resistance (RDirac) attained at the charge neutrality point increases with increasing VBG in both the positive and negative directions. Figure RDirac increases when increasing the displacement field (D), confirming the tunability of bandgap with perpendicular dipole electric field. We further extracted the lower limit of the bandgaps at each fixed D based on the Schottky barrier height inferred from the off-current at each charge neutrality point according to the method shown in ref. 2 , followed by using only 12CH4 in the second step with a shorter cooling time to form isolated second layer domains , followed by using only 12CH4 in the second step with a short cooling time to form isolated second layer domains (60 min). As shown in Supplementary Fig. 13C, without 12C being detected. This gives another evidence that the second layer was grown epitaxially through precipitation of the carbon atoms that were dissolved in the solid Pt core in the first step. Compared to the liquid Pt3Si above the Pt domains, the liquid Pt3Si nearby the GBs of Pt can get more carbon supply because of the locally enhanced carbon segregation30. As a result, the second layer preferentially nucleates and grows nearby the GBs of Pt in the beginning  than the BLG with non-AB stacking (8.4 meV per C atom32). Therefore, it is an energy favorable process to form AB stacking if the precipitated bottom graphene nucleus is easily movable on the substrate. Basically, the movement depends on two factors: one is the binding strength between graphene nucleus and the substrate, and the other is the energy for the movement (including rotation and translation) of graphene nucleus on the substrate. In order to understand the interlayer epitaxial growth mechanism of the bottom layer underneath the pre-formed top layer on liquid Pt3Si surface, we performed ab initio molecular dynamics (MD) simulations at 1500 K to study the movement of a small graphene nucleus consisting of 54 carbon atoms on Pt3Si (001). For comparison, we also studied the case on Pt (111), which has relatively low binding energy with graphene among the solid substrates that were used for graphene growth33. As shown in Supplementary Fig. 3Si (001) turns into liquid with Pt and Si atoms taking disordered distribution. DFT calculations show that the binding strength of graphene nucleus with solid Pt (111) and liquid Pt3Si is very similar  surface  surface. Further calculations show that a 1.5 Å translation of graphene nucleus on Pt (111) and liquid Pt3Si leads to an energy change of 5.75 and 1.47 eV (106.5 and 27.2 meV per C atom), respectively . Therefore, the precipitated bottom graphene can form energy favorable AB stacking with the pre-formed top graphene layer on liquid Pt3Si during the growth, showing interlayer epitaxial growth behavior as shown in Fig. 3Si/solid Pt substrate in Fig. Importantly, it is worth noting that the graphene nucleus takes a flatter morphology on liquid Ptace Fig. . Therefoles Fig. –16. The 40° Fig. . Figure 3Si/Pt substrate, forming high-quality ABA-stacked trilayer graphene domains , and then annealed for 12 h to obtain Pt3Si/Pt substrate.Pristine Pt substrate  was first rinsed with DI water, acetone and ethanol in sequence for 1 h each. A 500-nm-thick Si film was then deposited onto the pristine Pt by magnetron sputtering. After that, the Si/Pt substrate was loaded into a boron nitride holder (IMR BN laboratory) inside a fused-silica tube (inner diameter: 22 mm) of a tube furnace , heated to 1100 °C under the protection of H3Si/Pt substrate was shown in Supplementary Fig. 2 (500 sccm), CH4  was introduced to grow graphene for 10 min (the first step). Then, the temperature was decreased slowly to 1025 °C with a constant cooling rate of 12.5 °C h−1, while keeping the flow of H2 (500 sccm) and CH4 (5 sccm) mixture (the second step). After that, the substrate was quickly pulled out of the high-temperature zone, the furnace was shut down, and the CH4 and H2 flows were turned off when the furnace temperature was lower than 800 °C.A typical procedure for the CVD growth of AB-BLG films on Pt20. A piece of Pt foil  was loaded into a fused-silica tube (inner diameter: 22 mm) and heated to 1000 °C under H2 atmosphere (500 sccm) for 10 min to remove residual carbon or organic substances. Then CH4 (4.5 sccm) was introduced into the tube to initiate graphene growth. After 40 min, we stopped the feeding of CH4 and rapidly removed the Pt foil from the high-temperature zone to stop graphene growth.The single-crystal MLG domains for nanoindentation measurements were prepared as reported previously3Si/Pt (or Pt) substrate with the grown graphene was spin-coated with PMMA  at 2000 r.p.m for 60 s, and then cured at 110 °C for 10 min. After that, the PMMA/graphene/Pt3Si/Pt (or PMMA/graphene/Pt) stack was dipped in a 1 M NaOH aqueous solution as the cathode with a constant current of 0.2 A, and a Pt foil was used as the anode. After the PMMA/graphene film was separated from the Pt3Si/Pt (or Pt) substrate by H2 bubbles, it was stamped on the target substrate  and finally acetone was used to remove PMMA at room temperature.The Pt3Si/Pt substrates was characterized by optical microscope (Nikon LV100D), SEM , X-ray photoelectron spectroscopy  and XRD (Rigaku diffractometer with Cu Kα radiation).The structure of Pt2 (285 nm)/Si substrates were characterized by Raman spectroscopy and AFM . The CVD-grown single-crystal MLG domains were characterized by optical microscopy and Raman spectroscopy.The CVD-grown AB-BLG films were characterized by SEM, optical microscope (Nikon LV100D), UV-vis-NIR spectroscopy (Varain Cary 5000 spectrometer), Raman spectroscopy , and TEM . The mechanically exfoliated MLG and AB-BLG samples on SiO25. In general, AB-BLG films and single-crystal MLG domains were first transferred onto a SiO2 (285 nm thick)/Si substrate with circular wells  by electrochemical bubbling to obtain suspended membrane. The force curves were then measured by AFM  with a diamond tip . After the topography image was obtained, we changed the sample’s XY position to locate the membrane’s center right beneath the tip and performed nanoindentation measurement. The cantilever spring constant was 172.8 N m−1, which was calibrated by the Sader method34. The radius of curvature of tip was 11.07 nm. During an indentation, the sample moved up vertically with a constant rate of 50 nm s−1 to make contact with the tip and deflect the membrane according to the presetting upper/lower limits of z-piezo extension. In this procedure, only the change of the z-piezo extension and the change of cantilever deflection could be measured. The measured force was calculated by multiplying the cantilever deflection by the spring constant. The membrane deflection in z direction (Z) was further obtained through subtracting the cantilever deflection from the z-piezo extension. A force curve was finally acquired. After the tip made contact with the membrane, the displacement of the membrane in the middle could be acquired from the change of Z value. By adopting a non-linear model, the force-displacement behavior can be approximated24 asσ02D is the pretension accumulated in the membrane, E2D is the elastic constant with unit of force/length, R is the radius of the patterned holes  of the membrane, following q = 1/ (1.05–0.15ν–0.16ν2). We took ν = 0.165, such that q = 0.9824.The force curve measurements and data processing of CVD-grown AB-BLG and single-crystal MLG domains were the same as those reported previouslyσm as a function of breaking force F and tip radius r:The fracture strength of graphene was measured by loading the membranes to the breaking point. For MLG, it can be modeled in the elastic regime to yield an analytical expression for the breaking strain 25, it is difficult to find a correct model to extract the fracture strength. Since the fracture strength of BLG varies approximately with r1/2, we used the measured strengths of MLG, together with the tip radius used in each experiment, to scale the bilayer results as reported in ref. 25.Because the slip between the two layers of BLG2 (290 nm)/Si substrate. Then, the AB-BLG film was transferred onto the SiO2/Si substrate with source/drain electrodes by using electrochemical bubbling20. After 30 nm Al2O3 was deposited by atomic layer deposition (ALD) as the top-gate dielectric, the AB-BLG film was patterned with lithography, wet etching (Tetramethylammonium Hydroxide) and oxygen plasma etching. In order to avoid current leakage, we deposited another 30 nm Al2O3. Finally, the top gate was patterned and deposited with Ti/Au (5 nm/100 nm) by electron beam evaporation. The FET devices were measured using semiconductor analyzer (Agilent B1500A) and probe station (Cascade M150) at room temperature.First, we used a conventional lithography method to fabricate source/drain electrodes of Ti/Au (5 nm/50 nm) by electron beam evaporation on SiO35 within the framework of DFT. The ab initio MD simulation was conducted at 1500 K  with a 1.0 fs movement for each step in the canonical ensemble employing Nosé-Hoover thermostats (NVT)36. The simulation was performed for 8 ps  with the Gamma-point only. The exchange-correlation effects were treated within the generalized gradient approximation using the Perdew-Burke-Ernzerhof functional37. Electron-ion interactions were described using the frozen-core projector augmented wave approach39 and the Kohn-Sham one-electron valence states were expanded on the basis of plane waves with a cutoff energy of 300 eV. The system of graphene nucleus on Pt (111) surface, namely graphene (Gr)/Pt (111) for short, was constructed by three Pt (111) atomic layers (168 Pt atoms) and a graphene nucleus consisting 19 C-six member rings (54 C atoms). The system of graphene on Pt3Si (001) surface, namely Gr/Pt3Si (001) for short, was constructed by three Pt3Si (001) atomic layers (64 Si and 192 Pt atoms) and a graphene nucleus consisting 19 C-six member rings (54 C atoms). During the ab initio MD simulations, the bottom Pt atomic layer was fixed for both cases. A large vacuum thickness of 20 Å was used to avoid the periodic interaction at the z direction and provide sufficient space for possible structure changes of Gr/Pt (111) and Gr/Pt3Si (001) during the ab initio MD simulation.All the calculations were carried out with the Vienna ab initio simulation packageEb) were calculated by following equation:Etotal (Gr), Etotal (sub), and Etotal (Gr/sub) are the total energies of graphene nucleus, total energies of Pt (111) or Pt3Si (001), and total energies of Gr/Pt (111) or Gr/ Pt3Si (001) without geometry relaxation, respectively. According to this definition, larger Eb means stronger binding between graphene nucleus with substrate.The binding energies  or Gr/Pt3Si (001) after geometry relaxation, and EGr/sub (rotation/shift) is the total energies of Gr/Pt (111) or Gr/Pt3Si (001) with different rotation or translation movement after geometry relaxation.The relative energies were calculated by following equation:Supplementary InformationPeer Review File


Chronic kidney disease (CKD) is a worldwide public health problem. In stages III and IV of CKD, uremic toxins must be removed from the patient by absorption, through a treatment commonly called hemodialysis. Aiming to improve the absorption of uremic toxins, we have studied its absorption in chemically modified graphene nanoplatelets (GNPs). This study involved the reaction between GNPs and diamines with reaction times of 30, 45 and 60 min using ultrasound waves of different amplitudes and frequencies. Functionalized GNPs were analyzed by Fourier Fourier-transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), Scanning electron microscopy and energy dispersitive spectroscopy (SEM-EDS), and Thermogravimetric analysis (TGA). The analysis of the functional groups confirmed the presence of amide and hydroxyl groups on the surface of the GNPs by reactions of diamines with carboxylic acids and epoxides. Adsorption of uremic toxins was determined using equilibrium isotherms, where the maximum percentage of removal of uremic toxins was 97%. Dispersion of modified graphene nanoplatelets was evaluated in water, ethanol and hexane, as a result of this treatment was achieved a good and effective dispersion of diamines-modified graphene nanoplatelets in ethanol and hexane. Finally, the results of hemolysis assays of the modified graphene with amine demonstrated that it was not cytotoxic when using 500 mg/mL. The samples of modified graphene demonstrated low degree of hemolysis (<2%), so this material can be used for in vivo applications such as hemodialysis. A common health problem in different countries is chronic kidney disease (CKD), which has an increasing incidence and prevalence, and high cost of treatment. The main issue is that kidneys do not fulfill their function of purifying the blood. This illness leads to the accumulation of toxic molecules that are normally eliminated by healthy kidneys. These toxic molecules are known as uremic toxins, among which are uric acid, urea and creatinine, which are insoluble in water, and their removal is difficult due to their low molecular weight. To solve this problem, the patient is treated with hemodialysis with non-selective membranes, which are not completely efficient and present high cost for patients . The hemHigh levels of uremic toxins, such as uric acid and urea in the blood, can lead to the formation of solid crystals that locate in the joints, causing episodes of acute pain, in affected people. If high levels of uric acid and urea persist in the blood, they can cause cell death and even reach the stage of cancer . One of Nanoporous adsorbents with high surface area have offered a great potential as biomaterials for CKD. There are several different types of adsorbents, including activated carbon, resins, carbon nanotubes, mesoporous silica, zeolites, metal-organic frameworks, etc.Adsorbents from carbon-based materials  are some of the most promising materials for various applications, due to their high performance and relatively low cost. These materials have demonstrated high capability of adsorption for different organic and inorganic compounds and heavy metal ions ,4,5.Among the carbon-based materials, nanotubes, nanofibers and graphene sheets of micrometric and nanometric dimensions have been studied as adsorbents, due to their size and high specific surface area. Graphene is a carbon-based material that can be found in different arrangements, such as: graphene monolayers, graphene nanosheets and graphene nanoplatelets (GNPs) .2/g), compared to that of carbon nanotubes (1315 m2/g), makes GNPs an attractive candidate for adsorption applications , respectively [Jun et al., in 2015, when producing a graphene hybrid material for obtaining methane, reported nitrogen signals at the same binding energy values of 398.6 and 399.8 eV, corresponding to spectively . Table 2ectively . +NH3). A similar spectrum was reported in the formation of amides by the reaction of carboxylate function and cationic amine function [To corroborate the chemical environment, deconvolution of the N1s signal of the DD+DP30 sample was performed, because a stronger signal was seen through XPS in this sample. The N1s deconvolution spectrum  presentsfunction . The XPSTo observe the surface morphological change and the chemical elements present, Scanning Electron Microscopy and Energy Dispersive Spectroscopy studies were performed. 3+ Cl−).The EDS spectra for the samples DD+DP30, DD+DP45, DD+DP60 and GNPs  show sigThe thermogravimetric analysis of GNPs before and after surface modification are shown in The modified graphene thermograms presented a different thermal behavior; the functionalization of the surface of GNPs causes a lower thermal stability and makes the surface more hydrophilic. Weight loss at a temperature of 100 °C has values of 8.1–12.6%, which indicates that the modified graphene has a higher water adsorption capacity than unmodified GNPs, because the functional groups favor the interaction with water molecules through the formation of hydrogen bonds (-N ---- H).On the other hand, The dispersion test in different solvents provided information on the modification of the samples. Dispersion tests were performed using the unmodified GNPs as a control. In In distilled water, the sample that presented the best dispersion was that treated for 45 min, since it remained stable after 48 h. The DD+DP30 and DD+DP60 samples were adequately dispersed at the beginning of the test and after 48 h, a portion of the particles were deposited on the bottom, while others were suspended; this can be attributed to the time of modification not being optimal for the treatment of GNPs.The dispersion in distilled water of the modified GNPs was only favorable for the DD+DP45 sample, the same situation was observed in ethanol and hexane, the nanoparticles remained in suspension after 48 h of being dispersed, this can be attributed to the presence of alkyl chains of the amines used for the modification.The analysis of the results obtained by FTIR, XPS, SEM and TGA leads us to confirm the presence of different functional groups on the surface of GNPs . The amiThe cavitation generated by the ultrasonic horn under the influence of ultrasound waves of different amplitudes and frequencies, as well as the fluctuation of the pressure, help to carry out the chemical modification and to activate the surfaces of the GNPs. Defects in the edges of the graphene represent the most reactive areas of the material.The adsorption models consider data points within a period of 4 h . We aimed to evaluate the absorption of toxins and the behavior of the different modifications made over GNPs and the unmodified GNPs. The results of the removal percentage of urea from the 160 mg/L solution prepared with distilled water are presented in Modified graphene with amines showed an excellent adsorption capacity of urea; this was more effective for removing the urea in comparison to modified graphene with organic acid, as reported recently . This caThe removal percentage of uric acid is presented in et al. for the adsorption of heavy metals in graphene [graphene . In contgraphene .The Langmuir isotherm is adjusted for the unmodified graphene; therefore, there is monolayer adsorption. Similar results for the adsorption of creatinine and urea have been reported for graphene oxide . The samThe Freundlich isotherm proposes a multilayer adsorption. The application of nanoparticles in hemodialysis treatment involves contact with blood cells, although they are in a polymeric membrane. Hemolysis curves are illustrated in The new materials developed in the present investigation are good candidates for use in CKD; therefore, detailed study on their reusability and compatibility with polymeric membranes should be carried out.According to the results, the surface modification of GNPs with amine mixtures  under the influence of ultrasound waves of different amplitudes and frequencies was carried successfully out. The reaction of hydrophilic amine groups with carboxylic acids and epoxides present on the surface of GNPs proceeds by amidation and ring opening reactions. The functionalization was confirmed by FTIR, XPS, SEM-EDS and TGA. The functionalization of GNPs leads to a surface with greater hydrophilic character and improves its dispersion capacity in water, ethanol and hexanes.With respect to the study of the adsorption of uremic toxins, the percentage of removal was excellent, with sample DD+DP45 achieving a 97% adsorption of urea and uric acid. The values obtained were superior to conventional materials, which only achieve 60% elimination of uremic toxins.These results are attributed to the chemical modification of the surface of the GNPs, in addition to the interaction between the chemically modified nanoparticles and the physisorption process of the toxins evaluated. This investigation contributes to the absorption of uremic toxins using modified GNPs, the method of modification with ultrasound at variable frequency, can be considered as environmentally friendly and economically viable, so that these new materials are an excellent alternative for integration into filters of non-woven fabric for use in CKD.


Soluble guanylate cyclase (sGC) stimulators combined with cyclic AMP (cAMP) activators have been reported to reduce proliferation and matrix deposition in other conditions than IPF. Therefore, this drug combination may present a novel therapeutic concept for IPF. This study investigated the effect of BAY 41-2272 and forskolin on remodelling parameters in primary human lung fibroblasts. The study determined TGF-β induced proliferation by direct cell counts after 3 days; and deposition of collagen type-I, type III, and fibronectin. BAY 41-2272 significantly reduced TGF-β induced fibroblast proliferation, but did not reduce viability. This inhibitory effect was further supported by forskolin. Both BAY 41-2272 and forskolin alone reduced TGF-β induced collagen and fibronectin de novo synthesis as well as deposition. This effect was significantly stronger when the two compounds were combined. Furthermore, the TGF-β induced expression of fibrilar α-smooth muscle actin was reduced by BAY 41-2272 and this effect was strengthened by forskolin. In addition, BAY 41-2272 and forskolin reduced TGF-β induced β-catenin. All effects of BAY 41-2272 were concentration dependent. The findings suggest that BAY 41-2272 in combination with cAMP stimulation may present a novel therapeutic strategy to reduce tissue remodelling in IPF.Remodelling of the peripheral lung tissue and fibrotic foci are the main pathologies of idiopathic pulmonary fibrosis (IPF), a disease that is difficult to treat. TGF- Idiopathic pulmonary fibrosis (IPF) was considered a rare disease that was diagnosed mainly in patients older than 50 years of age , 2. Howeβ1 is a major cause of tissue remodelling in IPF and acts through tyrosine kinase receptors [Diffuse tissue remodelling of the alveolar wall is the major pathology of IPF and seems to be resistant to available therapies , 5. Studeceptors . Currenteceptors , 8.β1 stimulation compared to nondiseased fibroblasts. Furthermore, IPF derived fibroblasts produced a disease specific extracellular matrix (ECM), which modified the behaviour of nondiseased fibroblasts. As a result, healthy fibroblasts seeded onto ECM produced by IPF fibroblasts, expressed α-smooth muscle actin and phosphorylated Smad3 and STAT3. Furthermore, IPF derived ECM increased proliferation and migration of nondiseased fibroblasts [β and may explain the earlier described increase of TGF-β and its receptors in IPF [Isolated human IPF derived fibroblasts expressed markers, characterising the cells as myofibroblasts . IPF fibroblasts . These ds in IPF .β1 released from alveolar epithelial cells [The ECM produced by IPF derived fibroblasts showed a disease specific modified expression of collagen type I, type III, and type V and fibronectin. The expression of all three ECM components was reduced by pirfenidone and nintedanib, however, in a drug-specific pattern . In IPF,al cells . Furtheral cells . Togetheal cells . The twoal cells , but hav de novo synthesis of collagen type I was reduced by sGC due to the inhibition of TGF-β1 induced Erk1/2 signalling in human lung fibroblasts [In animal models of other types of fibrosis, soluble guanylate cyclase (sGC) stimulators reduced fibrotic events. Theroblasts , 17. In roblasts , 18. BAYroblasts . These sβ signalling [β induced remodelling and epithelial-mesenchymal transition [The role of cyclic-AMP (cAMP) in the pathogenesis of IPF had been investigated in a few studies and showed its interaction with TGF-gnalling , 21. In gnalling . In bronansition .In this study, the effect of BAY 41-2272 alone and in combination with cAMP activator (forskolin) on fibroblast proliferation and deposition of ECM components was investigated.α-smooth muscle actin (α-SMA).Nondiseased human lung fibroblasts (CC-2512) were obtained from Lonza, Switzerland, and were grown using standard protocols. Experiments were performed in 70%-80% confluent cell cultures between passages five to eight. Fibroblasts were grown in PRMI-1640 supplemented with 10% fetal calf serum, 20 mM HEPES, 8 mM L-glutamine (GlutaMAX), and 1 x nonessential amino acid mixture . Cells were characterised by their long stretched spindle phenotype which stained positive for fibronectin and inducible staining for Cell proliferation and cell viability were determined by direct cell count using Nexcelom Cellometer T4 ©  and Trypan blue staining after 24 and 72 hours of incubation according to the manufacturer's protocol [α-SMA, and β-catenin (TaqMan gene expression assays), Applied Biosystems . PCR conditions were 45 cycles with 95°C for 10 sec, 65°C for 30 sec, and 72°C for 5 sec. Human18s or Actin TaqMan Predeveloped Assay  was used as control. The target gene was normalized against the reference gene (18s/ACTIN mRNA) and data was expressed as relative increase or decrease from baseline values. Relative mRNA expression was determined using the ΔCT method. ΔCT indicates the difference between the amplification cycles of the target and the housekeeping gene [Total mRNA was isolated using the TRIZOL standard protocol and directly transcribed into cDNA using a reverse transcriptase kit  according to the manufacturer's instructions. Real-Time PCR was performed using a LightCycler480  to determine mRNA of the following genes: collagen type I and type III, fibronectin, ing gene .Deposition of collagen types I and III and of fibronectin was determined using a cell based ELISA as described earlier . All antα-SMA and β-catenin were determined by immunocytochemistry in cells which were grown on cover slips after 24-hour treatment. Cells were washed once with PBS, fixed in 4% formalin (2x5 minutes), followed by 1x wash with PBS, blocking in PBS, 0.01% Tween 20, 2% bovine serum albumin (30 minutes), before being incubated overnight (4°C) with the first antibody for α-SMA  or for β-catenin . After 3x washes with PBS, slides were incubated with a FITC labelled antibody , nuclei were stained by DAPI, and pictures were obtained after 3x washes in PBS by EVOS FL cell imaging system .Structure and protein levels of Data are presented as mean ± standard deviation (SD). The null hypothesis showed no difference between treated and untreated cells. Protein expression and mRNA transcription were compared by Student's t-test . Results were considered statistically significant with p value < 0.05.β1 significantly increased fibroblast proliferation up to 1.7-fold (p<0.01) within 72 hours, as compared to unstimulated cells  , the stimulatory effect was completely neutralised. In the presence of BAY 41-2272, the synthesis of collagen type I mRNA was reduced by 52% and in the presence of forskolin by 98%. When BAY 41-2272 was combined with forskolin, the collagen type I mRNA level was reduced by 80%, compared to TGF-β1 alone  reduced collagen type III mRNA to baseline level and in the presence of BAY 41-27272, a similar effect was achieved  as shown in α-SMA in TGF-β1 stimulated fibroblasts to baseline level (α-SMA mRNA expression by 80%, and when combined with BAY 41-2272, it completely prevented TGF-β1 induced α-SMA mRNA expression (TGF-ne level . Forskolpression .α-SMA in the cytosol as shown in β1 significantly increased the expression of α-SMA and changed the confirmation of the protein into contractile fibrils (β1 on the expression and arrangement of α-SMA was prevented (α-SMA (Unstimulated fibroblasts expressed a low level of  fibrils . When prrevented . Forskold (α-SMA .β-catenin mRNA was significantly increased by TGF-β1 within 24 hours, and this effect was nearly completely prevented in the presence of TGF-β1 inhibitor (β-catenin in TGF-β1 stimulated fibroblasts was reduced to baseline level by each of the treatments (The expression of nhibitor . Similareatments .β-catenin was expressed at a higher level in the cytosol of fibroblasts stimulated with TGF-β1, compared to unstimulated fibroblasts (β-catenin was significantly reduced in fibroblasts that were preincubated either BAY 41-2272 or forskolin.As shown by immunochemistry, roblasts . The expβ1, which were described in fibrotic tissues of IPF patients [IPF is characterised by the accumulation of fibroblasts and increased ECM deposition, which resembles features of scar formation, and were assigned as fibrotic foci . Both evpatients . The expβ1 in IPF [β-catenin signalling [β-catenin activation led to pathological features resembling IPF [β1 induced myofibroblasts generation [β-catenin reduced EMT in an immortalised cell line, A549, which was used as a model for pulmonary fibrosis [β1 induced β-catenin and α-SMA indicates that the combined drugs have the potential to limit myofibroblasts generation. Moreover, BAY 41-2272 prevented the arrangement of α-SMA in contractile fibrils, suggesting a reduced formation of contractile myofibroblasts. However, the combination of both drugs did not enhance this effect of BAY 41-2272. The above presented data showed that BAY 41-2272 and forskolin, are potent inhibitors of β-catenin and therefore may present novel therapeutic options for IPF. However, Wnt and β-catenin signalling are essential for the development and the function of the lung and its role in the pathogenesis of IPF has to be further studied [In IPF, increased proliferation of local resident lung fibroblasts has been suggested to result in fibrotic foci formation . In addi1 in IPF , 30 and gnalling . In humaling IPF . In an aneration . Inhibitfibrosis . Furtherfibrosis . The obs studied .−8 M for forskolin.The role of G-protein coupled receptors in the pathogenesis of fibrotic disease had been suggested in animal models earlier . In a reβ1 [With regard to IPF therapy, nintedanib and pirfenidone achieved their beneficial effects in part by inhibiting TGF-β1 , 40. Howβ1 . The samβ1 , 43. BAYβ1 induced de novo deposition of collagen types I and III. However, neither BAY 41-2272 nor forskolin had a significant effect on TGF-β1 induced fibronectin deposition, except at the highest concentration used in this study. Fibronectin has recently been indicated to play an essential role in wound repair and specifically in the recovery and function of epithelial cells [β1 induced fibronectin deposition may be regarded as beneficial and restore the function of the epithelial cells in IPF.Fibrotic foci in IPF tissue are characterised by increased deposition of ECM, which is composed differently compared to ECM in the healthy lung , 13, 26.al cells . TherefoThe presented data indicates that sGC presents a novel class of drug for IPF therapy; however, this study was limited by the fact that the drugs were only investigated in nondiseased human lung fibroblasts and therefore no information was available if they achieve the same effect as diseased fibroblasts. We did not consider including animal models, since bleomycin-induced fibrosis does not represent the pathogenesis leading to IPF. Future studies are needed to clarify the beneficial effect of sGC on wound repair and tissue regeneration in IPF.The data presented in this study indicates that BAY 41-2272, a sGC activator, may be considered as a novel therapeutic option for IPF. Furthermore, it may be beneficial to combine sGC activator with forskolin to improve their antifibrotic potential.


Inflammatory bowel disease (IBD) patients manifest symptoms of disturbed gut function, such as neural sensory-motor changes. Programmed cell death-ligand 1 (PD-L1), normally present in neural tissue, exists in close apposition to the mucosal immune system and intestinal epithelium, and a bi-directional communication is known to occur at these interfaces. Somatostatin has been shown to suppress the inflammatory reaction, and has been used in several clinical trials to treat inflammatory disorders, such rheumatoid arthritis. Recently, somatostatin receptor type 2A, that regulates neurotransmission, proliferation, and apoptosis, has been recognized in IBD. Although prominent abnormalities in the morphology of the enteric nervous system have been observed in idiopathic IBD, they are more marked in Crohn disease.A 55-year-old woman with recurrent Crohn disease, just surgically treated for ileal resection, have a stenotic complication.At surgery 5 cm of preterminal ileum with stenosis and anastomotic ileocolic block was removed.The histopathology showed a recurrent Crohn in fistulo-stenotic phase; the stenosis was mainly sustained by mass-forming, ganglioneuromatous hyperplasia. Normally very rare, fine nerve twigs extend up into mucosa but we found a new-formed fibrillary network, extending into the inflammation area at the subepithelial luminal site of the mucosa, that was positive to PD-L1 and somatostatin receptor type 2A (SSTR2A) immunostaining but not visualized in routinary stained slides.After surgery the patient was semestrally followed with clinical endoscopic evaluation that results uneventfully.Our case shows that before surgery neuromatous abnormalities can be predicted by immunostained new-formed twigs in the mucosa. Studying the role of programmed cell-death ligand 1 (PD-L1) in acute pain modulation, Chen revealed a mild inhibition of the first pain phase and an increased pain threshold in mice. PD-L1 has a great relevance in new oncological immunotherapy schemes, and is gaining importance in many other fields.,5 Moreover, PD-L1, normally present in neural tissue, has been shown to suppress spinal cord synaptic transmission in the pain circuit, acting as a unique neuromodulator. Somatostatin has been shown to suppress the inflammatory reaction and somatostatin has been used in several clinical trials to treat inflammatory disorders, such as psoriasis and rheumatoid arthritis. Recently, somatostatin receptor type 2A (SSTR2A), that regulates neurotransmission, proliferation, and apoptosis, has been recognized in IBD. Although prominent abnormalities in the morphology of the ENS have been observed in idiopathic IBD, they are more marked in Crohn disease (CD) than in ulcerative colitis (UC) and in some studies are considered as hamartoma.–13 Therefore, neuronal lesions have a true diagnostic role in CD, although it is debated whether the changes are merely secondary responses to chronic inflammation or have a direct pathogenic role.,14 Structural changes in the gut ENS can include an increased number (hyperplasia) and/or increased size (hypertrophy) of nerve bundles and ganglion cells. The neural plexuses abnormalities are prevalently associated with the extent of the inflammatory infiltrate but are reported in both involved and non-involved CD areas.,16 Moreover, the presence of plexitis has recently been shown to be predictive of CD recurrence and of an ominous disease evolution, suggesting that neuroprotection could decrease the disease severity. Well-oriented biopsies including muscolaris mucosae and a small portion of superficial submucosa are key material for a correct diagnosis of IBD. Ganglioneuronal involvement is well known as a diagnostic pattern of Crohn but like more commonly adopted diagnostic features such as transmural inflammation, aphthoid ulcers, fissuration, fistulas, lymphangiectasia, follicular hyperplasia, and fibrous stricturing, it is difficult to highlight in endoscopic grasp forceps biopsies. Mucosal suction biopsies, deep enough to include the submucosa plexus, are adequate to document nerve hypertrophy. However, it is difficult to visualize neurons and nerve bundles in conventionally stained haematoxylin and eosin sections, unless they are hypertrophic.The “intestinal brain” comprises extrinsic and intrinsic innervations, the latter organized in 2 major plexuses, myenteric, and submucosal. Patients with inflammatory bowel disease (IBD) manifest symptoms of disturbed gut function, such as neural sensory-motor changes and altered secretion. Neurons of the enteric nervous system (ENS) also exist in close apposition to cells of the mucosal immune system and the intestinal epithelium, and bi-directional communication is known to occur at these interfaces.2 and Tertychnyi, we immunostained for PD-L1 and SSTR2A to investigate its specific features in CD. Complete results and specific locations are listed in Table  and immunostained by PD-L1 and SSTR2A antibodies , many conditions, ranging from the undeclared use of local therapy to very early disease phases, as well as pediatric cases, can make differential diagnosis with UC a difficult task. In the initial biopsy diagnosis of Crohn, any other additional features,20 must be searched for, and will result extremely useful in distinguishing Crohn from other IBD or recent described CEAS (chronic enteropathy associated with the SLCO2A1 gene). It has been suggested that the diagnosis of CD should be based on the presence of 2 major or at least 3 minor criteria, in the absence of granulomas. In daily histological diagnostic practice in our specialized gastrointestinal institute, great attention is paid to searching for ganglionites, regarded as one of the minor diagnostic criteria, together with lymphocytostasis, lymphangectasia, and thickening of muscolaris mucosae, that are sometimes detectable in routine biopsies. However, ganglioneuromatous changes, not present in mucosal biopsies and in conventionally stained specimens, remain difficult to visualize. Further prospective studies of the utility of PD-L1 and SSTR2A immunohistochemistry (IHC) in cases of focal chronic active inflammation raising the suspicion of CD, where the expected intense staining of numerous mucosal nerve twigs may help in the diagnostic definition, are warranted. We believe evaluation of the immunostaining of numerous high density fragmented fibers in subepithelial luminal portions of inflamed mucosa deprived of glands may also be useful. In particular, we highlight for the first time the detection of PD-L1 in plexuses and, with increasing intensity, in hyperplastic neuromatous lesions in Crohn disease, present in bundles of nerve twigs lying in the thickened muscolaris mucosae containing pain-sensing nerve fibers. The PD-L1/PD-1 pathway can regulate pain sensitivity via non-immune modulation processes such as neuronal modulation. It is increasingly appreciated that primary nociceptive neurons share similarities with immune cells and can both listen to and talk to immune cells.–24 These results suggest that, given the high potency of PD-L1 in suppressing human nociceptors activities, local targeting of PD-L1/PD-1 signaling in sensory neurons may promote the development of novel analgesics.An electron microscopy study reported that normal intestinal nerves are infrequently identifiable, unless they are proven neuromatous lesions.–13 IHC for PD-L1 and SSTR2A supports the significance of new formed neural structures, long PD-L1 positive fibers parallel to the crypt and meshes consisting of neuronal fragments staining positive to SSTR2A, as exuberant repair processes secondary to chronic inflammation (Fig. Also mucosal twigs SSTR2A positivity can have a promising predictive role as a marker of underlying ganglioneuromatous lesions in Crohn because they are present only at the level of voluminous nodular submucosal trunks. Hence, the predictive value of subluminal SSTR2A positivity needs to be tested in different settings to evaluate its utility in clinical practice. Therefore, inflammation-related changes in ENS function likely involve neurodegeneration and neuroplasticity responsible for the main complication in CD, in some studies considered to be hamartoma.The authors are grateful to Antonio Cassano and Orazio Sabatelli for the professional technical support.A. Cassano and O. Sabatelli give permission to be named.Conceptualization: Maria Lucia Caruso.Data curation: Antonia Ignazzi, Sergio Coletta, Elisabetta Cavalcanti.Formal analysis: Maria Lucia Caruso.Methodology: Antonia Ignazzi, Sergio Coletta.Project administration: Maria Lucia Caruso.Resources: Maria Lucia Caruso.Supervision: Maria Lucia Caruso.Validation: Maria Lucia Caruso, Federica Di Pinto, Antonia Ignazzi, Sergio Coletta, Anna Maria Valentini, Elisabetta Cavalcanti, Francesco De Michele.Visualization: Maria Lucia Caruso, Federica Di Pinto, Antonia Ignazzi, Sergio Coletta, Anna Maria Valentini, Elisabetta Cavalcanti, Francesco De Michele.Writing – original draft: Federica Di Pinto, Antonia Ignazzi, Sergio Coletta, Anna Maria Valentini, Elisabetta Cavalcanti.Writing – review & editing: Federica Di Pinto, Anna Maria Valentini, Elisabetta Cavalcanti, Francesco De Michele.


Humidity sensors are indispensable for various electronic systems and instrumentations. To develop a new humidity sensing mechanism is the key for the next generation of sensor technology. In this work, a novel flexible paper-based current humidity sensor is proposed. The developed alternating current electroluminescent devices (ACEL) consist of the electroless plating Ni on filter paper and silver nanowires (AgNWs) as the bottom and upper electrodes, and ZnS:Cu as the phosphor layer, respectively. The proposed humidity sensor is based on ACEL with the paper substrate and the ZnS:Cu phosphor layer as the humidity sensing element. The moisture effect on the optical properties of ACELs has been studied firstly. Then, the processing parameters of the paper-based ACELs such as electroless plated bottom electrode and spin-coated phosphor layer as a function of the humidity-sensitive characteristics are investigated. The sensing mechanism of the proposed sensor has been elucidated based on the  Humidity monitoring is widely applied in the paper industry, tobacco industry, microelectronics, intelligent home, etc. Therefore, the development of fast and effective humidity detection technology has always been a concern for the community. So far various types of humidity sensors based on capacitance ,2,3, resPaper has been widely used in flexible electronic devices due to its attractive characteristics, such as chemically and mechanically stable under atmospheric conditions, low-cost, recyclable, light-weighted, and environmentally benign. For example, filter paper itself is a good sensing element candidate for the detection of gases and humidity due to its high flexibility and inherent porous structure, rough surface which facilitates to absorb the vapors. It was reported that it provides the large surface area comparable to the nonporous plastics . In a huZnS-based materials are also potential for luminescence. ACELs are very promising due to their intrinsic ability of uniform light emission, flexible architecture, low heat generation, and low power consumption ,27,28,29AgNWs solution (5 mg/mL) was purchased from Aladdin Industrial Corporation . ZnS/Cu phosphor microparticles with radius of 5~7 μm and PB glue were synthesized by Keyan Phosphor Technology Company . The amount of Cu activator in the phosphor is around 0.1 wt% ZnS, and the size of ZnS:Cu powder is around 4–10 μm. Filter paper was purchased from Whatman.The innovative ACELs based on filter paper substrates were fabricated via the all-solution processes, as shown in 2SO4 and in principle the maximum RH value yielded can be ~97% at room temperature at 25 °C. By adjusting the flow ratio between the dry/wet gases, a wide humidity range, ~20–90%, could be obtained. The current of ACELs was measured by a digital multimeter . During the humidity experiment, we measured the changes of current and light intensity with the % RH simultaneously. To precisely calibrate the sensing performance, we used a commercial humidity sensor  to monitor the chamber. During testing, each humidity input was kept constant for 10 min to obtain steady humidity environment. After test was finished, the flow rate of wet nitrogen was turned back to zero, and then the pure nitrogen was used to flush the chamber until the low humidity environment reached.The structural and morphological properties of as-received ACELs were examined by scanning electron microscopy . The power source for ACELs was a homemade Alternating Current (AC) system which could provide 1000 V with the frequency of ~10 Alternating current-driven inorganic powder electroluminescent devices were fabricated on the filter paper substrate via the all-solution processes. Nickel bottom electrodes were prepared from electroless plating solution, and the simple spin-coating method was used to fabricate the active layer and the top electrode. So, the whole fabrication processes were simplified greatly. In order to optimize the EL performance, we controlled the mixing ratio of the emitting-dielectric composite, which was composed of ZnS:Cu powder and an organic binder-PB glue. The EL sample with a phosphor-to-binder mixing ratio of 2:1 exhibited the highest EL performance. Here, it is worth noting that filter paper has multifunctional including: First, it acts as the carrier substrate for nickel electrode. Second, it uses the dielectric layer for ACELs. At last, the porous paper is a good absorber. Therefore, the luminance of paper-based ACELs can be used as the indicator to sense the humidity since the environmental water vapor can influence the electrical properties like dielectric constant remarkably.2O. Therefore, it is unquestionable to construct the humidity sensor via the electrical response of ZnS:Cu. More importantly, there also exists the potential to develop the new humidity detection technology which can integrate and employ both the electrical and optical responses simultaneously.The characteristics of the ACELs were measured by applying an AC voltage under ambient conditions. It is well known that the ZnS:Cu phosphor can give blue-green emissions corresponding to the transitions between Cu and ZnS materials. In principle, ACEL can be regarded as the serial of two capacitors. First, the capacitance response of ACELs without AC signal source applied was studied by a precision LCR meter at varied relative humidity values ranging from 20% to 90%. In order to improve the linearity, the characteristics of ACELs excited by the AC signal source in different humidity levels was studied. In order to simplify the testing, we measured the effective values of AC current instead of the capacitance. The digital multimeter in serial with the ACEL was employed as shown in First, the effect of nickel metallization on humidity-sensing characteristics was investigated. The current responses of ACELs with different ZnS:Cu phosphor layers were studied. The EN time was fixed at 10 min. The thickness was controlled by adjusting the spin-coating speed. The relationship between the current of ACELs with different phosphor layers and the humidity levels is shown in Through linear fitting the curve obtained above, the linear regression equation can be expressed as The time-dependent current response of the paper-based ACELs was tested by switching the device between the low humidity environment (11.9% RH) and the 80% RH environment. The dynamic current response of the device is shown in ® TBS 1052B, Shanghai, China), as shown in In principle, the change of AC current is attributed to the change of capacitive reactance in the humidity sensing behavior of paper-based ACELs can be explained roughly based on the electrical properties changes due to the water molecules adsorption/desorption, and adsorption of water vapor is totally responsible for changes in capacitive reactance of the ACELs. The porous and rough surface of the filter paper is favorable to accommodate water vapor which is beneficial to improve the sensitivity of humidity sensor. For a reasonable explanation of the capacitive modulation of the phosphor layer with the RH we have to consider semiconducting properties. It is known that ZnS is thermodynamically unstable in the presence of oxygen and should be converted to ZnO, one can expect oxygen promoted water molecule physisorption. In general, the formation of the SH- groups can be expected for the sulfide surface after interaction with water molecules. However, this effect has not been observed experimentally as a common trend . As a reThe ACEL is connected in series with a fixed capacitor echnique ,40. The o 87% RH . Of courThe flexibility is essential for humidity sensors applications in electronics. Thus, the static dynamic properties were measured. The results are shown in It is understood that low quantities of light can maintain the postharvest quality of vegetables and crops by mitigating senescence, and improving phytochemical and nutrient content in several species ,42,43. AWe have fabricated the AC-type ACELs with total solution processes on filter paper. It is found that the ACEL sensor exhibits an excellent linearity (


Graphene papers have great potential for various applications, such as electrodes in energy storage devices, protective coating, and desalination, because of their free-standing structure, flexibility, and chemical tunability. The inner structures of the graphene papers can affect their physical properties and device performance. Here, we investigated a way to fabricate graphene papers from crumpled reduced graphene oxide (rGO) spheres. We found that ultrasonication was useful for tailoring the morphology of the crumpled graphene spheres, resulting in a successful fabrication of graphene papers with tunable inner pore structures. The fabricated graphene papers showed changes in mechanical and electrical properties depending on their pore structures. In addition, the tailored pore structures had an influence on the electrochemical performance of supercapacitors with the fabricated graphene papers as electrode materials. This work demonstrates a facile method to fabricate graphene papers from crumpled rGO powders, as well as a fundamental understanding of the effect of the inner pore structures in mechanical, electrical, and electrochemical characteristics of graphene papers. The mass production of graphene powders has been enabled by the chemical oxidation of graphite, followed by the exfoliation of graphite oxide into individual graphene oxide (GO) and a reduction of GO with chemical and/or thermal treatments . A large2 [Among several methods to fabricate macroscopic structures from graphene flakes, a paper-like film presents an attractive form because of its flexibility, mechanical strength, electrical conductivity, and chemical tunability ,9. These2  or polym2 , have be2  or carbo2 ,24 have 2  or the f2 . SubsequIn this work, we investigated a simple method to fabricate a paper-like film from crumpled rGO spheres. In order to tailor the morphology of the crumpled graphene spheres, ultrasonication was utilized, providing successful fabrication of graphene papers with porous inner structures. The mechanical and electrical properties of the fabricated graphene papers were studied according to the sonication time. In addition, supercapacitors with a two-electrode configuration were assembled with the fabricated graphene papers as electrode materials, and their electrochemical performances were evaluated with respect to the sonication treatment.Commercially available rGO powders  were used for this work. The synthesis of the crumpled rGO powders was performed according to the following procedure. First, GO was obtained by the modified Hummer’s method  and thenExperiments started from the commercial, crumpled rGO powders. The shape of the rGO powders was controlled using ultrasonic treatments. The commercial rGO powders were used as received in this work, except the sonication treatments. The as-received rGO powders were mixed with dimethylformamide (DMF) at a concentration of 3 mg/40 mL and the mixture was placed in a bath sonicator with a power of 150 W. Each mixture was treated for different times: 2, 4, 6, 10, 14, and 20 h. Graphene papers were prepared by vacuum-filtration of the sonicated mixture with an anodic aluminum oxide (AAO) membrane filter .The morphology of the rGO powders was observed by scanning electron microscopy  before and after the sonication treatments. The chemical structures of rGO were characterized with X-ray photoelectron spectroscopy . The C 1s core-level spectra were deconvoluted with Gaussian-Lorentzian functions after the background signal was subtracted by the Shirley-background model. Raman spectroscopy  was also used to characterize the rGO powers before and after the sonication treatments.The mechanical properties of the fabricated graphene papers were characterized by a universal tensile machine after cutting the paper into rectangular strips ,16. In oCsp, was calculated from the CV curves by means of the following equation:I is the voltammetric current, m is the mass of a single electrode, V is the voltage of the supercapacitor, ∆V is the voltage window of the scan, and ν is the scan rate.The electrochemical performance of a supercapacitor was characterized with a symmetric two-electrode cell configuration by following the literature ,29,30,31I is the constant current, m is the mass of a single electrode, and dV/dt was obtained by the slope of the linearly fitted discharge curve.The specific capacitance of a single electrode was also obtained from the galvanostatic charge/discharge curves by using the following equation:The rGO powders had crumpled spherical shapes because a GO dispersion was spray-dried and thermally reduced after partial reduction by L-ascorbic acid in a GO solution. To observe the change of the morphology of individual rGO powders, a small amount of an rGO solution was dropped on an AAO membrane filter as shown in The lateral sizes of unfolded rGO flakes were characterized with the observed SEM images. 2-hybridized carbon (C=C) bonding positioned at the binding energy of 284.5 eV was modeled using the asymmetric Doniach-Sunjic line shape [3-hybridized carbon (C–C) at 285.1 eV, C–O at 286.3 eV, C=O at 287.5 eV, and O=C–O at 288.8 eV [XPS was used to further investigate the effect of the sonication treatments on the rGO powders. ne shape ,37,38,39288.8 eV ,41. In a288.8 eV . Based o−1, respectively [ID/IG, were 2.29, 2.35, 2.37, and 2.36 for the sonication times of 6, 10, 14, and 20 h, respectively, while the raw powders showed the ID/IG of 2.29. It has been known that ID/IG can indicate the ratio of disordered carbons in graphene nanosheets [Raman spectroscopy was also used to characterize the influence of the sonication on the rGO powders. nosheets . Based oThe rGO papers were fabricated by conventional vacuum filtration ,43. It wThe rGO papers with tailored pore structures were tested to understand their mechanical, electrical, and electrochemical properties. Mechanical properties were measured by tensile tests after cutting the papers in a rectangular shape. Additionally, electrochemical characteristics of the fabricated rGO papers were examined from supercapacitors based on EDLC. In general, porous structures are desirable for enhancing ion diffusion and adsorption in electrodes of supercapacitors. Since the rGO paper is free-standing, flexible, and electrically conductive, it is a good candidate for the binder-free electrode materials of supercapacitors, especially flexible supercapacitors. Graphene supercapacitors were assembled with two fabricated rGO papers as binder-free electrodes that are isolated by a porous separator. Since graphene powders are commercially available these days, the development of an appropriate method to directly use the graphene powders in a paper-like form is useful for certain applications, such as flexible supercapacitors, actuators, sensors, composites, and water purification. In this respect, this work demonstrates a simple but useful way to make graphene papers directly from crumpled graphene powders by using ultrasonication treatment. The simple sonication treatments tailored the morphology of the crumpled rGO spheres due to the strong cavitation effect, which generated unfolded and flatter rGO flakes. This leads to the successful fabrication of graphene papers directly from the rGO powders. The fabricated graphene papers showed the change of the pore structures depending on the sonication time, resulting in a change of the mechanical and electrical properties of the papers. In addition, supercapacitors with the graphene papers were evaluated as a function of the sonication time. It was found that more pore structures enhanced the specific capacitances of the supercapacitors. This work provides an efficient way to make graphene papers directly from rGO powders and enabled us to better understand the mechanical, electrical, and electrochemical properties of the fabricated graphene papers according to their inner pore structures.


Background: The immune infiltrate plays an important part in testicular germ cell tumors, but it remains scarcely studied. We aimed at thoroughly characterizing the immune infiltrate and expression of immune checkpoints PD-L1/CTLA-4 and mismatch repair (MMR) proteins in these neoplasms, seeking for associations with patient outcome. Methods: A total of 162 consecutively diagnosed patients (2005–2018) were included. Immunostaining for PD-L1, CTLA-4 and MMR proteins was independently assessed both in immune cells (ICs) and tumor cells (TCs) of primary tumors and metastases, and characterization of IC populations was pursued. Results: PD-L1 and CTLA-4 positivity in ICs was frequent (85.5% and 96.3%). Patients with absent PD-L1 positive ICs exhibited significantly worse relapse-free survival , both in univariable and multivariable analysis. Lower CD20 and CD3 IC infiltration in seminomas associated with higher disease stage . CTLA-4 TC intensity was significantly higher in yolk sac tumor, choriocarcinoma and teratoma, while PD-L1 TC positivity was significantly more frequent in choriocarcinoma. Both PD-L1 and CTLA-4 immunoexpression in ICs of metastatic samples was frequent (100% and 88.2%). MMR proteins were differentially expressed among the different tumor subtypes. Conclusions: Immune infiltrate/checkpoints associate with patients’ outcome, constituting novel  disease biomarkers. Testicular germ cell tumors (TGCTs) are the most common cancer type afflicting Caucasian men between 15–44 years-old. The overall prognosis is good, with outstanding cure rates and decreasing mortality ,3. Moreoin situ (GCNIS), and are grouped into two major families: the seminomas (SEs) and the various non-seminoma (NS) subtypes  ) . Given tma [TE]) ,11,12,13The Cancer Genome Atlas (TCGA) database, Shah et al. identified a surrogate signature of “T-cell inflamed genes” in 47% of TGCTs, and Siska et al. explored in depth the immune infiltrate in TGCTs [Immunotherapies have achieved important landmarks with clinical impact over the last years in many cancer models, including urological cancer . Howeverin TGCTs ,17. Sincin TGCTs ,19. Alsoin TGCTs ,21. Despin TGCTs , the rolRecently, mismatch-repair (MMR) deficiency has been strongly associated to PD-L1 expression, namely in colorectal and endometrial cancers ,24. MMR-Herein, we aim to evaluate and compare the immunoexpression of PD-L1, CTLA-4 and MMR proteins in a large and well characterized cohort of TGCTs, exploring their potential biological role and establishing important clinicopathological associations (namely impact on patients’ outcome). Furthermore, we aim to explore associations between abundance of specific IC populations and clinicopathological variables in a cohort of SE tumor samples.A detailed clinicopathological characterization of the TGCT cohort is depicted in Distribution of PD-L1 and CTLA-4 immunoexpression in ICs is depicted in p = 0.0345, p = 0.0419 and p = 0.0280, respectively, CTLA-4 positivity in ICs did not associate with clinicopathological variables . Howeverp = 0.013) (p = 0.028).Regarding PD-L1 positivity in ICs, it did not differ significantly among subgroups of patients with different clinicopathological variables  . However= 0.013) ; 2-year-= 0.013)  and alsop = 0.0216) and presence of rete testis invasion (p = 0.0079). Also, lower CD3 IC infiltration associated with higher disease stage (p = 0.0291) and pT stage (p = 0.0211) .Among the 86 pure SE patients, 43 (50.0%) and 59 (68.6%) were classified as being highly infiltrated with CD20- and CD3-positive ICs, respectively (specific criteria for considering IC infiltration are detailed in Methods section) . Lower C 0.0211) . CD20 anFurther characterizing the CD3-positive IC population, it consisted mostly of CD8-positive ICs; 61 (70.9%) and 80 (93.0%) SEs were regarded as highly infiltrated by CD4- and CD8-positive ICs. The CD4/CD8 ratio was <1 in 70 (81.4%) cases , with doIllustrative examples of different types of immune infiltrate are depicted in p < 0.0001, p < 0.0001); specifically, it was more intense in YST, CH and TE, when compared to EC or SE (Immunoexpression of CTLA-4 in TCs was depicted in 237/264 (89.7%) of available tumor samples; it is further detailed in EC or SE B.rete testis invasion, pT stage or N stage). CTLA-4 TC intensity did not have a significant impact on RFS (p = 0.934)  . p < 0.0001); specifically, PD-L1 positivity in TCs was significantly more common in CH and less common in TE  (p = 0.846) (Patients with evidence of PD-L1-positive TCs showed significantly more frequent lymphovascular invasion compared to those with no PD-L1 immunoexpression in TCs (= 0.846) .Immunoexpression of MMR proteins in primary tumors is depicted in p < 0.0001 and p = 0.0003, respectively, p < 0.0001, p = 0.0003, p = 0.0005 and p < 0.0001 for MLH1, PMS2, MSH2 and MSH6, respectively). A combined score ≥6 was significantly more common in SE when compared to all other subtypes (for MLH1) and when compared to TE (for PMS2) (SEs exhibited significantly higher immunoexpression scores for MLH1 and PMS2 when compared to NS (or PMS2) B,D. It wor PMS2) E–H. RemaWe found no significant association between the combined immunoexpression score for any of the MMR proteins and the presence of PD-L1-positive TCs or ICs. For SEs, also no association was found between immunoexpression score of any of the four MMR proteins and the infiltration by CD20- or CD3-positive ICs.Raw data regarding immunoscoring of metastatic samples is provided in p = 0.0801 and p = 0.0827) (PD-L1 immunoexpression in ICs and TCs was observed in 17/17 (100%) and 8/17 (47.1%) of available metastatic samples, both with higher frequency when compared to primary TGCT samples, although the difference was not statistically significant ( 0.0827) . Positivp = 0.0440, p = 0.0002, All nine residual mature TE samples disclosed immunoexpression of CTLA-4 in TCs, while this was observed in 3/6 (50%) of non-TE viable disease , and that this occurred across all subtypes. Higher PD-L1 immunoexpression scoring has already been indicated to associate with a better outcome in urothelial carcinoma, for which PD-L1 scoring in ICs has become routine in selected groups of patients . The onlGiven the prominent immune infiltrate across TGCT subtypes, it is surprising that few studies have attempted to better study this immune infiltrate. Previous works have been limited in cohort size and heterogeneity; some have focused on male fertility, comparing to biopsies of infertile patients or those with GCNIS; and, importantly, there is an absence of studies seeking for clinicopathological correlates, which is what we pursued in our work ,39,40,41in silico analysis of the TCGA database , which  samples ) but alsTo date, no assessment of either PD-L1 or CTLA-4 was reported in TGCT metastatic samples. Our findings show frequent immunoexpression (both in TCs and ICs) of PD-L1 and CTLA-4 in metastatic samples. Specifically, for PD-L1, immunoexpression was more frequent in metastases than in primary tumors (including positive ICs in 100% of the cases). Remarkably, all metastatic samples with non-TE cisplatin-resistant viable disease showed PD-L1 positivity in TCs. This could potentially indicate that drugs targeting these agents could be of use in the metastatic setting. However, importantly, data should be interpreted with caution given the failure of clinical studies employing targeted therapies as single agents thus far ,49,50. ADespite some controversy among initial studies  , the assA recent report found that methylation of homologous recombinant genes was associated with the expression of the immune checkpoint PD-L1 in squamous cell carcinoma of the head and neck, lung and cervix , and proLimitations of our work include its retrospective nature, the subjectivity inherent to immunohistochemistry scoring, and the modest number of metastatic samples available in the study. Further studies are needed to replicate our findings and validate them, particularly in the metastatic context.th edition of the American Joint Committee on Cancer (AJCC) staging manual [A cohort of TGCT patients  consecug manual  and the g manual . Patientg manual ,58. Foll2 of tissue and absence of extensive necrosis) were selected by a TGCT-dedicated pathologist. Consultation cases, cases corresponding to testicular type I or III tumors , and cases without adequate histological material available were excluded. Importantly, individual tumor areas were carefully demarcated  orchiectomy tissue samples (prior to any systemic treatment) and metastases  were available, and representative blocks  aThis study was approved by the ethics committee of Portuguese Oncology Institute of Porto (Comissão de Ética para a Saúde—CES-IPO-3-2018) and is within the scope of the project also approved by the same ethics committee (Comissão de Ética para a Saúde—CES-IPO-1-2018).The automated staining instruments, detection systems, antibodies, dilutions, respective clones and vendors used in the study are depicted in Immunostaining was assessed by a TGCT-dedicated pathologist with experience in routine reporting of PD-L1 immunoexpression in non-small cell lung cancer and urothelial carcinoma, blinded to clinicopathological data. For all the markers studied, no specific cutoff or methodology is validated for scoring immunoexpression. Hence, for the immune checkpoints PD-L1 and CTLA-4, both intensity  and percentage of positive cells (with 10% increments) were scored, both for tumor cells (TCs) and for immune cells (ICs). “Weak” was defined as slight staining only depicted at high power magnification (400×); “moderate” as staining discernible at low power magnification (40×) but better appreciated and characterized at 100× magnification; and “strong” as staining optimally and easily appreciated at low power magnification (40×). For PD-L1 evaluation in TCs, the same indications reported for use in non-small cell lung cancer were employed, with only membrane staining  of any intensity being considered . A minimIn all pure SE patients, the immune infiltrate within and at the periphery of the tumor was further characterized with CD20 (for B-lymphocytes), CD3 (for T-lymphocytes), CD4 (for T-helper lymphocytes), CD8 (for T-cytotoxic lymphocytes), CD68 (for macrophages) and CD56 (for NK cells). Cases were regarded as “highly” or “poorly” infiltrated by these cells in low-power assessment (proportion of available positive ICs in relation to the amount of viable TCs on the slide). “High infiltration” was defined as presence of true large follicle-shaped aggregates of immune cells throughout the tumor or completely filled septa overgrowing tumor cells, while “low infiltration” was considered when immune cells were found scattered throughout the tumor in septa, with only minor and few aggregate formation. To evaluate the predominance of each subtype of T-lymphocyte, the CD4/CD8 ratio was assessed. For MMR proteins, again, both intensity  and proportion of positive TCs  was scored. A combined score resulting of the sum of both intensity and percentage scores was calculated.In mixed tumor patients, the highest immunoexpression score for each of the markers was chosen as representative of the whole tumor (and the patient) for seeking clinicopathological correlates.p-values were adjusted to multiple testing by Bonferroni’s correction. Survival analyses were computed with Kaplan-Meier estimator and log-rank test. Relapse-free survival was assessed and hazard ratios with respective 95% confidence intervals were estimated using Cox regression models. Statistical significance was set at p-value < 0.05.Data was tabulated using Microsoft Excel 2016 and analyzed using IBM SPSS Statistics version 24 and GraphPad Prism 6. Associations between categorical variables were established by the Chi-square or Fisher’s exact test, as appropriate. Differences between distributions of continuous variables among groups were assessed by non-parametric Mann-Whitney U test and Kruskal-Wallis test, as appropriate. All To conclude, we have demonstrated an overall frequent expression of immune checkpoints in TGCTs, both primary and metastatic, and showed that heterogeneity in expression levels of these markers and variable amount and types of immune infiltrate associate with prognosis. Importantly, PD-L1 immunoexpression in ICs influenced patient outcome, when adjusting for various relevant clinicopathological features. To the best of our knowledge, expression of CTLA-4 in TGCTs has not been reported to date; our data should be validated in independent series and by techniques other than immunohistochemistry in future studies. Also, we have demonstrated differential expression of MMR proteins among tumor samples, both in primary and metastatic context, possibly indicating association with differentiation and resistance to chemotherapy, although validation in larger cohorts is warranted. Despite failure of recent clinical studies using targeted therapies against these proteins as single agents, they were performed in unselected populations. Also, there are preliminary results indicative of some clinical activity for the combination of durvalumab and tremelimumab . Given tOverall, the immunobiology of (testicular) germ cell tumors remains an understudied field , and the


As the IESM for the humidity sensor, the natural materials can be implemented in our daily life as they open a new gate way for bio-compatible devices.In this paper, we propose a novel bio-compatible organic humidity sensor based on natural inner egg shell membrane (IESM) with multilayer cross linked fiber structure that can be used as a substrate as well as a sensing active layer. To fabricate the proposed sensors, two different size inter digital electrodes (IDEs) with 10 mm × 4 mm for sensor 1 and 12 mm × 6 mm for sensor 2 are printed on the surface of the IESM through Fujifilm Dimatix DMP 3000 inkjet material printing setup, which have finger width of 100 μm and space of 100 μm. The fabricated sensors stably operates in a relative humidity (RH) range between 0% RH to 90% RH, and its output impedance and capacitance response are recorded at 1 kHz and 10 kHz. The response time (T In these features, a humidity sensing is widely investigated by many researchers8. Sensors based on impedance and capacitance responses are the best choice for humidity sensing due to low cost, easy fabrication, and nominal time response9, which can detect amount of water content in environment with following parameters like impedance10, capacitance11 and resistance14. To fabricate a humidity sensor, the various materials are studied like, graphene15, organic16, inorganic17, organic-inorganic nano-composite18, and other composites20.In environment sensing, there are various factors to be detected like humidity level21, piezoelectric nanogenerator using onion skin22, silk fiber applied in electronic devices and non-volatile resistive memory24, and fish scale based piezoelectric nanogenerator25. Hence, a biomaterial can be obtained from a hen egg and it is widely using as food in the world. It can be applied in different applications like egg albumen as an active layer for non-volatile resistive memory26, a high performance electrode for super capacitor using carbonized egg shell27, a bio-sensor to determine the glucose using inner egg shell membrane (IESM)28, and piezoelectric flexible nanogenerator using IESM29. The self-assembled nano fiber humidity sensor (based on donor and acceptor mechanism) can be used for monitoring purpose30. Although the IESM is structured with natural multilayer cross linked fibers, it is not applied for a humidity sensor. The natural biomaterials available in our daily life have potential to be employed in sensing applications as they can opens a new possibilities towards bio-compatible electronic devices.For an eco-friendly, low cost and high performance devices, many researchers are exploring natural materials, for example, humidity sensing layer and substrate using onion membrane31. Moreover, such substrates are used only to support device printed on them with no sensing properties and additional sensing layer is required to be top printed on electrodes. For these attributes, natural biological organic materials available in our daily life are very attractive for sensing in wearable electronic devices22. In this expectation, to achieve good results, the various printing methods are utilizing for high performance and transferable wearable devices33. The conventional printing methods utilized to transfer wearable electronic devices are very complex and damage may occur while transferring to arbitrary shaped substrate31. While an IESM is ultra-thin with thickness of ~19 μm, it is allowed to transfer to any arbitrary shaped substrate. The IESM can be transfer in a single step to an arbitrary shape substrate without causing any damage.Many researchers are focusing on transferable, wearable, and implantable electronic devices on thin substrates, which are highly flexible and light in weight35. Especially, the IESM has fibrous and porous structure, which is a suitable to absorb a permeable solvent like water; hence it can be used as a humidity sensing active layer. To get a signal from the IESM, we fabricated inter digital electrodes (IDEs) pattern with finger width of 100 μm and spacing of 100 μm through Fujifilm Dimatix DMP-3000 material as shown in Fig. res) of ~1.99 sec and a recovery time (Trec) of ~8.76 sec using sensor 1 and Tres of ~2.32 sec and Trec of ~9.21 sec using sensor 2. The surface morphology and cross sectional image of the proposed sensor is analyzed with NV-2000 Universal non-contact surface profiler, Scanning electron microscopy (SEM) Jeol JSM-7600F and TESCAN MIRA 3 scanning transmission electron microscope (STEM), and Fourier transform infrared spectroscopy (FTIR) is performed with Bruker IFS 66 V spectrometer. To find the material characteristics of the IESM, its elemental composition is performed with energy dispersive X-ray (EDS) spectrometer.The proposed humidity sensor is fabricated by using the IESM as an active sensing layer and substrate. The IESM is contacted with the egg albumen and an external contact with the egg shell minerals36. A network-like structure is perceived in the natural IESM with 10 μm scale at ×500 magnification as shown in Fig. 2) of 0.89 µm. These results shows that the thickness of the dried IESM is almost uniform with small value of standard deviation as shown in Fig. Hen egg is shown in Fig. The 2D and 3D nano profile of the printed IDEs is shown in Fig. −1, amide II shows NH in plane bending and CN stretching at 1440 cm−1, and amide A shows NH stretching vibration at 3400 cm−1. An absorption band shows stretching vibration of C-S bonds appeared at 670 cm−1, which is associated with cysteine-rich proteins of an IESM fibers37. A sharp absorption peak of eggshell was also present at 875 cm−1 which shows out of the plane bending vibration of (CO3−2)38.The Fourier transform infrared spectroscopy-potassium bromide (FTIR-KBr) spectra of IESM showed the characteristic absorption peaks related to the organic structure of the membrane. In the FTIR spectra of an IESM shown in Fig. The elemental composition on the surface of IESM can be confirmed by energy dispersive X-ray spectroscopy (EDS) as depicted in Fig. f, and c represent the resistance, test frequency, and capacitance of SUT, respectively.The impedance response of the humidity sensor was recorded on different levels of humidity in the environment. The effect of humidity on the surface of the IESM was recorded on different frequencies as shown in Fig. The fabricated sensor was mounted/transferred on the different arbitrary surfaces as a substrate. The target substrates have different uneven surfaces with different curvature, which includes plant leaf and stem, computer mouse, and conical flask as shown in Fig. γ) and capacitance at higher frequencies. The capacitance of SUT with leakage conduction is given in Eq. 40. The uronic acid is a product of monosaccharide in which the terminal carbon’s hydroxyl group has been oxidized to carboxylic acid. It contains both aldehyde and carboxylic acid moieties and lot of hydroxyl, carbonyl, and amino functional groups40 are present on the protein fibers of IESM39 to interact with moisture content present in the environment37 and can be used as a humidity sensing layer. Hence, the humidity sensing mechanism can be explained as shown in Fig. 37 and it can easily can catch humidity. The structure of IESM also can observed using NV-2000 Universal non-contact surface profiler, which indicate that IESM has surface roughness of 1.35 μm and it also confirm the average thickness between ~18 to 21 μm as shown in Fig. 21 for humidity sensing as shown in Table The proposed bio-compatible humidity sensor has been reported that the main constituent of the IESM are collagen and saccharidesThe main reason of the hysteresis in the IESM is due to the porous structure and chemical absorption. The proposed senor 1 and sensor 2 was analyzed for the hysteresis characteristics at 1 kHz as shown in Fig. For weather monitoring applications, the paper proposed the bio-compatible humidity sensor based on IESM. This IESM applied as a humidity sensing layer and substrate using IESM. Here, Inter digital electrodes (IDEs) with finger width of 100 μm and spacing of 100 μm were deposited on the surface of IESM through Fujifilm Dimatix DMP 3000 inkjet material printer for sensor 1 and sensor 2. The proposed sensor achieved a good response and recovery time, and its impedance and capacitance change are recorded from 0% RH to 90% RH. Hence, the 19 μm thin IESM could be used to replace flexible and ultra-thin substrate based on PDMS. The Possible areas of IESM were applied as ultra-thin substrate for electronic skin applications, which could be conformably transferred to different arbitrary shape surfaces. The IESM is an environment friendly and can be used as a disposable substrate for environment sensing applications.−1. EDS mapping is performed to confirm the element by element composition of IESM using TESCAN MIRA 3 STEM.Different characterization techniques are used to investigate the chemical and structure composition of IESM as shown in Figs 2) was used as dehumidification.Fresh egg was processed using white vinegar. The humidity sensing electrodes were patterned on the surface of the IESM using the Ag nanoparticle ink with 50% dispersion in triethylene glycol monomethyl ether (TGME). Ethanol and de-ionized water was used for cleaning purpose and purchased from sigma Aldrich. The silver (Ag) conductive epoxy CW2400 was used for connecting wires and purchased from circuitsworks. Dry nitrogen gas 44. Then, the naked rubbery egg will remain at the end after 3 days. We make a small hole on the surface of the IESM and pour out the yolk and albumen. The IESM was cleaned with 20 ml ethanol and 30 ml de-ionized water, and place it on a flat surface in a high tension wet state to remove all the water trapped inside the membrane and make it dry at room temperature.The manual separation of the IESM can be done, but it is difficult to obtain the IESM as it is tightly bonded to the mineral shell. The egg shell is made of calcium carbonate crystalsTo fabricate the proposed sensor 1 and sensor 2 on the surface IESM, the IDEs were designed using EAGLE version 7 in dxf format. The design file was converted into bmp file format using ACE 3000 and exported to Dimatix Drop manager software, which converts bmp into ptn format. The ptn file was loaded into software controlled Fujifilm Dimatix DMP-3000 material inkjet printer. The Ag ink was loaded in 10 pL 16 nozzles cartridge with diameter of 9 μm. The 30 V were applied on cartridge nozzles for a stable printing with drop spacing of 20 μm as shown in Fig. 2) are used, respectively. Cool term software is used for reference sensor data logging and OrignPro 8.0 is used for graph plotting. The fabricated sensor 1 and sensor 2 data is logged with built-in software of KEYSIGHT Digital U1700C handled LCR meter.Humidity setup consist of airtight homemade humidity box, HTU21D sensor with resolution 0.04% RH, accuracy ±2% RH, temperature coefficient −0.15% RH/°C and response time <5 sec was used as a reference sensor. Arduino UNO is used as a controlling board and PC is used for data acquisition. The impedance of the fabricated sensor was measured with KEYSIGHT Digital U1700C handled LCR meter, for humidification and dehumidification humidifier and dry nitrogen gas (N2 is injected for dehumidification level from 40% to 0% and the commercialized humidifier is used to increase the humidification level from 0% to 100%. The external valve is used to control the level of humidity inside airtight box using humidifier and N2 gas. The temperature of the experiment setup was controlled at 25 °C. Figure Figure The targeted surface was wetted with water and non-printed side of IESM was placed on the wet surface as shown in Fig.


A wide-band and high gain circularly polarized (CP) graphene-based reflectarray operating in the THz regime is proposed and theoretically investigated in this paper. The proposed reflectarray consists of a THz CP source and several graphene-based unit-cells. Taking advantages of the Pancharatnam Berry (PB) phase principle, the graphene-based unit-cell is capable of realizing a tunable phase range of 360° in a wide-band (1.4–1.7 THz) by unit-cell rotating, overcoming the restriction of intrinsic narrow-band resonance in graphene. Therefore, this graphene-based unit-cell exhibits superior bandwidth and phase tunability to its previous counterparts. To demonstrate this, a wide-band (1.4–1.7 THz) focusing metasurface based on the proposed unit-cell that exhibits excellent focusing effect was designed. Then, according to the reversibility of the optical path, a CP reflectarray was realized by placing a wide-band CP THz source at the focal point of the metasurface. Numerical simulation demonstrates that this reflectarray can achieve a stable high gain up to 15 dBic and an axial ratio around 2.1 dB over the 1.4–1.7 THz band. The good radiation performance of the proposed CP reflectarray, as demonstrated, underlines its suitability for the THz communication applications. Moreover, the design principle of this graphene-based reflectarray with a full 360° phase range tunable unit-cells provides a new pathway to design high-performance CP reflectarray in the THz regime. Terahertz (THz) science and technology that is undergoing an unprecedented rapid development has attracted considerable attention in the scientific community. As a promising technology, it has enormous potential in applications such as communication , sensing2/Vs at room temperature [−6 Ω·cm [λ/10–λ/100) and tight field confinement on the graphene sheet [Reflectarrays ,10,11,12perature , and a l[−6 Ω·cm ,22 in THne sheet ,24, whilne sheet ,29,30,31ne sheet ,39,40,41ne sheet . Soon afne sheet ,44,45,46ne sheet  are essene sheet ,42,43,44In this article, we propose a graphene-based wide-band CP reflectarray operating in the THz regime. The proposed reflectarray consists of a THz CP source and several graphene-based unit-cells. Based on PB phase principle , the graz direction) illuminates the surface, with an incident electric field expressionxr and yr represent the reflectivity of x-polarized and y-polarized waves, respectively. Similarly, xφ and yφ are the reflected phases of x-polarized and y-polarized waves, respectively.To have a clear physical insight of the proposed CP reflectarray, firstly, we have deduced the principle of PB phase, obtaining the necessary phase requirement of the unit cell. As shown in θ, the relationship between the uv coordinate and the xy coordinate can be represented byAs shown in uvz coordinate, we substitute Equation (3) into Equation (1), and the incident waves can be expressed asThus, in the ur and reflected phase φu in the uvz coordinate are equal to the reflectivity xr and reflected phase φx in the xyz coordinate, respectively. In a similar way, the reflectivity vr and reflected phase φv in the uvz coordinate are equal to the reflectivity yr and reflected phase φy in the xyz coordinate, respectively. Therefore, u = rxr, φu = φx, v = ryr, and φv = φy. Then, Equation (5) can be expressed asFrom x = ryr, it is found that the reflected waves consist of two components, RHCP and left hand CP (LHCP), respectively.x − φy| = π, Equations (7) and (8) change toFrom Equation (6), if θ, equal to two times of the rotation angle. Contrary to the metallic plate, the reflected waves of the PB unit-cell based metasurface only contain the co-polarized components. It is worth noting that the conditions of |φx − φy| ≈ π and xr ≈ yr must be fulfilled.Obviously, there is only an RHCP component inside the reflected wave, and the phase variation is 22) substrate. When we extend these unit-cells periodically along both the x and y directions, a 2-D graphene-based metasurface can be obtained, as shown in w or l, as shown in xE component of the incident wave can only excite the plasmonic resonance in the x direction, and yE component of the incident wave can only excite the plasmonic resonance in the y direction [x−φy| ≈ π and xr ≈ yr.Graphene can strongly interact with electromagnetic waves in THz regime through plasmonic resonance ,24. Howeirection . Based oe is the elementary charge, Bk is the Boltzmann’s constant, T is temperature, τ is the relaxation time, ω is the radian frequency, and fE is the Fermi energy. When fE is much larger than the thermal energy B Tk, the complex conductivity of graphene can be simplified to τ is the function of the carrier mobility μ, the Fermi energy fE, and Fermi velocity fv. It can be expressed as μ will reduce the loss and enhance the efficiency of the device. The carrier mobility μ often has a variation range from ~1000 cm2/Vs to ~230,000 cm2/Vs with varied fabricated technologies [f =E 0.64 eV σ(ω)nologies . In our nologies , it is r in Ref. ), carrie2 substrate. The SiO2 substrate has a relative permittivity of rε = 3.75, and a loss tangent tanδ = 0.0184. The parameter w and l are the width and length of the graphene patch in the x and y directions, respectively, and p = 15 µm denotes unit-cell side-length which also equal to a periodicity to form the 2-D metasurface. Besides, t = 10 nm and d = 26 µm are the thickness of the metallic ground and quartz glass (SiO2) substrate, respectively. In our simulation, the master and slaver boundary condition was added to the unit-cell to modeling an infinite array. Meanwhile, Floquet port is placed at z = 6d and utilized to interact with the periodic unit-cell structure. In addition, the reflectivity and phases are obtained by the parametric sweep module of HFSS solver. The center frequency is set to 1.52 THz.The reflectivity and phases of the proposed unit-cell were full-wave simulated using Ansoft HFSS 2017 software . The unit-cell depicted in w = 13.39 μm, and l = 3.2 μm. x- and y-polarized waves, respectively. The reflectivity of x- and y-polarized waves are almost equal, with values near 1, indicating high efficiency. A nearly constant 180° reflected phase difference between the x- and y-polarized incident waves can be achieved in a wide frequency band (1.4–1.7 THz), realizing exactly 180° at 1.52 THz, as shown in Such a PB unit cell can be looked as an anisotropic scatterer, which has a polarization dependent response. In normally incident case, we optimize the geometric parameters of the unit-cell, and choose the geometric parameters RRr and LRr indicate the co-polarized conversion ratio and cross-polarized conversion ratio, respectively.According to the principle of PB phase, the proposed graphene-based unit-cell can realize co-polarized conversion, which can convert the illuminated CP wave into a co-polarized CP wave efficiently. After verifying the wide-band high efficiency co-polarized converting capability of the proposed PB unit-cell, we can compensate arbitrary phase in a range of 0°–360° by element rotating.f represents focal length, λ0 represents wavelength in free space, φ0 represents initial phase at the original point. Following Equation (12), we design a focusing graphene metasurface with 51 × 51 unit-cells at 1.52 THz, and the focal length is set to 190 μm. The phase distribution is illustrated in −z direction, obtaining energy distribution in the xoz plane at 1.4, 1.52, 1.6 and 1.7 THz, respectively, as depicted in To focus the reflected CP wave of a normally incident plane wave, a phase distribution must be fulfilled, where f = 190 μm of the proposed metasurface.Previously, we have proposed a graphene metasurface which has prominent focusing capability in a wide-band of 1.4–1.7 THz. Then, according to the principle of optical path reversibility , we can We have systematically investigated the spectral responses of graphene-based PB phase unit-cell and demonstrated a practical implementation of wide-band high gain CP reflectarray based on this configuration in the THz regime. Based on PB phase principle, an arbitrary reflected phase controlling can be realized, extremely extending the phase modulated ability of conventional graphene-based unit-cells that only have a phase controlling range of 300°. Finally, a focusing graphene-based metasurface and a reflectarray were designed, simulated and optimized. Simulation results exhibit excellent performances as theoretical expectations. The proposed reflectarray has a stable gain of 15 dBic and axial ratio of 2 dB over the 1.4–1.7 THz bands, demonstrating high gain and CP characteristics, simultaneously. This reflectarray may have a promising application in future THz communications. Meanwhile, for practical fabrication, we can design the graphene-based unit-cell which is consist of five layers, such as graphene layer, alumina layer, polysilicon layer, quartz glass layer, and ground. The polysilicon can be applied as an electrode. The Fermi energy is related to the conductivity of graphene, and can be dynamically tuned by varying the DC voltage (VDC) between the graphene and the polysilicon. Detailed techniques to fabricate these kinds of graphene reflectarray can be found in Reference , support