Graphene, owing to its inherent chemical inertness, biocompatibility, and mechanical flexibility, has great potential in guiding cell behaviors such as adhesion and differentiation. However, due to the two-dimensional (2D) nature of graphene, the microfabrication of graphene into micro/nanoscale patterns has been widely adopted for guiding cellular assembly. In this study, we report crumpled graphene, i.e., monolithically defined graphene with a nanoscale wavy surface texture, as a tissue engineering platform that can efficiently promote aligned C2C12 mouse myoblast cell differentiation. We imparted out-of-plane, nanoscale crumpled morphologies to flat graphene via compressive strain-induced deformation. When C2C12 mouse myoblast cells were seeded on the uniaxially crumpled graphene, not only were the alignment and elongation promoted at a single-cell level but also the differentiation and maturation of myotubes were enhanced compared to that on flat graphene. These results demonstrate the utility of the crumpled graphene platform for tissue engineering and regenerative medicine for skeletal muscle tissues. A technique that introduces instabilities onto normally flat graphene can enhance the engineering of muscle fibers. Researchers have recently found that applying cyclic electric stimuli to conductive graphene films promotes the growth of tubular myoblast cells that comprise muscle tissue. SungWoo Nam at the University of Illinois at Urbana-Champaign in the United States and colleagues have now developed a ‘crumpled’ form of graphene to further control the morphology of myoblasts. The team transferred graphene film onto a pre-stretched elastomeric tape, and then gradually released the tape’s tension. Microscopy revealed graphene’s transformation into an accordion-like surface featuring nanoscale ridges and valleys. Comparisons with flat graphene showed the new material improved the assembly of myoblasts into longer myotubes by promoting growth along the direction of the crumpled valleys. For example, skeletal muscles have longitudinally aligned myofibrils presenting directional contractile functions in response to stimuli2. Ligaments and tendons have unidirectionally aligned collagen fibers and thus absorb sudden mechanical forces by the shearing mechanism of the protein matrix3. In addition to those muscular tissues, the hippocampus in brain tissue has directionally sprouting neuronal networks, allowing a unidirectional signal transfer through the CA1-CA3 neural circuit4. Because geometrical directionality has a close relationship with function, the recapitulation of anisotropic orientation has been conceived as a key factor in the fabrication of in vitro tissue models6. One simple yet robust method is the fabrication of anisotropic structures and the seeding of tissue-specific cells on top of tissue scaffolds. Using this method, cells recognize the orientation and surface materials of the underlying substrates and adapt their behaviors based on physicochemical cues. To prepare directional micro/nanostructures, various microfabrication methods, such as photolithography, soft lithography, and electrospinning, have been developed9.Native human tissues possess inherent anatomical directionality, and such directionality endows tissue-specific functions12. Even in the absence of electrical stimuli, simply cultivating C2C12 mouse myoblast cells and neurons on conductive materials enhances differentiation13. These results clearly show the importance of the electrically conductive nature of the matrix in the development of electrically active tissue models. In this regard, graphene has substantial potential in the recapitulation of physiological electrical properties because it is a very thin material with high electrical conductivity14. Furthermore, graphene shows promise in the regulation of cell functions, such as promoting cell adhesion and enhancing stem cell differentiation17 because the adhesion of growth factors on a graphene film is promoted by interactions with π–π stacking18 and enhanced graphene-plasma membrane interaction, which regulates downstream cell signaling pathways19.In the fabrication of in vitro tissue models, the material properties are also an important issue. Skeletal muscle, heart, and brain tissues have electrical stimuli-responsive characteristics. The introduction of cyclic electrical stimuli during the culture period can promote the differentiation and functional maturation of C2C12 mouse myoblast cells20. As a different approach, a suspended graphene sheet was fabricated by transferring a graphene film on top of engineered nanogrooves21. On the graphene film, human mesenchymal stem cells showed an aligned morphology due to the nanogrooves beneath the graphene film, and the morphology promoted neurogenic and osteogenic differentiation. The transfer printing of graphene oxide flakes on the substrate allowed the fabrication of strip and grid graphene oxide patterns and demonstrated the graphene-specific adhesion and controlled differentiation of adipose-derived stem cells on the graphene patterned substrates22. A graphene-nanofiber hybrid scaffold created by electrospinning polycaprolactone nanofibers on a graphene oxide film was used to explore the potential of this material in oligodendrocyte differentiation23. Micrometer-scale graphene oxide wrinkles fabricated by an unconventional fabrication method were shown to be useful in their system in guiding cell alignment24. A microgroove-patterned methacrylated gelatin substrate showed enhanced differentiation capabilities compared to a nonpatterned methacrylated gelatin substrate25. Moreover, recent research has demonstrated that conductive three-dimensional graphene foam bioscaffolds improve the growth and differentiation of C2C12 myoblasts into functional myotubes26. Although previous approaches showed the potential of graphene-based tissue engineering platforms, they usually adapted complex and multistep fabrication techniques for topographically patterned graphene substrates. To utilize novel material properties in tissue engineering applications, a simpler yet robust fabrication method is required.To utilize the characteristics and topographical effect of graphene in tissue engineering, a variety of approaches have been introduced. As a straightforward approach, rectangular graphene patterns with a large area of 150 × 1500 μm were fabricated using standard photolithography methods, and myogenic differentiation of C2C12 mouse myoblast cells was demonstrated on the patternsIn this study, we report a tissue engineering platform that can utilize the topographical features and material properties of graphene by inducing mechanical instability in a graphene film. For this purpose, we transferred graphene onto a prestretched elastomer film and subsequently released the stretch so that the film could spontaneously form anisotropic geometrical features via wrinkling phenomena. We then cultured C2C12 mouse myoblast cells on the crumpled graphene and observed myotube formation and maturation in response to the anisotropic geometrical features. On the crumpled graphene substrate, C2C12 myoblasts showed a highly aligned morphology following the wrinkled structures and exhibited enhanced myotube formation compared to those on the flat graphene substrate.x-direction and y-direction depending on the orientation to induce anisotropic mechanical instability. The major axis was stretched by 150–300%, while the minor axis was stretched by 50% , whereas the amplitude (displayed as the root mean square (RMS) height) increased in response to increased prestrain   intensity remained constant as the prestrain increased -grown graphene onto a prestretched elastomeric substrate (VHB tape) using a polydimethylsiloxane (PDMS) block as a temporary stamp. The stretchable substrate was prestretched by different amounts in the 50% Fig. . We applly) Fig.  and S2. ly) Fig. . Raman ssed Fig. . Further32, showing contact guidance in response to the underlying surface topography.To demonstrate the utility of crumpled graphene in skeletal muscle tissue engineering, C2C12 mouse myoblast cells were seeded on various crumpled graphene platforms Fig. . Here, tp-value indicates that the angular distribution is significantly different from the ‘uniform’ distribution, with given statistical reliabilities corresponding to the p-values33. In the 150 and 300% prestrain cases, p-values smaller than 0.01 indicate a significantly different distribution of angle values from the uniform distribution. The Mardia–Watson–Wheeler test was further used to confirm the equality of the two angular populations34. A sufficiently small p-value indicates that the angular populations of two data sets are significantly different with p-value-specific statistical reliabilities. The Mardia–Watson–Wheeler test between the data sets, such as flat:150% and flat:300%, showed significantly small p-values, indicating a significant difference between the cases in terms of the angular populations. These statistical analysis results consistently showed the utility of the anisotropically crumpled graphene platform for the unidirectional alignment of C2C12 cells.To compare the differences in those cell alignment behaviors, we conducted a statistical analysis Table . The cir36. In this regard, the higher amplitude in the case of 300% prestrain is more beneficial than that for 150% prestrain, and thus, cell alignment and elongation were promoted in the 300% prestrain case.The morphological indices, including the cell length, cell width, and aspect ratio, were different depending on the various platforms Fig. . For exap < 0.01). The Mardia–Watson–Wheeler test demonstrated significant differences between the angular populations on various samples, such as flat:150% and flat:300% (p < 1e−12). The morphological indices of myotubes were also quantified. The length, width, and aspect ratio of myotubes were larger in the case of the crumpled graphene substrates than the flat substrates . These four indices were higher for crumpled graphene substrates (150 and 300%) than for flat substrates. Similar to the results of the morphological indices of the myotubes, there were no significant differences in those four indices between the 150 and 300% prestrain cases. According to previous studies, neither too large (>20 μm)37 nor too small (<70–80 nm)29 size patterns affect myotube fusion, implying the limited effect of feature size control over C2C12 differentiation.The myogenic differentiation efficiency was evaluated with the fusion index, maturation index, myotube fraction, and myotube density Fig. . The fus38, such stiffness-dependent elongation was not observed in our study, presumably because of the small difference in stiffness.To examine the change in stiffness with respect to the applied prestrain and ultimately to the cultured cell behaviors on the crumpled graphene substrates, we measured the elastic modulus of the crumpled graphene using a nanoindenter Fig. . As the From the quantification, we found that at the single C2C12 mouse myoblast cell level, crumpled graphene with a uniaxial topography increases the cell length while decreasing the width. Furthermore, as the prestrain increases from 150 to 300%, the length and aspect ratio of the individual cells tend to increase due to the increased height of the surface texture. However, after myotube formation , such morphological differences between the 150 and 300% prestrain samples decreased.40. As reported in previous studies41, an ultrathin membrane of a rigid material has a quite limited effect on the overall mechanical properties when layered on a soft substrate. Therefore, we believe that the similarities in the mechanical properties of VHB and graphene on VHB resulted in marginal differences in the morphological indices of C2C12 mouse myoblast cells before and after differentiation.Furthermore, C2C12 mouse myoblast cells and differentiated myotubes on bare VHB and flat graphene on VHB Figs.  do not s31. However, in this study, we fabricated a nanoscale  topography using a CVD-grown monolayer graphene. Although, as shown in Fig. 29 support that the imperfections of crumpled graphene have a negligible effect on cell behaviors. The micron-scale topography is known to have the ability to align cells towards a topographical orientation but not to effectively guide cell differentiation because the feature size is much larger than that of focal adhesions42. Conversely, the nanoscale topography can control both alignment and differentiation because the sizes of the features and focal adhesions are comparable43. Second, although previous studies reported the superior properties of graphene in the myogenic differentiation of C2C12 cells20, the synergistic role of the surface topography and the material properties of graphene have not been clearly demonstrated.Our crumpled graphene platform is a significant departure from previous approaches for the following reasons. First, the geometrical size (wavelength and amplitude) of our platform is much smaller than that of the platforms used in previous studies, and our platform thus can stimulate cell behaviors and functions more precisely. For example, previous studies fabricated wrinkled graphene oxide films with a wavelength and amplitude of a few micrometers to a few tens of micrometers due to the thick graphene oxide film44. Such morphology-controlled cell behaviors are also commonly observed in other cell types, such as immune cells and human mesenchymal stem cells46. Therefore, we believe, although further in-depth studies are needed, that the morphology control can control the differentiation and maturation of mouse myotube cells. As reported in previous studies, culturing mouse myoblast cells  on a nanotopography upregulates multiple genes related to myogenic differentiation, such as Myf5, MyoD, and MyoG. The enhanced gene expression of myogenic differentiation was evident irrespective of the surface characteristics, including the gelatin-coated poly(lactic-coglycolic acid) (PLGA)6, sphingosine-1-phosphate-conjugated PLGA47, and gold-coated polyurethane acrylate (PUA) substrates13. In addition, microgroove patterns also upregulated the expression levels of focal adhesion-related, actin cytoskeleton-related, and MAPK signaling-related genes48. Furthermore, on multiscale micro-patterns and nano-patterns, aligned nanofibers on top of microgrooves with orientations perpendicular to each other, C2C12 cells preferentially aligned following the nanofibers rather than the microgrooves49. These results indicate, at least in adhesion and alignment aspects, C2C12 cells prefer nanoscale patterns over microscale patterns. Based on these earlier findings, future investigations on the upregulation of various myogenic genes induced by the crumpled graphene substrate will provide further evidence of the impact of the crumpled topography on myogenic differentiation. The observation of cross-striation will also provide evidence of myotube maturation. In addition to gene expression, the effect of extracellular matrix components on differentiation and subsequent production by the cells can be studied in the future. Previous researchers reported that the laminin protein significantly promotes cell adhesion and differentiation of C2C12 cells51. Furthermore, we expect that these electrically active, nanotopographic crumpled graphene substrates can electrically stimulate cells. According to previous studies, consistent electrical stimulation can enhance the differentiation and maturation of skeletal muscle cells54. Although, in this work, there was no significant difference in the experimental results between ‘bare’ and ‘flat’ substrates due to the absence of electrical stimulation and the similarity of the elastic moduli  foil  by a chemical vapor deposition (CVD) system . Under a hydrogen (H2) gas environment (50 sccm) at 150 mTorr, the chamber was heated to 1050 °C. After annealing at 1050 °C for 2 h, methane (CH4) and H2 gases (CH4: 100 sccm and H2: 50 sccm) were introduced into the chamber at 520 mTorr for 2 min. Then, the chamber was cooled down to room temperature slowly under an argon (Ar) gas environment (500 sccm) at 330 mTorr. After the synthesis of graphene, a polydimethylsiloxane (PDMS) block was attached on the graphene side of the Cu foil, and then Cu was etched with 1 M FeCl3 solution. For a flexible substrate, a very high bond (VHB) substrate  was used. After stretching the VHB substrate in biaxial directions, with a larger prestrain applied to one direction  to induce uniaxial deformation, the graphene on the PDMS block was transferred onto the stretched VHB substrate. Due to the mechanical limitation of the VHB substrate, the stretching of the VHB substrate was controlled to be less than 400%. After transfer, the stretched VHB substrate was released, and the crumpled graphene structure was obtained. The surface structure of the crumpled graphene was monitored with a scanning electron microscope  and an atomic force microscope , and the integrities of the crumpled graphene at various tensile strain conditions between 0 and 300% were evaluated with a Raman spectroscope  using a 633 nm wavelength laser. Nanoindentation was carried out with the same AFM system used for imaging.The crumpled graphene structure was fabricated by modifying the protocols of previous studiesεxpre, : 300 and 150%) and a flat graphene substrate (50% prestrain). The prepared substrates were sterilized with alcohol and ultraviolet (UV) light. Then, mouse skeletal myoblast C2C12 cells were seeded at a density of 5000 cells/cm2 on sterilized graphene/VHB substrates. The cells were maintained in growth media  in a humidified atmosphere with 5% CO2 at 37 °C. The GM was replaced every 24 h. After 3 days of cell culture, the morphology and angular orientation data of the cells were analyzed using cytoskeleton staining. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with TRITC-phalloidin  and DAPI . The actin filaments and nuclei were observed using a fluorescence microscope . From the obtained fluorescence images, cell width, length, aspect ratio, and angular orientation data were analyzed with ImageJ software. We analyzed 5 different samples from four kinds of cell substrates and monitored over 100 cells from each sample. We conducted Rao’s spacing test to evaluate the uniform distribution of angular orientation data of cells and the Mardia–Watson–Wheeler test to evaluate the identical distribution of the angular orientation data of cells.Three types of cell substrates were prepared: two crumpled graphene substrates  and a flat graphene substrate were prepared and sterilized with alcohol and UV. Mouse skeletal myoblast C2C12 cells were seeded at a density of 5000 cells/cm2 on sterilized graphene/VHB substrates. The seeded cells were grown in GM at 37 °C and 5% CO2. GM was replaced every 24 h. When the cells covered 80% of the substrate, GM was replaced with differential media . The cells were incubated at 37 °C and 5% CO2, and the DM was replaced every 12 h. After 7 days of differentiation, cells were fixed, permeabilized and blocked with 4% paraformaldehyde, 0.1% Triton X-100 and 1% bovine serum albumin. For immunostaining, cells were incubated with the primary antibody MF20 (anti-myosin heavy chain (MHC)), Developmental Studies Hybridoma Bank ((DSHB), University of Iowa) overnight at 4 °C, followed by incubation with the secondary antibody (fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG, Thermo Fisher Scientific) and DAPI (Thermo Fisher Scientific) for 2 h at 37 °C. Myotube formation and alignment were observed using a fluorescence microscope  and analyzed with ImageJ software. We analyzed 5 different samples from four kinds of cell substrates and monitored over 100 myotubes from each sample. To evaluate the uniform distribution and identical distribution of myotube alignment data, Rao’s spacing and Mardia–Watson–Wheeler tests were conducted. To evaluate the myogenic differentiation efficiency, we analyzed the fusion index (the ratio of the nuclei number in myocytes with two or more nuclei), maturation index (the percentage of myotubes having five or more nuclei), myotube area fraction and cell density from each sample.Two types of crumpled graphene substrates (p-values smaller than 0.05 were considered to be significant.All of the quantitative data are expressed as the mean ± standard error of the mean. Statistical analysis was performed by means of one-way analysis of variance. For the determination of statistical significance, Supplementary Information


In this work, we grew transfer-free graphene-like thin films (GLTFs) directly on gallium nitride (GaN)/sapphire light-emitting diode (LED) substrates. Their electrical, optical and thermal properties were studied for transparent electrode applications. Ultrathin platinum (2 nm) was used as the catalyst in the plasma-enhanced chemical vapor deposition (PECVD). The growth parameters were adjusted such that the high temperature exposure of GaN wafers was reduced to its minimum (deposition temperature as low as 600 °C) to ensure the intactness of GaN epilayers. In a comparison study of the Pt-GLTF GaN LED devices and Pt-only LED devices, the former was found to be superior in most aspects, including surface sheet resistance, power consumption, and temperature distribution, but not in optical transmission. This confirmed that the as-developed GLTF-based transparent electrodes had good current spreading, current injection and thermal spreading functionalities. Most importantly, the technique presented herein does not involve any material transfer, rendering a scalable, controllable, reproducible and semiconductor industry-compatible solution for transparent electrodes in GaN-based optoelectronic devices. Gallium nitride (GaN) has attracted remarkable attention as an important material for application in optoelectronic and microelectronic devices, such as light-emitting diodes (LEDs), laser diodes (LDs), solar cells (SCs), and high electron mobility transistors (HEMTs) ,2,3,4. I2 mesa pattern and was fabricated by two steps of photolithography. The manufacturing schematic of the LED devices is shown in 4 and Cl2 gas mixture to reach the heavily doped n-GaN layer, forming the mesa arrays. The surface GLTF and ultrathin platinum were etched and patterned together. After mesa fabrication, iron trichloride was used to remove the rest of the nickel. Finally, Ti/Au (15 nm/300 nm) p and n metal electrodes were fabricated together by lift-off lithography and sputtering. No annealing was conducted in the metal contacts. For a comparison study, GaN LED devices without GLTF were also fabricated with a similar process (samples 2 and 3).The plasma-enhanced chemical vapor deposition (PECVD) system used in this experiment was a Black Magic Pro system from AIXTRON Nanoinstruments Ltd. . Unlike the traditional tubular furnace growth, a vertical cold wall CVD system was used here, wherein the only heated area was the middle part of the heater while the rest of the area was “cold”. Faster growth rate and less energy consumption are the main advantages of this system, a schematic of which is shown in −1 and 1601 cm−1, corresponding to the D and G peaks, respectively, which are the signatures of sp2-C graphitic carbon. The D peak is relatively big, indicating that the material grown in this manner contains a number of defects or disorder, which is a subject of future improvement. The Raman spectrum is not that of standard graphene, but rather a nanographitic structure, and therefore in this paper we term it as GLTF for scientific rigor.In order to determine whether or not the platinum was affected by the high temperature process, gallium nitride samples with a 2 nm platinum deposit were annealed for 25 min in an argon environment at 600 °C and 40 W-DC plasma. These conditions were nominally the same as the conditions for GLTF growth, except that no carbon source was added. Photographs of the four samples prepared for this study are shown in 2H2 to form GLTF, the performance of the device appeared to be drastically improved under the same operating conditions. In other words, although the Pt film in this device also underwent a high temperature deteriorating process, the addition of GLTF compensated for this effect and made the device outperform the other two types of devices. In fact, we found the turn-on voltage was strongly affected by the sheet resistance and work function of the transparent electrode materials. In sample 1, which did not have a transparent electrode, it was difficult to inject current uniformly and thus it required a large voltage to turn it on. For sample 2, which had the ultrathin Pt film added, its low sheet resistance and relatively good match to p-GaN’s Fermi level resulted in a reduction of the turn-on voltage. However, after annealing in sample 3, the Pt became islandic and the sheet resistance (hence the turn-on voltage) went up. The turn-on voltage was brought back down by the addition of GLTF in sample 4, as a result of the reduced sheet resistance.As shown in Finally, in order to study the thermal management characteristics of the as-grown GLTF, we welded and encapsulated three types of LEDs. The package welding diagram of the GaN LEDs is shown in As shown in As seen in The overall performance of the devices prepared by our method is reasonably good. However, at this stage, it is still not able to compete with commercial GaN LEDs. For example, the work voltage of standard blue LEDs at 20 mA is only slightly above 3 V. Despite this, compared to graphene-based devices, our devices are very competitive with a 3.9 V work voltage at 20 mA. Commonly, graphene-on-GaN LEDs have forward work voltages of 6–7 V or even more than 10 V ,27.2, GaN is relatively prone to damage and requires subtle control over the growth process [Based on graphene’s high electrical and thermal conductivity, high optical transmittance, rich raw materials, broad spectrum and good chemical stability, there have been a considerable number of papers suggesting graphene is a promising candidate for substitution of ITO transparent electrodes in GaN-based optoelectronics. However, as early as 2012 , we reco process ,30. In o process , yet was


As the fundamental concept of surface plasmon resonance (SPR) sensor that relies on the refractive index of the sensor chip surface, this research focused on the SPR signal when the DENV and Co2+ interact with the graphene-based material sensor chip. The results demonstrated that the proposed sensor-based graphene layer was able to detect DENV and Co2+ as low as 0.1 pM and 0.1 ppm respectively. Further details in the detection and quantification of analyte were also discussed in terms of sensitivity, affinity, and selectivity of the sensor.The emergence of unintentional poisoning and uncontrolled vector diseases have contributed to sensor technologies development, leading to the more effective detection of diseases. In this study, we present the combination of graphene-based material with surface plasmon resonance technique. Two different graphene-based material sensor chips were prepared for rapid and quantitative detection of dengue virus (DENV) and cobalt ion (Co Aedes mosquito, Aedes aegypti. Although recent laboratory techniques have contributed to treating the disease, the efficient and rapid detection for DENV is still of great significance to detect the dengue virus within the viremia and early febrile phase of infection [In this era of ever-increasing populations, the demand for the improvement of living standards may cause some serious environmental issues, e.g., chemical exposure, electromagnetic radiation, and air and water pollutions ,2, and, nfection ,15,16,17nfection ,22,23,24The next important health problem to address is metal toxicity, which can be found in canned drinks and foods ,26, vehiA rising surface-sensitive technique, surface plasmon resonance (SPR)-based biosensors, has drawn much attention from many researchers due to its ease of preparation, real-time detection, high sensitivity, fast measurements, and label-free method ,42,43,44Mycobacterium tuberculosis DNA (deoxyribonucleic acid) hybridization in SPR sensor was introduced by Prabowo et al. (2016), and yielding the best detection limit of 28 fM [2 = 0.973 with a detection limit of 0.01 pg/mL [2 of 0.996 was achieved in the concentration range of 0.1 nM to 1 µM [In view of biological sensing, Wang et al. (2011) have developed the graphene-based SPR sensor with the aptamer binding to monitor alpha-thrombin in the detection chamber . The novof 28 fM . In 2017of 28 fM . Next, t01 pg/mL . The fir01 pg/mL . Today,  to 1 µM . As can be noticed from 2+ using Au-chitosan-graphene oxide (Au/CS/GO)-based SPR sensor. They compared the performance of the developed SPR sensor with and without graphene oxide (GO). Even though both thin films can detect Pb2+ as low as 0.03 ppm, a rougher surface was observed when GO was added to Au/CS, which was believed it could improve the adsorption of Pb2+. As a result, Au/CS/GO showed wider Pb2+ range with higher sensitivity [2+ detection when exposed to different concentrations of Pb2+, ranging from 0.03 ppm to 3 ppm [2+, and there was a shift in SPR angle increased up to 3.5° compared to the Au-CS-GO layer only. A high sensitivity value of 2.05° ppm−1 using the SPR sensor was obtained [2+. They observed higher sensitivity of the previously developed sensor towards Pb2+ compared to Hg2+, thus, they calculated the binding affinity and found that CS-GO was more favorable to Pb2+ [+ detection using SPR sensor was reported by Zainudin et al. (2018) where valinomycin-doped chitosan-GO (C-GO-V) was used as an active layer. A preliminary test with Au thin film for K+ detection showed no shift in the resonance angle. However, when replaced with Au/C-GO-V, they successfully yielded the lowest detection limit of 0.001 ppm with a sensitivity value of about 0.00948° ppm−1 [2+. A high sensitivity value of 3.271° ppm−1 was obtained. It was found that the sensor could detect Cu2+ as low as 0.01 ppm [2+ detection. Experimental results showed that CTA-NCC/GO can detect Ni2+ as low as 0.01 ppm with a calculated sensitivity value of 1.509° ppm−1 [2+. The resonance angle was directly increased with Hg2+ concentrations. The Au/Cs/CGQDs can detect Hg2+ as low as 0.5 ppm with a sensitivity of 0.00062° ppm−1 [Meanwhile, in the first work on heavy metal ion detection, Lokman et al. (2014) successfully detected Pbsitivity . Furtherto 3 ppm . Anotherobtained . Once ag to Pb2+ . Besides8° ppm−1 . Daniyal0.01 ppm . Another9° ppm−1 . As can 2° ppm−1 . Table 2Briefly, the studies on graphene-SPR-based material for heavy metal ion detection remains inchoate, thus, further work on heavy metal ions detection, using other graphene composite material as an active layer in SPR, should be conducted since graphenes have the potential to enhance SPR performance. In the present work, chitosan-graphene oxide-cadmium sulfide quantum dots (Chitosan-GO-CdS QDs) composite thin film was developed as a sensor layer for cobalt ion detection via the surface plasmon resonance sensor. The objective of this work was to improve the detection limit of the metal ion and the sensitivity of the detection. xk = sp,k where xk is spk is approximated as pn is the refractive index of the prism, Ɛ2 is the dielectric constant for the dielectric medium in contact with the metal surface. The coupling of two waves vector of the evanescent wave, xk, with that of the surface plasmons, spk, results in a sharp dip of SPR signal at resonance angle (SPRθ) as shown in The optical phenomenon of SPR occurs when the incident He-Ne laser light is reflected from the metal-dielectric interface under certain resonance conditions. Basically, when an electromagnetic wave of incident light hits a metal film, the free electrons on the metal collectively oscillate in waves, thus producing charge density waves propagating along the metal-dielectric interface a. These  surface ,86. In oAs illustrated in Further, the developed sensor film was exposed to various concentrations of DENV solution ranging from 0.1 pM to 100 pM, as shown in Δθmax is the maximum SPR shift at the saturation, C is the concentration of DENV, and KD is the equilibrium dissociation constant. As shown in D value of 9.13 pM with R2 of 0.98. Moreover, the binding affinity, AK, of the sensor film to the DENV antigen, determined as the inverse proportional to DK, was 0.10948 pM−1. The sensitivity of the sensor film was then determined by plotting a linear graph from 0 pM to 10 pM DENV solution. The linear fit showed a good response with the sensitivity value of 0.0055°/pM (R2 = 0.62). The selectivity test was also tested by detecting 100 pM human serum albumin (HSA), and the mixture of HSA and DENV solution. As shown in The reaction between the sensor film and the DENV antigen was evaluated using the Langmuir model, which can be expressed as follows:The surface plasmon resonance technique integrated with graphene oxide-based material can also be applied for metal ion detection, as illustrated in AK and the maximum angle shift at the saturation of the SPR curves, Δθmax. From the Langmuir fitting, the sensor layer showed a high binding affinity toward the cobalt ion, with an affinity constant of 5.4357 ppm−1 with an R2 value of 0.8224. The high binding affinity of the sensor layer toward the cobalt ion may due to the electrostatic force between the graphene oxide sheets and also the CdS QD element. Complementary to this, the Langmuir fitting also showed that the saturation value of the sensor layer occurred when the concentration of the cobalt reached 10 ppm, with a maximum angle shift of 0.1865. In addition, the sensitivity of the sensor was 0.01937 ° ppm−1, which was determined by obtaining the slope of the linear fitting of the data from 0 to 10 ppm. These results show that the sensor layer has a great potential to detect cobalt ion.The angle shift value was then calculated and a graph was plotted for the degree of the resonance angle against concentration, as shown in 2+ was developed using graphene-SPR-based materials. The graphene-based materials of Au/CdSQDs-rGO/Ab and Chitosan-GO-CdS QDs were able to differentiate the concentration of DENV and Co2+ from 0.1 to 100 pM and from 0.1 to 100 ppm, respectively. Besides that, the graphene-SPR-based sensor showed a good sensitivity of 0.0055° pM−1 and 0.0193° ppm−1 for DENV and Co2+, respectively. The reaction between the graphene-based sensor film with DENV and Co2+ were also evaluated using the Langmuir model. Interestingly, both the DENV and Co2+ have a good binding affinity towards the graphene-based film, i.e., 0.10948 pM−1 and 5.4537 ppm−1, respectively. On the other hand, the graphene-based material showed a higher sensitivity towards DENV compared to HSM. The DENV was also able to be differentiated in the mixture of HSM, and, hence, verified that the Au/CdSQDs-rGO/Ab had a good selectivity towards DENV. Therefore, the above results proved that the graphene-SPR-based sensor was successfully developed for health and environmental monitoring application in sensing DENV and Co2+.In this study, a quantitative technique for the rapid and quantitative detection of DENV and Co


This method can also be applied to the roll-to-roll preparation of large area high-quality graphene.In this work, sharp wrinkles on graphene films, caused by graphene duplicating the grain boundary cracks of copper foil during the preparation process, were carefully explored. A secondary-transferring graphene film process was proposed to re-transform the “Peak” morphology of graphene surface into “Valley” form. The process we have developed is highly effective and almost nondestructive to the graphene through testing the surface morphology and photo-electric properties before and after the secondary-transferring process. Flexible organic light-emitting device (FOLED) with PEDOT:PSS/SLG/NOA63 framework as a targeted application was fabricated to illustrate the value of our proposed method in fabricating stable devices, the maximum luminance can reach about 35000 cd/mThe online version of this article (10.1186/s11671-018-2767-z) contains supplementary material, which is available to authorized users. Graphene, arranged by single-layer carbon atoms in the shape of a unique hexagonal honeycomb lattice structure, is a promising two-dimensional transparent conductive material for flexible organic light-emitting device (FOLED) due to its excellent conductivity, high transmittance, and flexibility –3. Jong 2 hybridized graphene [Graphene can be prepared by micromechanical exfoliation , electrographene . Once thHowever, it is well known that there are various atomic scale defects, large number of wrinkles, and especially artificial cracks and impurity residues on the copper foil-based SLG films –17. PrevIn this paper, we used a fast and efficient bubble transfer method which can nondestructively transfer graphene from Pt or Cu substrate and no residual impurities while comparing with other transfer methods , then we2 gas flow in the CVD chamber. CH4, used as the carbon source, was injected at a flow rate of 60 sccm for 30 min, then samples were fast cooled to room temperature, as shown in Fig. 2 bubbles generated at the interface between the graphene and Cu foil and removed the graphene from the copper substrate. After electrolysis, graphene was transferred from copper foil to PET/HRA. Figure Figure 2/Si, a double-frequency Nd:YAG laser (532 nm) as excitation source. G band caused by the in-plane vibration of sp2 carbon atoms with a peak position ~ 1590 cm−1, and the G’ originated from two-phonon double resonance Raman process with a peak position ~ 2686 cm−1. Here, the intensity ratio of the G’ band to G band (IG’/IG) was 1.75 ± 0.015  quantified the structural defects and disorders of graphene; its value was just ~ 0.065, illustrating high quality of the prepared SLG [To determine the quality of the graphene obtained, we performed optical microscope test and Raman measurements. Figure ared SLG .Fig. 2ThWe further precisely explored the height of sharp wrinkles and the changes of surface morphology and photo-electric properties before and after the secondary-transferring. Figure We fabricate the FOLEDs as a targeted application to explore the effectiveness of our secondary-transferring progress where the graphene acts as an anode. Figure 2, this was attributed to the decrease of sharp winkles of graphene film after the secondary transfer. What is more, we can see that the secondary transfer process almost did not reduce the performance of FOLED by comparing the current efficiency of D1 and D2; a series of repetitive experiments also support this conclusion. We further improved the brightness and efficiency of FOLED by introducing the modified layer PEDOT:PSS as the D3 showed. The luminance of the D3 can reach 35000 cd/m2, and the maximum current efficiency was 16.19 cd/A, which was higher than the D2 of 10.74 cd/A. That is because PEDOT:PSS played a role as work function stair and enhanced the sheet conductivities. Furthermore, it also smoothed the surface of secondary-transferring SLG film by filling part of the “Valley,” making the FOLED more stable.The optoelectronic characteristics, including current density-voltage-luminance (J-V-L) and current efficiency-voltage (CE-V) of the FOLEDs with/without secondary-transferring graphene electrode structure, are shown in Fig. 2, and maximum current efficiency was 16.19 cd/A with PEDOT:PSS/SLG/NOA63 framework. This method can also be applied to prepare large area high-quality graphene by roll-to-roll way.In this paper, we explored in detail the sharp wrinkles of graphene duplicating the grain boundary cracks of copper foil after the first bubble transfer; the sharp wrinkles can cause a large surface roughness, resulting in deterioration even breakdown of FOLED. We proposed a secondary-transferring method to re-transform the wrinkles on graphene surface into “Valley” form to fabricate the stable FOLED; the graphene film is almost nondestructively transferred by controlling the different adhesiveness. The maximum luminance can reach about 35000 cd/mAdditional file 1:Figure S1. The count of Ig’/Ig as shown in the following figure. Most of the Ig’/Ig values are concentrated at 1.75 and the standard deviation was 0.015, which shows the consistency of the sample quality, while somewhere the Ig’/Ig was ~ 0.8, which illustrates bilayer of graphene sample and the monolayer rate of our graphene sample is > 90%. (DOCX 106 kb)


The mobility is significantly affected by the grain size in graphene, and there exists a relatively stable region between 500 and 800 pulses. The observed phenomena originate from competition between decreasing resistance and increasing carrier concentration. These studies should be valuable for regulating grains sizes for thermoelectric applications of graphene.Improving the thermoelectric efficiency of a material requires a suitable ratio between electrical and thermal conductivity. Nanostructured graphene provides a possible route to improving thermoelectric efficiency. Bi-layer graphene was successfully prepared using pulsed laser deposition in this study. The size of graphene grains was controlled by adjusting the number of pulses. Raman spectra indicated that the graphene was bi-layer. Scanning electron microscopy (SEM) images clearly show that graphene changes from nanostructured to continuous films when more pulses are used during fabrication. Those results indicate that the size of the grains can be controlled between 39 and 182 nm. A detailed analysis of X-ray photoelectron spectra reveals that the sp With increasingly serious environmental pollution and an energy crisis, it is very important to reduce environmental pollution and convert waste heat into electrical energy. For this reason, it is necessary to find efficient thermoelectric conversion materials. Excellent thermoelectric efficiency requires high electrical conductivity and low thermal conductivity. Nanostructured materials  limit thBi-layer graphene was prepared using PLD in this study. The effect of pulse numbers on the size of graphene grains was studied. In this case, the growth process of bi-layer graphene grains could be sufficiently controlled. Kα radiation. The electrical properties of graphene were determined from Hall measurements.Graphene grains were deposited on single crystal Cu (111) substrates by PLD. An excimer KrF laser was used for ablation. The specific experimental parameters are listed in −1 was induced by disorder in the atomic arrangement, the edge effect of graphene, or ripples and charge puddles. This means a significant number of defects appeared in the graphene thin films. The G peak at approximately 1580 cm−1 originates from highly oriented graphite induced by the doubly degenerate zone center E2g mode. The 2D peak at approximately 2700 cm−1 originates from the double resonance Raman excitation of two-photon near two mutually nonequivalent K points at the center of the first Brillouin zone. The intensities of IG, I2D, and their ratios are useful indicators of the quality and number of layers in the graphene samples. The specific peak information, ID/IG, and I2D/IG ratios are shown in I2D/IG fell in between 0.79 and 0.94, implying the graphene layers have a bi-layer structure [−1  is a dual-phonon process peak originating from one intravalley and one intervalley phonon scattering [tructure ,29,30,31tructure . Anotherattering . This pe#–5#, respectively. Sample 1# contains small and discontinuous graphene grains. The corresponding grain size distribution is shown in #, it was found that graphene nanograins are connected to each other to form graphene grains with an average size of 66 nm when the number of pulses increased to 500. Although the small grains are connected to each other to form larger graphene grains, it can be seen from the figure that there are still many discontinuities. Sample 3# was grown using 700 pulses, resulting in large graphene grains with an average size of approximately 182 nm. In sample 4#, the number of pulses increased to 800. It can be seen that the graphene grains are almost all connected together to form a continuous film in #, full coverage is achieved, indicating that a completely continuous graphene film was formed. The size of bi-layer graphene grains was controlled by adjusting the number of pulses. Nanostructured graphene prepared by PLD growth gives hope that one would have a much better control of the thermal properties of supported bi-layer graphene since the grain size has an effect on K (T) of graphene. Recently, studies [K (T) ~T1.4 or ~T1.5. That means K can be adjusted within a certain range by controlling the graphene structure.The morphology of graphene is clearly shown in the SEM image in  studies ,35,36 sh2 carbon bonds, which agrees with the component of graphene [3 carbon atoms. Peak C exhibits much smaller intensity at about 286.3 eV and is attributed to some C–O contamination at the surface of the films due to exposure to air [3 bonds into sp2 bonds in graphene. These results indicate the presence of growth defects during graphene preparation using PLD.XPS measurements  can provgraphene . It is we to air . The XPSσ is the electrical conductivity [ρ is the resistivity. Therefore, the graphene mobility formula can be simplified as RΩ is the sheet resistance and n is the carrier concentration. The relationship between RΩ and n and the number of pulses used during growth was studied in order to better understand its effect on mobility. n is ~1013, which is one to two orders of magnitude higher than graphene with good mobility [n also increased. However, ΩR decreases with increasing grain size. The mobility μ becomes relatively stable when the competition between RΩ and n is balanced. This is the reason why the mobility of graphene is basically stable when grown using 500 to 800 pulses.uctivity . σ=1/ρ, mobility . The mea3 bonds exist in graphene, which lead to many defects during graphene growth. Electronic mobility can be affected by grain size and becomes relatively stable between 500 and 800 pulses. These results may occur due to competition between resistance and carrier concentration. These findings can be used to tune the grain size of graphene, and the results are beneficial for thermoelectric applications.In conclusion, we prepared bi-layer graphene from a solid carbon source using PLD. The grain size of graphene can be controlled between 39 and 182 nm by varying the number of pulses from 300 to 900. Regarding the chemical structure, sp


An angle-insensitive broadband absorber of graphene covering the whole visible spectrum is numerically demonstrated, which is resulted from multiple couplings of the electric and magnetic dipole resonances in the narrow metallic grooves. This is achieved by integrating the graphene sheet with a multi-grooved metasurface separated by a polymethyl methacrylate (PMMA) spacer, and an average absorption efficiency of 71.1% can be realized in the spectral range from 450 to 800 nm. The location of the absorption peak of graphene can be tuned by the groove depth, and the bandwidth of absorption can be flexibly controlled by tailoring both the number and the depth of the groove. In addition, broadband light absorption enhancement of graphene is robust to the variations of the structure parameters, and good absorption properties can be maintained even the incident angle is increased to 60°.The online version of this article (10.1186/s11671-019-2937-7) contains supplementary material, which is available to authorized users. Graphene has been demonstrated as a good candidate for optoelectronic devices because of its remarkable electronic, mechanical and tunable optical properties –3. For mIn recent years, various approaches have been suggested to enhance light absorption of graphene in the visible and near-infrared regions, and the physical mechanisms behind the absorption enhancement of graphene include epsilon-near-zero effect , cavity In this work, a novel angle-insensitive broadband absorber of graphene covering the whole visible region is proposed by integrating the graphene sheet with a multi-grooved metasurface. The enhanced absorption band of graphene has arisen from the multiple couplings of electric and magnetic dipole resonances confined in the groove cavity. The absorption band of graphene can be flexibly controlled by tailoring both the number and the depth of the grooves. High absorption efficiency can be maintained even if the structure parameters and the incident angle are significantly altered.y-axis) for angle-insensitive broadband absorption enhancement of graphene. The unit cell of the structure consists of a planar graphene sheet and a patterned silver film with five grooves separated by a polymethyl methacrylate (PMMA) spacer. The PMMA layer is functioned as a buffer layer which controls the coupling between graphene and the patterned silver film, and it can also be easily transferred onto the multigrooved surface by spin-coating in application. The period of the unit cell is Λ, the thickness of the PMMA spacer is t, the thickness of the bottom silver film is D, and the substrate is silica. The geometry of the groove is described by both its width w and its depth. The width of the five grooves is equal, and their depths are d1, d2, d3, d4, and d5, respectively. The refractive index of PMMA is 1.49 [N = 10 [σg calculated from Kubo formula [Figure  is 1.49 , and the is 1.49 . The pla [N = 10 , 27. The formula , 39. At e and ħ are the elementary charge and reduced Planck’s constant, respectively. kB is the Boltzmann constant, μc is the chemical potential, Γ = 1/2τ is the phenomenological scattering rate, and τ is the momentum relaxation time. The physical parameters of the graphene are set as μc = 0.15 eV, T = 300 K, and τ = 0.50 ps.x directions, while boundaries in the z direction are adopted as perfectly matched layers (PMLs). Reflectivity (R) and transmissivity (T) are obtained by two monitors at the top and bottom of the structure. The bottom silver film is chosen to be optically thick enough (D = 100 nm) to prevent light transmission; therefore, total absorption (A) of the structure can be reduced as A = 1–R. The absorption of graphene (Ag) can be calculated as [Pup (λ) and Pdown (λ) are the powers passing through the upside and downside planes of the graphene sheet at the wavelength λ, respectively. Pin (λ) represents the incident power at the wavelength λ. In simulation, Pin (λ) is the power of the light source, and two power monitors are inserted at the top and bottom planes of the graphene to obtain Pup (λ) and Pdown (λ). These powers are extracted from the total field in the FDTD simulations.In simulations, the finite-difference time-domain (FDTD) method  is adopted to calculate the absorption properties of the graphene-based multi-grooved metasurface. Periodic boundary conditions (PBCs) are employed in the lated as :4\documeFigure x-axis, corresponding to an electric dipole resonance mode [xoz-plane, corresponding to a magnetic dipole resonance mode [To gain insights into the effect of broadband absorption enhancement of graphene under the TM wave illumination, the electric and magnetic field distributions of the structure for different wavelengths are investigated. As can been seen in Fig. nce mode , 45. On nce mode , 46. TheM is the mode number, and M = 1 in calculation; neff is the effective refractive index of the groove cavity, which can be equivalent to the mode refractive index of the metal-insulator-metal (MIM) waveguide. Only the fundamental mode of TM0 can be supported because the groove width is far smaller than wavelength, and the corresponding neff can be determined by using the even mode dispersion of the MIM waveguide [εd and εm are the dielectric constants of PMMA and silver, respectively; k0 is the wave vector of incident light, β is the propagation constant of the MIM waveguide mode, and neff = β/k0.To further identify the location of the absorption peak of graphene of the multi-grooved metasurface, resonant properties of the single-grooved structure is studied. For the single-grooved structure shown in the inset of Fig. aveguide :6\documeAs can be seen in Fig. To further evaluate the absorption performance of graphene integrated with the multi-grooved metasurface, we first investigated the influence of the thickness of the spacer layer on light absorption of graphene. As can be seen in Fig. N and w. In Fig. N > 10 and it becomes saturated as N is increased to 30. Light absorption of graphene is not always increased with the increase of the number of monolayer graphene, and similar phenomenon can also be observed in the graphene-based waveguide-resonance gratings [w = 30 nm for both the total structure and graphene in the visible region. Because the electromagnetic coupling of the electric and magnetic dipole resonances is mainly confined in the groove, deviation from the design value of groove width with ± 10 nm will distinctly affect the absorption performance of the multi-grooved metasurface.Figure gratings . In Fig.θc = 60° within the spectral range of 450–800 nm, and the absorption band is kept almost the same although the incident angle is significantly altered. This is because that the broadband absorption enhancement of graphene integrated with the multi-grooved metasurface is originated from the coupling of the electric and magnetic dipole resonances in the groove cavity, which is almost immune to the variation of the incident angle. The angle-insensitive absorption performances are very important because the absorption performances of most graphene-based absorbers are generally depended on the incident angle [We also investigate the angular robustness of the proposed graphene absorber integrated with the multi-grooved metasurface. In Fig. nt angle , 28–35. In conclusion, an angle-insensitive broadband absorber of graphene integrated with a multi-grooved metasurface is proposed and its light absorption property is numerically investigated. The absorption band of graphene covers the whole visible region, and an average absorption efficiency of 71.1% can be realized in the spectral range from 450 to 800 nm. The extended absorption band of graphene has arisen from the multiple couplings of electric and magnetic dipole resonances confined in the groove cavity, and its mechanism can be verified by using the single-grooved structure. The location of the absorption peak of graphene can be tuned by the groove depth, and the absorption bandwidth of graphene can be flexibly controlled by tailoring both the number and the depth of the groove. Broadband absorption properties of graphene are almost unaffected by the variation of the thickness of the spacer layer, the number of monolayer graphene, and the groove width. In particular, the light absorption spectra of graphene remain almost the same even at large angles. The idea of using multi-grooved metasurface to widen the interaction band between light and graphene could be also adopted in near-infrared region and other graphene-based optoelectronic devices.Additional file 1:Figure S1. Absorption spectra of the total structure, graphene, and silver for the multi-grooved metasurface with graphene under the TE wave illumination. Figure S2 Absorption spectra for the graphene-based metasurface with different number of groove. (DOCX 380 kb)


Evidence suggests that alterations in BDNF/TrkB, including TrkB transactivation by A2aRs, can occur in several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Although enhancing BDNF has been a major goal for protection of dying motor neurons (MNs), this has not been successful. Indeed, there is emerging in vitro and in vivo evidence suggesting that an upregulation of BDNF/TrkB can cause detrimental effects on MNs, making them more vulnerable to pathophysiological insults. For example, in ALS, early synaptic hyper-excitability of MNs is thought to enhance BDNF-mediated signaling, thereby causing glutamate excitotoxicity, and ultimately MN death. Moreover, direct inhibition of TrkB and A2aRs has been shown to protect MNs from these pathophysiological insults, suggesting that modulation of BDNF/TrkB and/or A2aRs receptors may be important in early disease pathogenesis in ALS. This review highlights the relevance of pathophysiological actions of BDNF/TrkB under certain circumstances, so that manipulation of BDNF/TrkB and A2aRs may give rise to alternate neuroprotective therapeutic strategies in the treatment of neural diseases such as ALS.Brain derived neurotrophic factor (BDNF) is well recognized for its neuroprotective functions, via activation of its high affinity receptor, tropomysin related kinase B (TrkB). In addition, BDNF/TrkB neuroprotective functions can also be elicited indirectly via activation of adenosine 2A receptors (A Amyotrophic lateral sclerosis (ALS), the most common form of motor neuron disease (MND), is a fatal adult onset neurodegenerative disease resulting in progressive and preferential degeneration and death of upper motor neurons  of the motor cortex, and alpha lower motor neurons (LMNs) of the brain stem and the spinal cord . The inc2+ dysregulation within the pre-synaptic compartment  . The onlG93A ALS model mice, while upper MNs have been shown to be hyper-active prior to their death, these neurons display reductions in dendritic length and spine density, suggesting a homeostatic response to heightened pre-synaptic activity  that links abnormal neuronal activity to neuronal death? One proposed mechanism is the activity-dependent synthesis and release of neurotrophins . Neurotr2+) concentration via activation of NMDA glutamate receptors or L-type voltage gated calcium channels (L-VGCC) following a depolarizing stimulus initiates transcription of the BDNF gene, predominantly at exon IV (2+ response elements (CaRE) – CaRE1 and CaRE3, which regulate transcription  where the pro domain is proteolytically cleaved off to form pro-domain and mature BDNF and is secreted into the extracellular space (hence forth termed “BDNF”)  . The pro “BDNF”) . Intrace “BDNF”) . The pro “BDNF”) . The ext “BDNF”) .2+-dependent mechanisms. BDNF is secreted pre-synaptically via increased intracellular influx of Ca2+  of the sorting receptor family  to triggThe third product, BDNF, binds with high affinity to TrkB of the Trk family of tyrosine kinases and with lower affinity to the p75 receptor . Activat2+ increase  with similar affinity . ExactlyBinding of BDNF to TrkB initiates two different categories of cascades: a fast acting BDNF/TrkB cascade that excites neurons or a slow acting occurring over minutes to hours. The action of BDNF markedly differs between these two categories . AdditioActivation of the TrkB receptor at its Tyr490 and Tyr515 residue causes the docking of Shc adaptor protein (Src- homology 2-domain) at these tyrosine sites and recruits growth factor receptor bound protein 2 (grb2) which binds with GTPase Ras to form a complex, and initiates extracellular signal regulated kinase (ERK) activation . ERK act2+ influx via L-type voltage gated calcium channels (L-VGCC)  and GSK-3β (glycogen synthase kinase 3β), essential for neuronal survival, growth and differentiation, and is activated by Ca(L-VGCC) . Activat(L-VGCC) .3), and diacylglycerol (DAG) which is important for survival, neurite outgrowth and synaptic plasticity. BDNF via the PLCγ/IP3 pathway results in calcium release from intracellular stores activating CaMKII  which in turn activates CREB phosphorylation (The phosphorylation of the TrkB receptor at its Tyr816 residue activates the phospholipase C γ (PLC γ) pathway, generating inositol-1,4,5-triphosphate (IPrylation . The genrylation  which isrylation .The BDNF-TrkB complex not only activates on its transmembrane surface (described above) but it also internalizes via endosomes (both early and late endosomes) to activate downstream pathways. This BDNF-TrkB signaling via endosome also determines the cellular fate of BDNF-TrkB complexes, which can be transported retrogradely, recycled back to the membrane, or prepared for degradation by lysosomes .G93A mice is capable of increasing BDNF secretion, which in turn can increase release of glutamate to trigger excitotoxicity, leading to MN degeneration. Indeed, BDNF has been shown to enhance MN death by glutamate excitotoxicity, via activation of TrkB  terminal and a post-synaptic  membrane. BDNF elicits rapid effects on synaptic transmission and membrane excitability, via activation of pathways in both the pre- and post-synaptic compartments. In the pre-synaptic compartment, BDNF causes release of glutamate and GABA, via the TrkB-ERK mediated pathway . Enhance of TrkB . TogetheBrain derived neurotrophic factor also modifies neurotransmission by altering the expression of pre-synaptic proteins that regulate neurotransmitter release . For exa2a adenosine receptor (A2aR) present both pre- and post-synaptically , such as the Aptically . Adenosiptically . Extraceptically . Adenosiptically . Pre-synlutamate  and the lutamate , thus fah as Fyn , in a sl pathway . Post-syDNF mRNA  and BDNFDNF mRNA   leading to migration of early cortical neurons to form a differentiated cortical layer  known for its activity-dependent interplay with neurotrophic factors during development and in mature neurons. Post-synaptically, the effect of glutamate is mediated by activation of two major ionotrophic glutamate receptors; AMPA receptors and NMDA receptors . Pre-syn+, Ca2+, and K+ channels through intracellular cascades  which releases Ca2+ from IP3-sensitive stores, activating PKC.In addition to these pre- and postsynaptic effects on glutamatergic transmission, TrkB activation also modulates ion channels that can alter neuronal excitability, including Nacascades . For exaa Nav1.9 . Metabot2+, enhancing BDNF release, which could trigger further release of glutamate. This proposed mechanism would perturb the neuron’s ability to regulate its activity, leading to glutamate excitotoxicity and neuronal death  mediated inhibition in CNS by binding to their post-synaptic receptors. The strength of synaptic inhibition critically depends on intracellular Cl– concentration, hence on Cl– homeostasis   and glycine are the primary neurotransmitters regulating chloride (Cleostasis . A key rr (KCC2)  has beenr (KCC2) , contribr (KCC2)  and BDNFr (KCC2) . Thus, By in MNs , 2004, s balance . Indeed, balance , eventuaeostasis . Besideseostasis  making min vivo (in vitro following exogenous BDNF application (in vitro (in vitro (2a receptors has also been reported to enhance survival of MNs in culture (in vivo (G93A mice (G93A mice (G93A mice improved the disease symptoms rescuing hippocampal interneurons and regulating long term potentiation (The ability of BDNF/TrkB to promote neuronal survival and resistance to toxic insults is well characterized . Severalin vivo  and funclication . Contrarin vitro  and imprin vitro . BDNF poin vitro  and in oin vitro . Additio culture . However(in vivo . This su(in vivo . Moreove(in vivo . Several(in vivo . Additio(in vivo . Further(in vivo . BDNF al(in vivo , and Trk(in vivo . Additio(in vivo . Fryer e(in vivo , 2000 al(in vivo . Additio93A mice  which fu93A mice . Furtherntiation  found tontiation .2aRs, which co-localize with and directly transactivate TrkB, protected cultured MNs from these detrimental effects (2aRs also prevents toxicity following expression of the G85R or G37R SOD1 mutations, which are highly toxic to cultured MNs (in vivo conditional deletion of TrkB in MNs of adult transgenic mice carrying a G85R SOD1 mutation attenuates SOD1 toxicity, resulting in extension of life span and motor function, slowing MN loss and causing persistence of neuromuscular junctions (G93A rats, phrenic motor neurons displayed an increased expression of BDNF and phosphorylated ERK at end stage disease, consistent with possibly increased BDNF function and basal protein levels (In addition to the above BDNF-TrkB signaling effects, blocking A effects . Similarured MNs . These punctions . Furthern levels .2aRs in epilepsy has been shown to reduce seizures and neuronal damage (Hyper-activity induced activation of BDNF and TrkB have also been observed in other disease states, such as epilepsy and traumatic brain injury . Upregull damage , 2009. DAmyotrophic lateral sclerosis is an incurable multi-factorial disease state where synaptic and intrinsic hyper-activity of MNs is a significant early factor . TherapeAll authors contributed to the writing and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Graphene-based materials are a family of carbonaceous structures that can be produced using a variety of processes either from graphite or other precursors. These materials are typically a few layered sheets of graphene in the form of platelets and maintain some of the properties of pristine graphene . In this work we present melt mixed graphene-based polypropylene systems with significantly reduced percolation threshold. Traditionally melt-mixed systems suffer from poor dispersion that leads to high electrical percolation values. In contrast in our work, graphene was added into an isotactic polypropylene matrix, achieving an electrical percolation threshold of ~1 wt.%. This indicates that the filler dispersion process has been highly efficient, something that leads to the suppression of the β phase that have a strong influence on the crystallization behavior and subsequent thermal and mechanical performance. The electrical percolation values obtained are comparable with reported solution mixed systems, despite the use of simple melt mixing protocols and the lack of any pre or post-treatment of the final compositions. The latter is of particular importance as the preparation method used in this work is industrially relevant and is readily scalable. Graphene-based polymer nanocomposites attracted significant scientific and commercial interest in recent years ,3,4,5,6.The introduction of a filler in a polymer matrix can lead to an increase of mechanical properties  and in the case of carbonaceous fillers an increase of electrical conductivity. Both these properties are tightly linked to the type of filler and polymer combination. Depending on the targeted application the filler amount might be significant , something that has been seen to lead to an increase of the overall viscosity with potential processing and performance difficulties . The wayThe most economically advantageous method of dispersing a filler in a matrix, especially in a large scale industrial environment, is by melt mixing. Such methods have been successfully used for traditional carbonaceous fillers like carbon black , but dueHere we must note that although graphene is commonly seen as a pristine monolayer of hexagonal carbon atoms, in reality, there is a whole class of materials that can be considered graphene-like. The initial distinction comes from the different preparation methods that can be classified as bottom-up and top-down. Bottom-up graphene can be produced via a variety of methods that include chemical vapor deposition , epitaxi3. In previous studies on PP the electrical percolation threshold for melt mixed systems using a variety of mixing protocols and pre/post mixing procedures has been reported in the range 6–15 wt.% γ peak in γ phase was observed in that work. The reasons for this difference can be tentatively attributed to differences in the type of filler used and possibly in the iPP used, since nucleation of γ phase is not surprising for metallocene PP, due to the presence of the effects of regiodefects and for PP copolymers with ethylene, however nucleation of γ phase for this iPP in particular in this work, is not something that is commonly observed [Polypropylene has a very rich polymorphism and it has been reported that filler inclusion has an effect of the overall morphology of filled systems . The iPPobserved ,68.maxT is increasing with increasing filler loading, indicating an improvement in the thermal stability of the composition. This improvement in thermal stability can be associated with the inherent property of graphene to act as a mass transport barrier that inhibits the transport of polymer degradation by-products to the surface [Thermal stability and the amount of filler in the different compositions used in this study were evaluated from TGA and differential thermo-gravimetric (DTG) curves see . From th surface . Traditi surface ,56,69. I surface ,3, there−1K−1 for pure polypropylene to 0.37 Wm−1K−1 for a system with 4 wt.% filler exhibiting an increase of ~37%. Heat propagation in graphene/iPP nanocomposites is generated mainly due to acoustic phonons and electrons scattering processes. The effective heat conduction of the composite depends on the thermal resistance between filler and the polymer matrix and scattering processes on its interface as well as potential thermally conductive paths created by the existence of the filler network. We can observe a rapid increase in thermal conductivity with increasing filler loading below the electrical percolation threshold indicating that although the overall network is still being created, the dispersion process is efficient enough to allow for such processes to occur. Above the electrical percolation threshold, the increase in thermal conductivity is more gradual, something that can be associated with the possibility of agglomeration and aggregation processes that increase with the extension of the network formation in the matrix. In composite materials, the scattering of phonons is due to the existence of a thermal barrier, originating from an acoustic mismatch at the interface between the organic polymer and the inorganic filler. Therefore, high filler loadings compared to the electrically conductivity threshold  are typically necessary to achieve an appropriately high level of thermal conductivity. Such an increased loading has the potential to represent a significant processing challenge and can in certain conditions cause mechanical properties of interest to deteriorate [eriorate .3/1I, is shown in 3/1I scaling in the MAOS region becomes angular frequency dependent with angular frequencies greater than 2 rad/s reverting to The addition of fillers can significantly increase the melt viscosity and affect polymer chain mobility . Therefolsewhere .δ)) behavior as a function of temperature and filler loading. For the case of pure polypropylene, three molecular relaxations have been reported in the literature, and these transitions namely α, β, and γ are observed with decreasing temperature [In perature . For comperature ,76. In tgT. Furthermore, it is worth noticing that there appears to be a gradual decrease in the observed glass transition temperature below the electrical percolation threshold. This rate of change remains relatively unchanged in the region above the electrical percolation, indicating that despite the increased amount of available filler no further interaction can be observed, probably due to agglomeration or localized saturation of the available graphene surfaces. Previous work in different PMMA isomers with different graphene types and preparation conditions has indicated a correlation between the level of dispersion and the strength of the interaction between the filler and the matrix to the observed glass transition temperature [The β-transition is thought to being related to segmental relaxation mechanisms in the amorphous region, and has been associated with the glass transition , somethiperature .1 and α2 that are located at lower and higher temperatures respectively [1 and α2 can be associated with specific intralamellar motions and other intracrystalline processes, originating from the existence of anisotropies on the crystal lattice potential [In comparison to polyethylene ,81,82, tectively . The exaotential . The α rotential .1 and α2 almost disappear. This behavior can be associated with the overall morphological changes discussed previously, namely the suppression of the β phase and the increased nucleation activity [From this work we observe a significant suppression of the overall α-process with increasing filler loading, something that can be associated with the morphological changes the introduction of the filler initiates in the matrix See . As we hactivity . Since bIn this work, we have presented an industrially relevant melt-mixed system of polypropylene and hierarchical reduced graphene oxide. The filler allows for the creation of a three-dimensional extended structure, something that leads to efficient dispersion. This efficiency can be seen via the low electrical percolation threshold observed by this study. Furthermore, it must be noted that we have kept the entire mixing procedure as simple as possible, something that was seen to have adverse effects on the overall filler dispersion in the past ,2,3. In


The maximum discharge specific capacitance is 1634.2 F/g when the current density is 3.5 mA/cm2 and it can keep at 463 F/g after 500 charging–discharging cycles when the current density increased about three times.Phosphorus oxide modified graphene was prepared by one-step electrochemical anodic exfoliation method and utilized as electrode in a redox supercapacitor that contained potassium iodide in electrolytes. The whole preparation process was completed in a few minutes and the yield was about 37.2%. The prepared sample has better electrocatalysis activity for I The advantages of this strategy are the heteroatoms could tailor the electronic properties and average electronic charge distribution of adjacent carbon atoms of graphene. Among various heteroatom-doped graphene materials, nitrogen-doped (N-doped) graphene has been widely studied in supercapacitors [x) in electrochemical energy storage field. Recently, Karthika et al. prepared a high electrochemical activity P-doped graphene by reducing graphene oxide with phosphoric acid. The prepared sample exhibited a specific discharge capacitance value of 367 F/g, which is much larger than that of graphene [In recent years, graphene and its composite materials have attracted great attention due to their excellent performance in various fields. Many researchers have presented that graphene plays an important role as electrodes, catalysts, and supports in energy-storage fields, such as supercapacitors, ions batteries, fuel cells and so on ,2,3,4. Tpacitors  and battpacitors . Howevergraphene . Howevergraphene . Xu et agraphene . To over−/I−3, 2I−/I2, 2I−3/I2, I2/IO−3, etc. [Currently, supercapacitors (SCs) play an important role in the field of energy storage and have been applied in many areas due to high power density, good rate capability and excellent cycling stability . Differe−3, etc. ,17,18,19In recent studies, some researchers have presented that heteroatom-doped graphene has excellent electrocatalytic activity for the reduction of iodine, due to the electrons conjugation effect, charge redistribution and defect sites . However−/I−3 via various electrochemical analysis. We present that the introduced P–O group in graphene could effectively improve its electrochemical performance. The whole preparation process can be completed within 30 min and the yield is about 37.2%. Owing to the catalytic effect of P–O group, the PO-graphene has a better electrochemical performance than graphite in promoting the redox reaction of I−/I−3.In this paper, we present a facile method to prepare high quality PO-graphene and study the electrochemical property. The PO-graphene is prepared by the one-step electrochemical anodic exfoliation method and used as the electrode in a redox supercapacitor, whose electrolyte contains KI. We investigated in detail the catalytic effect of P–O group on redox reaction of 3I2SO4) and potassium iodide (KI) were obtained from Aladdin Co. Ltd., Shanghai, China.High purity graphite rod was obtained from C Nano Technology Co. Ltd., Beijing, China. Ammonium phosphate, Poly(vinylidene fluoride) (PVDF), Sulfuric acid (H3) were used as the anode and cathode, respectively, and the distance was fixed at 1.5 cm. The exfoliation was performed at 10 V in 1 M ammonium phosphate aqueous. After the preparation, the sample was collected by centrifugation at 5000 rpm for 30 min and purified repeatedly with distilled water until PH = 7.In a typical procedure, high purity graphite rod and platinum plate , Transmission electron microscopy (TEM) images are performed on a Tecnal G2 20STWIN microscope operating  at 200 kV. Fourier transform Infrared spectroscopy (FT-IR) analyses were accomplished on Thermal Nicolet 6700 . Laser Raman spectra were carried out using Raman spectrometry . X-ray diffraction analysis (XRD) was carried out by Bruker D8 Advance , Cu Kα = 0.154 nm with a voltage of 40 kV. 2SO4. The cyclic voltammetry (CV) tests were performed from −0.5 to 0.7 V (vs. SCE). Electrochemical impedance spectroscopy (EIS) measurements were performed under open circuit conditions over a frequency region from 0.01 Hz to 100 kHz by applying an AC signal of 5 mV in amplitude throughout the test. The galvanostatic charge–discharge (GCD) analyses were performed in a potential range of 0–0.7 V (vs. SCE). The CV, GCD and EIS tests were all performed on a CHI660D electrochemical workstation .The electrochemical measurements were performed by a three-electrode cell. In detail, the working electrode was composed of PO-graphene (90 wt %) and polyvinylidene fluoride PVDF (10 wt %). A Pt foil electrode and a saturated calomel electrode (SCE)  were used as the counter electrode and reference electrode, respectively. The electrolytes were composed of 0.1 M KI and 0.2 M HThe schematic diagram of the preparation and the photo of PO-graphene are shown in −1, respectively. Different types of oxygen functionalities are presented at 3434 cm−1 (O–H stretching), 1076 cm−1 (C–O stretching), 980 and 861 cm−1 (P–O–C stretching). The peaks at 1649  and 2856 cm−1 (CH2 stretching) are also detected [The chemical composition of PO-graphene was determined by FT-IR, XPS and Raman spectra. As presented in detected . These cThe high resolution XPS spectra could provide detailed bonding and chemical information for the PO-graphene. As presented in −1 and strong G band at 1563 cm−1. The D band is related to the defects and disorder in the structure of the graphite due to the intervalley scattering, while the G band corresponds to the E2g vibration of sp2 of C atoms. In contrast to the origin graphite, the PO-graphene shows a clearly strong D band. The intensity ratio of D to G band (ID/IG) increases from 0.1 to 0.87, manifesting that the disorder degree and structural defect (decrease in the average size of the sp2 domains) in PO-graphene are enhanced due to the oxidization during the preparation [−1.The Raman spectra of PO-graphene and graphite are presented in paration . The decThe XRD patterns of PO-graphene and graphite are presented in −↔ I−3 and the interaction between P–O groups and iodide ions. Clearly, the PO-graphene possesses three pair of redox peaks. The peaks centered at 0.27 and 0.12 V are due to the transformation of 3I−–2e ↔ I−3, the peaks at 0.53 and 0.22 V could be assigned to the transformation of 5I−–4e ↔ I−5 and the peaks at 0.65 and 0.29 V may be attributed to the interaction between P–O and iodide ions.The electrochemical performance of PO-graphene is investigated by CV analysis. As shown in To investigate the charge transfer process between PO-graphene and iodide ions, CV tests at different scan rates were performed. As exhibited in 2, respectively. This result could be ascribed to the strong redox interaction between P–O groups and iodide ions, and this conclusion agrees well with the CV results. The charge transfer performance is also investigated by EIS analysis. The Nyquist plots and equivalent circuit are shown in 2 to 16.5 mA/cm2 due to the polarization. The maximum discharge specific capacitance was calculated as 904.3 F/g when the current density was 6.5 mA/cm2 and it reached 342.5 F/g at 16.5 mA/cm2. This result manifests that the PO-graphene possesses a good rate performance and this discharge performance is also better than the other electrochemical systems, as shown in 2. As shown in 2 compared to pristine during the charge–discharge process (as presented in Because of the convenient charge transfer character, the PO-graphene has a better charging–discharging performance than that of graphite. As shown in 2. The PO-graphene possesses good rate performance and cycling stability.In summary, we applied electrochemical anodic exfoliation method to prepare phosphorus oxide modified graphene and used it as an effective electrode in a redox supercapacitor. The PO-graphene has excellent electrochemical performance due to the significant catalysis to iodide ions and convenient charge transfer capability between phosphorus oxide and iodide ions. The maximum discharge capacitance is 1634.2 F/g when the current density is 3.5 mA/cm


Prescribing anti-programmed death-1 (PD-1) immunotherapy for advanced melanoma is currently not restricted by any biomarker assessment. Determination of programmed death-ligand-1 (PD-L1)-expression status is technically challenging and is not mandatory, because negative tumours also achieve therapeutic responses. However, reproducible biomarkers predictive of a response to anti-PD-1 therapy could contribute to improving therapeutic decision-making.This retrospective study on 70 metastatic melanoma patients was undertaken to evaluate the relationships between clinical, histological, immunohistochemical and/or molecular criteria, and the 6-month objective response rate.P = 0.04), PD-L1 tumour- and/or immune-cell status (P = 0.01), CD163+ histiocytes at advancing edges (P = 0.009) of primary melanomas and NRAS mutation (P = 0.019). Moreover, CD163+ histiocytes at advancing edges (P = 0.04) were associated with longer progression-free survival (PFS), and metachronous metastases with longer overall survival (P = 0.02) and PFS (P = 0.049).Better objective response rates were associated with metachronous metastases (Combining these reproducible biomarkers could help improve therapeutic decision-making for patients with progressive disease. Nivolumab or pembrolizumab anti-programmed death-1 (PD-1) monoclonal antibodies have been reported to significantly increase advanced melanoma patients’ response rates, progression-free survival (PFS) and overall survival (OS), compared to anti-cytotoxic T-lymphocyte antigen-4 antibodies and/or chemotherapy.5 Overall response rates (ORRs) in pivotal clinical studies on nivolumab or pembrolizumab, respectively, were 28%6 or 38%.7 Anti-PD-1 is currently indicated as first-line treatment for BRAF–  metastatic melanomas or sometimes BRAF+ ones with few metastases or slow evolution.Melanomas are highly immunogenic cancers,P = 0.0002; and 2.22, P < 0.00001).8 Moreover, because all studies reported significant response rates of PD-L1– melanomas,9 PD-L1–status determination is not mandatory for the prescription of these agents to melanoma patients, unlike lung adenocarcinomas.10Tumour-cell programmed death-ligand-1 (PD-L1) expression has been associated with better outcomes under anti-PD-1 immunotherapy: a recent meta-analysis highlighted better odds ratios of ORRs for the 1%- and 5%-positive tumour-cell thresholds 13 or CD163+ histiocytes,14 that play important roles immune-response regulation, could be involved in immunotherapy efficacy.Biological and technical challenges in immunohistochemically evaluating PD-L1 expression might explain, at least partially, PD-L1 status-therapeutic response discordances,We hypothesised that PD-L1-expression status combined with other clinical, histological and/or molecular criteria might be able to better predict responses to anti-PD-1 therapy than PD-L1 status alone. Therefore, we retrospectively analysed characteristics of 70 anti-PD-1-treated, metastatic melanoma patients, to try to identify markers contributive to therapeutic decision-making.15 We also collected acute (first month) and chronic (thereafter) immune adverse events (IAEs), defined as all AEs, most frequently skin, bowel, liver or endocrine symptoms potentially attributable to immunotherapy.This two-centre (Toulouse and Bordeaux), retrospective, biomarker analysis concerned a cohort receiving routine care. Seventy patients with advanced and/or metastatic cutaneous melanoma and available formalin-fixed, paraffin-embedded (FFPE) tumour blocks were identified. All primary and/or metastatic lesion specimens were collected before any anti-PD-1 administration. All patients provided written informed consent for the use of the samples, and the local Ethics Committee approved the study. Clinical and histological parameters and follow-up data were retrieved from medical files. The primary outcome measure was the 6-month ORR, determined radiologically with iRECIST criteria.µm-thick sections of FFPE blocks with E1L3N . As stipulated in REMARK criteria, for each case, anti-PD-L1–antibody 22C3-immunolabeling  was run in parallel on an automated Dako immunohistochemistry apparatus, for comparison with the more novel E1L3N.An automated Leica immunohistochemistry instrument labeled 3-16 Positivity was defined as PD-L1 cell-membrane labeling of >5% of tumour area and cells, and >10% of PD-L1 and CD163+ histiocytes at advancing edges , and PD-L1+ cells and CD163+  histiocytes at advancing edges (immediate tumour periphery) of invasive melanomas, were determined as previously described.18 All criteria were independently assessed by three experienced dermatopathologists  blinded to clinical-radiological information. Between-observer scoring mismatches were resolved by case review.PD-L1 expressions in each patient’s primary melanoma and metastases were compared. TILs, their intratumoural or peritumoural distribution pattern  and CD8  expression were analysed according to the literature.χ2 or Fisher’s exact tests and Kruskal–Wallis tests. All survival parameters were measured from the date of the first anti-PD-1 infusion. Kaplan–Meier analyses of PFS and OS used the following first-event definitions, respectively: progression or death, and death. Univariate analyses used the log-rank test, with P ≤ 0.05 defining significance. Statistical analyses were computed using STATA and R software. Given the exploratory nature of this study, no adjustments were made for multiple comparisons.Categorical variables, expressed as numbers, (%) and continuous variables, as median [range], were compared respectively using Via recursive partitioning (rpart package in R v4.1–10), classification-and-regression trees created a decision tree indicating how well different variables predict 6-month objective response class (confusion matrix available in supplementary figure The intraclass-correlation coefficient (ICC) assessed interobserver variability of PD-L1-expression scores.Seventy patients’ 118 melanoma samples were examined; 48 had primary and metastatic specimens available Table  and 7.TaBRAF, neuroblastoma-RAS oncogene (NRAS) or cKIT mutation was found in 62.9% of the lesions and 22.7% were NRAS-mutated. Although NRAS-mutated lesions (regardless of mutation type) were associated with better ORR than those NRAS–, their corresponding OS  and PFS  were comparable. Non-significant trends towards better ORR and PFS were observed for patients with tumours harboring at least one mutation (Kaplan–Meier plots available in supplementary figures At least one P = 0.02). Neither PD-L1 status at advancing edges nor the percentage of PD-L1+ tumour cells was associated with ORR, OS or PFS , and CD163+-histiocyte analysis at the advancing edges (ICC = 0.78)P < 0.05) were combined to create a decision-tree algorithm .According to our results, PD-L1 status alone cannot be used as a reliable biomarker of therapeutic response to anti-PD-1 immunotherapy but could be combined with other criteria, e.g., synchronous metastases, > 10% CD163+ histocytes at advancing tumour edges or 12 Evaluating PD-L1-positivity on tumour and immune cells is more reliable and reproducible than tumour cells alone. Although double-labeling was not done, analysing only tumour-cell PD-L1–positivity (with a 5% cut-off) is perhaps less reliable, especially at the advancing tumour edges, where inflammatory immune cells are usually extensively intermingled.16We observed that PD-L1 was often expressed on histiocytes closely intermingled with tumour melanocytes, which made the distinction between PDL1+ histiocytes and PD-L1+ tumour cells more difficult. The pathophysiological significance of such histiocyte PD-L1 expression remains unclear but has been reported for several cancers.PD-L1 status appears to be relevant in primary melanomas. If that observation is confirmed, it could be helpful, especially because metastases are often difficult-to-access or analyse .20 Although surprising, our results are consistent with histiocytes playing a critical role in immune resistance.To the best of our knowledge, CD163+ histiocytic infiltrates at advancing tumour edges have not yet been thoroughly evaluated in melanomas and might be a potentially helpful biomarker. CD163+ histiocytes are usually considered M2 macrophages and associated with poorer outcomes.In conclusion, identification of patient subgroups responding less well to anti-PD-1 immunotherapy with our algorithm will not avoid these agents, but might obtain better monitoring, and hence more quickly identify progressive disease. Our findings highlighted the potential biomarker role of combining CD163+ histiocytes in melanomas and their metastases with other variables but need to be validated with a larger prospective cohort, including assessment with available anti-PD-L1 monoclonal antibodies, before definitive conclusions can be drawn.Supplementary figures