The study on durability of carbon fiber reinforced plastics (CFRP) in complex environments is critical because of its wide applications. Herein, mechanical behavior of carbon fiber reinforced epoxy composites in the fatigue process were investigated under different humidity via molecular dynamics (MD) simulation method. Transversely isotropic atom based models were established to simulate the structure of CFRP at the atomistic level. Owing to the weak performance in vertical fiber direction, mechanical behavior in a 90° orientation was investigated. Mean stress and energy were both employed to describe the evolution of mechanical performance while mean squared displacement (MSD), radius of gyration (Rg), and free volume were performed to describe the evolution of structural change during the fatigue process. The results show that the humidity led to a weakened interfacial adhesive performance. Free volume became larger under cyclic load, which caused the water molecules to diffuse into the inside of epoxy resin. The distance between the matrix and fiber became larger in the dry system while it reduced because of the diffusion of water molecules in wet system. The rate of performance degradation decreased with the increase in humidity because of poor initial performance at high humidity.  Owing to their excellent material performance such as extremely high specific modulus, large specific strength, and great conductive properties, carbon fiber reinforced plastics (CFRP) are widely used in the aerospace, civil, construction, power, shipping, and other industries. Therefore, several researchers have proposed that it is necessary to study the fatigue properties of CFRP because of its specific applications and complex fatigue behavior. CFRP can be used in the moving structures such as airplanes and helicopters . When itTill now, the fatigue performance analysis has primarily been experimental. The general procedures for fatigue tests are similar. The evolution of specimen stiffness is monitored by the response of stress or strain and the microstructural transformation can be observed by checking the sliced samples under electronic microscope . The strMa et al.  investigHowever, unexpected problems arise in our study on influence of humidity on fatigue performance of CFRP in laboratory. The performance of the specimens varied so much that more parallel experiments were required to eliminate data volatility. Also, from our experience with fatigue testing, the occasional power break, machine failure, and other reasons have different degrees of impact on the experimental data. All of these problems lead to a significant increase in the time and money requirements. Otherwise, only structural changes are observed at the micrometer scale and the observation is discontinued. With the development of computer science and theory of molecular simulation, the molecular dynamics (MD) method has been widely used in the field of computational materials science. Materials behavior can be simulated by the partial movement based on Newton’s theory of motion and potential function based on quantum mechanics. The MD method is widely used in studying the basic performance of materials, such as tensile properties, shear properties, Young’s modulus , glass tHerein, we have studied the performance degradation in vertical fiber orientation and explored the influence of humidity on the fatigue performance of composites via MD method. Besides, some of the phenomenon during fatigue are explained at atomic scale. Multilayer molecular model is widely used to simulate the composites ,30,31 anThe consistent-valence force field (CVFF) was used in the simulation of graphene-epoxy nanocomposites . CVFF cobK, θK, ΦK, and χK are the force constants. b0, θ0, Φ0, and χ0 are the equilibrium bond length, bond angle, torsion angle, and improper angle, while n is the periodicity parameter. ijA and ijB are the square root of product between iA with jA and iB with jB. C and ε are the energy-conversion constant and permittivity, while ijr is the distance between the atom i and j with charges iq and jq. Herein, an all-atom model was employed as it is easy to implement. The model employed in Yu’s research  is used There are two methods to build epoxy resin models. Yu et al.  compared3 is shown in An amorphous cell composed of 30 representative molecules can be obtained by using the method chosen above. The crosslinking model with an initial density of 0.8 g/cmThe method of constructing graphene reinforced epoxy resin is using the Build Layer module in the commercial software Materials Studio. The build Layer module was used to build multi-layers graphene and epoxy resin as a multi-layered system. Polymer-water-graphene-water-polymer model was used to simulate the system with humidity to make the modeling more intuitive and simple. The model for water molecule was TIP3P. Parameters of TIP3P are listed in The moisture content of systems with different humidity are listed in Before performing the strain-control fatigue cyclic loading, an equilibrium system should be first set-up. The Langevin thermostat was applied on the initial structure within NVE ensemble for 2 ns at 298 K to perform Brownian dynamics simulation of a molten polymer. The system was then equilibrated at the NPT ensemble for 2 ns at 298 K with the time step of 0.5 fs. Finally, a stable model was obtained. The NPT ensemble was controlled by the Nose-Hoover thermostat and Nose-Hoover barostat. The time integration of the motion equations was conducted using Velocity-Verlet integration. Final density of equilibrium systems with different humidity are listed in Molecular models with different humidity are shown in −6/ fs and the time step was 1 fs. Stress calculation method was based on the Virial theory that is widely used in the atomic scale. The whole process was completed using open source software LAMMPS. Herein, we primarily investigated the impact of humidity on the fatigue performance and ignored other factors which can be considered in further work. Before applying the fatigue loading, the model was monotonically deformed to the initial strain within the NPT ensembles. The pressure in the unloaded direction was kept at 1 atm. Strain rate was a constant value of 10The stress-strain curves are shown in ε0 is the initial strain, B is the amplitude, t is the time, and T is the period. Herein, ε0, B, and T were 5%, 0.5%, and 1000 fs, respectively. Mean stress [minσ and maxσ are the minimum stress and maximum stress per cycle. Some researchers have predicted the long-term performance using short-term performance with a certain function. Mallick et al. [S is the mean stress per cycle, N is the cyclic number, and a and b are the constant values. Notably, b can be regarded as the speed of the performance degradation and a can be regarded as the initial performance. Fatigue load controlled by strain was applied to the system and herein, primarily, the fatigue behavior was studied in vertical fiber direction (90°). Fatigue cyclic loading was performed using sinusoidal function in the loading direction, the pressure in the unloaded direction was kept at 1 atm. The loading curve can be expressed as:n stress  is widelk et al.  found thk et al.  used an k et al.  employedAs shown in Comparing value a and b in each model with increase in humidity in For the composites, the total performance and interfacial performance are equally important. Evolution of energy can be used to characterize some microscopic phenomena. In this section, total potential energy and interfacial energy were relatively studied. Total energy includes bond, angle, dihedral, improper, van der Waals, and coulomb energy. Among them, bond, angle, dihedral and improper belong to the bond energy, van der Waals and coulomb belong to non-bond energy. The cause of performance degradation can be analyzed by analyzing the evolution of energy. Herein, systems with different humidity were studied. The results of systems with humidity were almost the same as those of the system without humidity. Therefore, we have only displayed the results of the dry system to avoid figures too jumble. The evolution of individual potential energy is shown in From Interfacial energy can reflect the change in distance between the epoxy resin and graphene. The evolution of interfacial energy is shown in As It is critical to investigate the change in structure because macroscopic mechanical response and microscopic energy change are both dependent on the microscopic structural change. In this section, we primarily studied the radius of gyration (Rg), mean-squared displacement (MSD), and free volume fraction (FVF). ir(t) is the current position of the atom i in the group and ir(0) is the original position of the atom i in the group. The x, y, and z components of the mean-squared displacement tensor can be determined using the same formula.The mean-squared displacement was calculated as the root mean square distance between its centre of mass position of fracture and each monomer position in this fracture:As shown in gR2 value in the formula below:M is the total mass of fracture and cmr is the center of mass position of the fracture. The x, y, and z components of the radius of the gyration tensor can be determined using the same formula. This property indicates the level of deformation. To further clarify the results, the initial value of the radius of gyration was set to 0 and relative mean value of radius of gyration was employed to describe the structural change during the fatigue process. The radius of gyration was a measure of the size of the group of atoms, and was computed as the square root of the As per Free volume fraction which reflects the porosity in a molecular system was explored to study the diffusion conditions of water molecules. A molecular model consisting of pure epoxy resin was established and same fatigue cyclic loading method was applied to the pure epoxy system as before. The number of representative units here was same as it was in the composites molecular model above. The free volume fraction was calculated using Multiwfn, an open source software package. (1)Presence of water in the polymer-graphene interface weakened the stiffness and interfacial adhesive performance in the vertical fiber direction. Higher humidity led to a weakened stiffness and adhesive performance.(2)During the process of transversal fatigue, molecular fragments tended to align along the loading direction leading to performance degradation. With regards to microscopic energy, this phenomenon is primarily reflected by the decline of van der Waals, while in terms of macroscopic performance, it is mainly reflected by the decrease in mean stress. (3)During the process of fatigue, interfacial energy of dry system decreased with the cycle numbers. This phenomenon indicates that the distance became larger. However, different results and increase in interfacial energy were observed in the wet system. To understand this phenomenon, another molecular model consisting of pure epoxy resin was established and the free volume of the model before fatigue and after fatigue were relatively calculated. Free volume amplified slightly after fatigue. Increase in the free volume in epoxy resin made it easy for the water molecules to diffuse into the inside of epoxy resin. The diffusion of water molecules reduced the water layer in the polymer-graphene. The thinner water layer led to a stronger interfacial adhesion properties. This conclusion can be confirmed by the evolution of interfacial energy and the mean squared displacement of the epoxy resin and water molecules during fatigue. Besides, interfacial energy of the dry system decreased with the cycle numbers. This phenomenon indicates that the distance became larger. (4)Higher humidity led to a slower rate of performance degradation during fatigue as the performance degraded fast in first several cycles and less prominently in the last several cycles. The performance of composites with high humidity was so poor that no more significant degradation occurred. However, performance continues to decrease because molecular arrangement is the main cause of the performance degradation, even if the interfacial performance improved slightly. In addition, initial performance and rate of performance degradation show a linear relationship with the change of moisture content.Molecular dynamics simulations were used to assess the effect of humidity on the fatigue performance of composites. Four molecular models of composites with different moisture contents were formulated, and for comparison, a molecular model without moisture content was also established. Mean stress was calculated to describe the change in mechanical performance. Interfacial energy was used to describe the interfacial adhesive performance. Mean square displacement and radius of gyration were both used to explain the microstructural change in the process of fatigue. Main conclusions are as follows:This research can provide reference for prospective anti-fatigue design for composites in humidity environment.


Protein inclusions containing the RNA-binding protein TDP-43 are a pathological hallmark of amyotrophic lateral sclerosis and other neurodegenerative disorders. The loss of TDP-43 function that is associated with these inclusions affects post-transcriptional processing of RNAs in multiple ways including pre-mRNA splicing, nucleocytoplasmic transport, modulation of mRNA stability and translation. In contrast, less is known about the role of TDP-43 in axonal RNA metabolism in motoneurons. Here we show that depletion of Tdp-43 in primary motoneurons affects axon growth. This defect is accompanied by subcellular transcriptome alterations in the axonal and somatodendritic compartment. The axonal localization of transcripts encoding components of the cytoskeleton, the translational machinery and transcripts involved in mitochondrial energy metabolism were particularly affected by loss of Tdp-43. Accordingly, we observed reduced protein synthesis and disturbed mitochondrial functions in axons of Tdp-43-depleted motoneurons. Treatment with nicotinamide rescued the axon growth defect associated with loss of Tdp-43. These results show that Tdp-43 depletion in motoneurons affects several pathways integral to axon health indicating that loss of TDP-43 function could thus make a major contribution to axonal pathomechanisms in ALS. In neurons, mechanisms for subcellular mRNA transport and translation in dendrites and axons allow the regulation of localized responses to cellular stress conditions. Alterations in these RNA processing mechanisms can lead to neural dysfunction and neurodegeneration –3. LikewSubcellular mRNA transport is mediated by messenger ribonucleoprotein particles (mRNPs) which contain RNA-binding proteins as essential components. One of these RNA-binding proteins is the transactive response DNA-binding protein (TARDBP or TDP-43) . Since tALS pathology is generally characterized by degeneration of upper and lower motoneurons, resulting in a dramatic loss of motor function . DisruptIn order to investigate how TDP-43 is involved in modulation of axonal functions, we determined the consequences of Tdp-43 depletion in primary mouse motoneurons both on the morphological level and by analysis of alterations in the somatodendritic and axonal transcriptomes. Tdp-43 knockdown significantly impaired axon elongation. RNA-seq analysis revealed that many transcripts relevant for axonal cytoskeletal integrity, mitochondrial function and translation were dysregulated in the axonal compartment upon Tdp-43 depletion. In agreement with this finding, protein translation and mitochondrial functionality were specifically impaired in axons of Tdp-43-deficient motoneurons. Finally, we found that treatment with nicotinamide, a precursor for the cofactor NAD+ that is required for oxidative phosphorylation, restored axon growth in Tdp-43 knockdown motoneurons to normal levels. Our results therefore suggest that reduction of Tdp-43 function dysregulates RNA transport processes that are vital for axon growth, local protein synthesis and energy production.CD-1 mice were housed in the animal facility of the Institute of Clinical Neurobiology at the University Hospital of Wuerzburg. Mice were maintained at a 12 h/12 h day/night cycle under controlled conditions in 55–65% humidity. Food and water were available in abundant supply. Each experiment was conducted following the regulations on animal protection of the German federal law and of the Association for Assessment and Accreditation of Laboratory Animal Care, in agreement with and under control of the local veterinary authority.2. 50% of the culture medium was replaced at 1 d in vitro (DIV1) and then every second day. For nicotinamide treatment, medium was supplemented with nicotinamide (Applichem) at the indicated concentrations. Motoneuron cultures in compartmentalized chambers were performed as previously described  and 20 nM TMRM or MitoTracker™ Orange were loaded into cells by incubation for 15 min at 37 °C and 5% CO2. Then, motoneurons were washed twice with pre-warmed Tyrode’s buffer and imaged in 2 ml Tyrode’s buffer supplemented with 5 ng/ml BDNF. Live-cell imaging was conducted on an inverted epifluorescence microscope  equipped with 60× 1.4-NA objective, Perfect Focus System (Nikon), a Cool LED pE-4000 light source and an Orca Flash 4.0 V2 camera (Hamamatsu Photonics). During live-cell imaging, cells were maintained at 37 °C and 5% CO2 on a heated stage chamber . TMRM and MitoTracker™ Orange CM-H2TMRos were excited at 560 nm illumination using 2% light intensity and 12-bit images of 1024 × 1024 pixels were acquired for 5 min at 30 s intervals. Mitochondria labeled with MitoTracker™ Orange were counted in axons of control and Tdp-43 knockdown motoneurons and normalized to axon length. ImageJ was used to measure the mean grey value of TMRM fluorescence intensity per mitochondrion and average background intensity was subtracted. Median intensity values of mitochondria per axon were normalized to the average intensity of the corresponding control group. In control experiments with FCCP, mitochondria labeled with TMRM were imaged first for 1 min with 30 s intervals. Next, 10 μM FCCP  was added and axons were imaged for another 8 min. FCCP depolarizes the mitochondrial membrane potential, resulting in a rapid and significant decrease in the fluorescent intensity of TMRM upon 1 min addition.Mitochondria were labeled with MitoTracker™ Orange CM-H2TMRos  and TMRM  was used to determine the mitochondrial membrane potential (Δψm). Motoneurons were cultured on laminin-111-coated  35 mm Ibidi μ-dishes  for 6 d. Cells were washed twice with pre-warmed Tyrode’s buffer [125 mM NaCl, 2 mM KCl, 30 mM glucose, 2 mM CaClRNA was reverse-transcribed with random hexamers using the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Reverse transcription reactions were diluted 1:5 in water. Reactions were set up with Luminaris HiGreen qPCR Master Mix (Thermo Fisher Scientific) on a LightCycler® 96 thermal cycler (Roche). Primers are listed in Supplementary Table In order to characterize the subcellular distribution and in particular the location and function of Tdp-43 in axons we first investigated the Tdp-43 distribution in spinal cord of adult mice by immunostaining Fig. a. An antTdp-43.1-4) targeting the Tdp-43 transcript and cloned them into a lentiviral expression vector. The lentiviral constructs also expressed GFP for visualization of virus infection. We transduced primary motoneurons with these different Tdp-43 knockdown viruses or a control virus expressing a Luciferase shRNA (shLuc) and cultured them for 7 d  for all the subsequent Tdp-43 knockdown experiments and used it also for confirmation of the specificity of the Tdp-43 immunolabeling procedure  dysregulated in the somatodendritic compartment  (Supplementary Table r = 0.87) , Tubulin beta-2A (Tubb2a) and Tubulin alpha-1B (Tuba1b). We validated the downregulation of these transcripts in axons of Tdp-43 knockdown motoneurons by qPCR  term analysis of transcripts upregulated in the somatodendritic compartment of Tdp-43 knockdown motoneurons revealed an enrichment of transcripts with functions in neuron projection development, synapse and cytoskeleton Fig. . Among tpatients , 36. FurIn contrast, the group of transcripts downregulated in the somatodendritic compartment of Tdp-43-depleted motoneurons showed a high association with GO terms related to mitochondrial function and energy production Fig. . Among tFor transcripts downregulated in axons of Tdp-43 knockdown motoneurons the GO term “translation” is the most significantly affected biological process Fig. . The traBeyond transcripts related to translation, we found an enrichment of GO terms related to mitochondrial functions for transcripts downregulated in the axonal compartment of Tdp-43 knockdown motoneurons Fig. . These GOur data point towards the possibility that a lack of energy supply due to disturbed mitochondrial function contributes to the axon growth defect associated with Tdp-43 depletion in motoneurons. To investigate this possibility we treated Tdp-43 knockdown and control motoneurons with nicotinamide (NAM), a precursor for nicotinamide adenine dinucleotide (NAD+) that acts as a key cofactor in mitochondrial pathways for the production of ATP. NAM is converted to NAD+ through the salvage pathway . We founWe then tested whether NAM treatment had an impact on the defective axonal protein synthesis which we observed upon Tdp-43 depletion. Control and Tdp-43 knockdown motoneurons were cultured in the presence or absence of 0.1 mM NAM for 6 d followed by puromycin exposure to label nascent proteins Fig. b. In theIn this study, we have investigated transcriptome alterations in the somatodendritic and axonal compartments of motoneurons after Tdp-43 depletion. Subcellular transcriptome alterations in ALS cell models have been investigated before using different approaches. In the study by Rotem et al. mouse motoneurons were transduced with lentiviruses for expression of GFP-tagged SOD1(G93A) or TDP-43(A315T) and cultured in modified Boyden chambers to obtain neuritic RNA . While tIt has been suggested that, in addition to the toxicity exerted by TDP-43 insoluble inclusions, loss of TDP-43 functions contributes to the etiology of ALS . Given tTXNIP transcription directly [Txnip we observed after Tdp-43 depletion could cause inhibition of thioredoxin activity, leading to enhanced ROS levels. This, in turn, could induce the observed mitochondrial dysfunction and thus contribute to the pathological alterations in mitochondria seen in Tdp-43-associated motoneuron disease models. The rescue effect by treatment with NAM, which has been shown to reduce ROS levels [After Tdp-43 depletion, several transcripts are upregulated in the soma that appear highly relevant for neuronal integrity. For example, upregulation of Txnip is expected to result in an inhibition of thioredoxin leading to enhanced oxidative stress. The Pi3K/AKT pathway, which is important for neuronal survival  and propdirectly , 53, anddirectly  indicateS levels , supportGO term analysis of transcripts downregulated in the axonal compartment of Tdp-43-deficient motoneurons revealed that pathways related to mitochondrial functions might be particularly affected. Mitochondria are a known target of Tdp-43 function , 56–60 aTdp-43 transcript. The reduced axon lengths we observed for Tdp-43-depleted motoneurons stand in contrast to previous observations [We found that reduced axon growth is a key morphological phenotype of Tdp-43-deficient motoneurons. We observed this phenotype with three different shRNAs targeting the rvations , but ourrvations , 68. Beyrvations . Reducedrvations –73. In mrvations . This raIn summary, our data have revealed that multiple pathways are dysregulated in Tdp-43-depleted motoneurons, many of them having been characterized before in the context of other neurodegenerative diseases such as Alzheimer’s, Huntington’s and Parkinson’s disease. Thus, it might be the combination of defects in these pathways that underlies the vulnerability and axonal degeneration of Tdp-43-deficient motoneurons. A multitude of targets could be conferred from these pathways that might need to be addressed in combination for therapy development in ALS.Additional file 1: Supplementary Table 1. List of primers for cloning of shRNAs into pSIH. siRNA sequences are underlined. Supplementary Table 2. List of qPCR primers. Supplementary Table 3. List of transcripts significantly (p<0.05) upregulated in the somatodendritic compartment of Tdp-43 knockdown (T_sd) motoneurons relative to control (G_sd) motoneurons. Supplementary Table 4. List of transcripts significantly (p<0.05) downregulated in the somatodendritic compartment of Tdp-43 knockdown (T_sd) motoneurons relative to control (G_sd) motoneurons. Supplementary Table 5. List of transcripts significantly (p<0.05) upregulated in the axonal compartment of Tdp-43 knockdown (T_ax) motoneurons relative to control (G_ax) motoneurons. Supplementary Table 6. List of transcripts significantly (p<0.05) downregulated in the axonal compartment of Tdp-43 knockdown (T_ax) motoneurons relative to control (G_ax) motoneurons. Supplementary Table 7. List of transcripts significantly (p<0.05) deregulated in the somatodendritic (sd) and axonal (ax) compartment of Tdp-43 knockdown motoneurons relative to control motoneurons.


Such non-linearly enhanced strain exceeds the values predicted for harmonic vibrations with the same root mean square (RMS) amplitude by more than one order of magnitude. Our work holds promise for dynamical strain engineering and dynamical strain-mediated control of light-matter interactions in 2D materials and related heterostructures.Graphene and related two-dimensional (2D) materials associate remarkable mechanical, electronic, optical and phononic properties. As such, 2D materials are promising for hybrid systems that couple their elementary excitations  to their macroscopic mechanical modes. These built-in systems may yield enhanced strain-mediated coupling compared to bulkier architectures, e.g., comprising a single quantum emitter coupled to a nano-mechanical resonator. Here, using micro-Raman spectroscopy on pristine monolayer graphene drums, we demonstrate that the macroscopic flexural vibrations of graphene induce dynamical optical phonon softening. This softening is an unambiguous fingerprint of dynamically-induced tensile strain that reaches values up to ≈4 × 10 Here, the authors use Raman spectroscopy on circular graphene drums to demonstrate dynamical softening of optical phonons induced by the macroscopic flexural motion of graphene, and find evidence that the strain in graphene is enhanced under non-linear driving. These efforts triggered the study of 2D resonators beyond graphene, made for instance from transition metal dichalcogenide layers18 and van der Waals heterostructures21. In suspended atomically thin membranes, a moderate out-of-plane stress gives rise to large and swiftly tunable strains, in excess of 1%23, opening numerous possibilities for strain-engineering24. These assets also position 2D materials as promising systems to achieve enhanced strain-mediated coupling28 of macroscopic flexural vibrations to quasiparticles  and/or degrees of freedom . Such developments require sensitive probes of dynamical strain. Among the approaches employed to characterise strain in 2D materials, micro-Raman scattering spectroscopy29 stands out as a local, contactless and minimally invasive technique that has been extensively exploited in the static regime to quantitatively convert the frequency softening or hardening of the Raman active modes into an amount of tensile or compressive strain, respectively33. Recently, the interplay between electrostatically-induced strain and doping has been probed in the static regime in suspended graphene monolayers34. Dynamically-induced strain has been investigated using Raman spectroscopy in bulkier micro electro-mechanical systems36, including mesoscopic graphite cantilevers37 but remains unexplored in resonators made from 2D materials.Since the first demonstration of mechanical resonators made from suspended graphene layers38 and dynamical strain-mediated control of light-matter interactions.In this article, using micro-Raman scattering spectroscopy in resonators made from pristine suspended graphene monolayers, we demonstrate efficient strain-mediated coupling between "built-in” quantum degrees of freedom  of the 2D resonator, and its macroscopic flexural vibrations. The dynamically-induced strain is quantitatively determined from the frequency of the Raman-active modes and is found to attain anomalously large values, exceeding the levels of strain expected under harmonic vibrations by more than one order of magnitude. Our work introduces resonators made from graphene and related 2D materials as promising systems for hybrid opto-electro-mechanics2 substrate. The resulting graphene drum is capacitively driven using a time-dependent gate bias Vac ≪ Vdc and Ω/2π the drive frequency. The DC component of the resulting force  and integrated intensity of a Raman feature42 . Biaxial strain is expected around the centre of circular graphene drums22 and the large Grüneisen parameters of graphene 32 allow detection of strain levels down to a few 10−5. The characteristic slope 42. This difference allows a clear disambiguation between strain and doping (see “Methods” section for details).The Raman spectrum of graphene displays two main features: the G mode and the 2D mode, arising from one zone-centre  phonon and from a pair of near-zone edge phonons with opposite momenta, respectively see Fig. 29. Both 0/2π ≈ 33.8 MHz for Vdc = −6 V . At Vdc = −9 V, the 4 ± 0.5 cm−1 2D-mode downshift relative to its value near εs = 3 ± 0.3 × 10−4 that agrees qualitatively well with the value εs = 2 ± 0.2 × 10−4 estimated from the gate-induced upshift of Ω0 , with Vac increasing from 0 to 150 mV and applied at a drive frequency that tracks the Vac-dependent non-linear softening of the mechanical resonance frequency zrms. The correlation plot between ω2D and ωG reveals a linear slope near 2 ; in our experimental geometry they indicate an equilibrium position upshift Δξeq by up to ≈12 nm  and arises from the time-averaged resonant vibrations of the graphene drum. Starting from a reference recorded at Vdc = −8 V and Vac = 0 mV, εd recorded under resonant driving at Vac = 150 mV (where zrms ≈ 9 nm) is as high as the static strain εs induced when ramping Vdc from 0 V to  −8 V (where ξ ≈ 60 nm). Along these lines, the small yet observable broadenings ΔΓG,2D of the Raman features  under Vdc = −6 V with and without resonant driving. Very similar results are observed when performing a line-scan along the perpendicular direction  SiN membranes51 showing RMS displacement saturation similar to Fig. 44, geometrical52 or electrostatically-induced53) may lead to internal energy transfer towards harmonics of the driven mode  are rectified under strong non-linear driving, an effect that further increases the discrepancy between the time-averaged εd we measure and The large values of entclass1pt{minimaentclass1pt{minima,2D Fig.  is smallεd may require microscopic models that may go beyond elasticity theory54 and explicitly take into account the ultimate thinness and atomic structure of graphene56.Combining multi-mode opto-mechanical tomography and hyperspectral Raman mapping on larger graphene drums  would allow us to test whether localisation of harmonics occurs in graphene and to possibly correlate this phenomenon to the dynamically-induced strain field. More generally, unravelling the origin of the anomalously large −4. Realistic improvements of our setup, including phase-resolved Raman measurements36 could permit to probe dynamical strain in finer detail, including in the linear regime, where the effective coupling strength28 could be extracted. For this purpose, larger resonant displacements may be achieved at cryogenic temperatures. In addition, graphene drums, as a prototypical non-linear mechanical systems, can be engineered to favour mode coupling and frequency mixing, which in return can be readout through distinct modifications of their spatially-resolved Raman scattering response.Concluding, we have unveiled efficient coupling between intrinsic microscopic degrees of freedom  and macroscopic non-linear mechanical vibrations in monolayer graphene resonators. Room temperature resonant mechanical vibrations with  ≈10 nm RMS amplitude induce unexpectedly large time-averaged tensile strains up to  ≈ 4 × 1033 could be used as invaluable probes of in-plane and out-of-plane dynamical strain, respectively. Strain-mediated coupling could also be employed to manipulate the rich excitonic manifolds in transition metal dichalcogenides57, as well as the single photon emitters they can host59. More broadly, light absorption and emission could be controlled electro-mechanically in nanoresonators made from custom-designed van der Waals heterostructures60. Going one step further, with the emergence of 2D materials featuring robust magnetic order and topological phases61, that can be probed using optical spectroscopy, we foresee new possibilities to explore and harness phase transitions using nanomechanical resonators based on 2D materials63.Our approach can be directly applied to a variety of 2D materials and related van der Waals heterostructures. In few-layer systems, rigid layer shear and breathing Raman-active modes2/Si substrates, using a thermally assisted mechanical exfoliation scheme as in ref. 64. The pattern is created by optical lithography followed by reactive ion etching and consists of hole arrays (5 and 6 μm in diameter and 250 ± 5 nm in depth) connected by  ~1 μm-wide venting channels. Ti(3 nm)/Au(47 nm) contacts are evaporated using a transmission electron microscopy grid as a shadow mask34 to avoid any contamination with resists and solvents. Our dry transfer method minimises rippling and crumpling effects65, resulting in graphene drums with intrinsic mechanical properties (see ref. 31 and Supplementary Note Monolayer graphene flakes were deposited onto pre-patterned 285 nm-SiO−5 mbar). The drums are capacitively driven using the Si wafer as a backgate and a time-dependent gate bias a is the drum radius, ϵ0 the vacuum dielectric constant, ξ the static displacement, dvac the graphene-SiO2 distance in the absence of any gate bias, 2 layer. This force contains a static component proportional to ξ and a harmonic driving force proportional to Electrically connected graphene drums are mounted into a vacuum chamber , with m0 the pristine mass of the graphene drum. As discussed in details in Supplementary Note 66, as expected for a resist-free fabrication process.A 632.8 nm HeNe continuous wave laser with a power of  ~0.5 mW is focused onto a  ~1.2 μm-diameter spot and is used both for optomechanical and Raman measurements. Unless otherwise stated,  and charge inhomogeneity67. Considering our experimental geometry, we estimate a gate-induced doping level near 3 × 1011 cm−2 at the largest 66. In the dynamical regime, the RMS modulation of the doping level induced by the application of Vac is typically two orders of magnitude smaller than the static doping level and can safely be neglected. Similarly, the reduction of the gate capacitance induced by equilibrium position upshifts discussed in Figs. The Raman scattered light is filtered using a combination of a dichroic mirror and a notch filter. Raman spectra are recorded using a 500 mm monochromator equipped with 300 and 900 grooves/mm gratings, coupled to a cooled CCD array. In addition to electrostatically-induced strain, electrostatically-induced doping might in principle alter the Raman features of suspended graphene≲1011 cm− and char68 is more than three orders of magnitude shorter than the mechanical oscillation period, Raman scattering processes provide an instantaneous measurement of εd. However, since our Raman measurements are performed under continuous wave laser illumination, we are dealing with time-averaged dynamical shifts and broadenings of the G-mode and 2D-mode features. Raman G-mode and 2D-mode spectra are fit using one Lorentzian and two modified Lorentzian functions, as in refs. 67, respectively ps68 is mSupplementary InformationPeer Review File


The incorporation of graphene nanoplatelets (GnPs) within a polymer matrix can play an important role in the physical properties and the functionality of the composite material. Composites consisting of low-density polyethylene (LDPE) and GnPs of different concentrations were developed by mixing GnPs with a molten form of the polymeric matrix. The effect of the GnPs content on the morphological, structural, and electrical properties of the composites were investigated. As shown, graphene presence and its concentration significantly modified the polymer matrix properties, a behavior that can be employed for tailoring its applicability in electrical applications. It was found that the increase of the graphene platelets concentration seems to promote the formation of graphene agglomerates, air gaps, and inhomogeneities, while higher dielectric constant/lower dielectric losses can be achieved. Polymers have been proved to be suitable matrices for the development of composite materials due to their ease of production and processing, resistance to corrosion, good mechanical properties, and light weight. In polymer-based nanocomposites, the type and size of the filler, the morphology of the filler network, the thermal properties, and the conductivity have been considered to be important variables influencing its final properties ,2. PolymPresently, graphene, has become one of the most exciting materials , becauseSubstantial amount of research has been carried out in the field of nanocomposites incorporating graphene, some of them regarding electrical applications. In general, the increasing demand of energy is a technical challenge for the generation, transmission, storage, and distribution energy systems. This challenge often requires contradictory features such as increasing voltage levels in combination with more compact designs in urban environments. This leads to an increased electric stress on the insulation systems, which can be addressed by using insulating materials with tunable properties, as well as high dielectric constant and low losses, especially for electric field grading applications. In this particular research field, the reports in the literature on graphene nanocomposites concern, improvement of conductivity in polyethylene (PE) matrices , advanceThis work concerns nanocomposite materials made using commercial widely available ingredients using methods that can easily undergo direct further scale up, to become compatible with the industrial needs. In particular, in this work, low-density polyethylene (LDPE) was chosen as the basic matrix material for the fabrication of graphene nanocomposites, due to its significant importance in various applications such as squeeze bottles, toys, carrier bags, high frequency insulation, chemical tank linings, heavy duty sacks, general packaging, gas and water pipes . In addi3.Nanocomposite specimens were fabricated by employing commercially available LDPE from Sigma Aldrich GmbH, Darmstadt, Germany with average molecular weight—MW of 35,000 determined by gel permeation chromatography (GPC) and average numerical mass—Mn 7.700 GPC. Graphene nanoplatelets were provided by EMFUTUR Technologies Ltd., Hillsborough, Spain, with ~5 μm lateral size, 5 nm thickness, and a bulk density of 0.03 to 0.1 g/cmThe melting method was used since this is rather simple for fabricating polymer nanocomposites consisting of low molecular weight polymer matrices. This method involves melting of the polymer to its viscous form, followed by the addition and the mechanical mixing of the filler within the polymer matrix. Graphene concentration in the composite material was varied in the range 0% (%wt.) up to 5% (%wt.) at a step of 0.5%. For each composition 5 replicas were made and characterized. All the obtained formulations were characterized as follows:2 flow and a temperature increment of 20 °C/min. Finally, the electrical characterization of the nanocomposite samples was conducted by means of Broadband Dielectric Spectroscopy (BDS), using a TH2829C LCR precision bridge  capable of taking measurements in the frequency range of 20 Hz to 1 MHz.SEM characterization was performed using a (FE-SEM) Nova NanoSEM 630 , equipped with an energy-dispersive X-ray spectroscopy—EDX detector , in order to investigate and understand the formation and the structuring of the obtained nanocomposite materials. All samples were examined in the high vacuum mode, low landing energies and fast dwelling time, without any conductive coating in order to preserve the samples in their natural state for further analyses. XRD was also performed using a Rigaku Ultra high-resolution triple axis multiple reflection SmartLab X-ray Diffraction System . Moreover, Raman analysis was done using a WiTec alpha 300S GmbH Germany system , employing an Nd-YAG laser at 532 nm and confocal Raman microscopy . Raman spectra were recorded under ambient conditions, using a 532 nm diode-pumped solid-state laser  with a power of 10% (10 mW) to avoid local drying of the samples. A 20× objective and a 25 μm slit aperture were used in order to obtain quite detailed spectra, while the total acquisition time 0.5, 10 and 20 s (1 s exposure × 20 exposures) and 600 grooves/mm grating was chosen for each Raman spectrum. The only notable difference observed in spectra was a higher overall spectral intensity when the acquisition time increases. Severally spectra, in different points were recorded per sample and a representative typical spectrum for each sample was chosen. The instrument calibrations were performed on a silicon probe prior to measurements to ensure reproducible results. The spectrometer scanning data collection and processing were carried out using the WiTec Project Five software  and appropriate background corrections were performed on all spectra in Origin software . The thermal response characterization was studied using a Diamond  differential scanning calorimeter (DSC) operated at a scan rate of 10 °C/min in order to characterize the thermal transitions of employed polymer nanocomposites. Samples were prepared inside aluminum shielded pans while an empty aluminum shielded pan was used as a reference. Experimental temperature range was set from ambient to 200 °C. For thermogravimetric analysis (TGA), a Diamond  TGA was used under 200 mL/min N2 areas of the sample in order to form a capacitor. Two metallic pins were attached to the silver paste plates, and these pins were connected to an HIOKI 3532-50 LCR HITESTER , through its specialized cables. Impedance Z and corresponding loss angle δ are simultaneously measured, as a function of frequency, in the frequency range 10 Hz–1 MHz. The presented results are representative for the specific kind of samples.For the low energy impedance spectroscopy measurements, samples of a rectangular shape were used, with dimensions 10 mm × 10 mm × 0.5 mm. Conductive silver paste was smeared onto both the 10 × 10 mmSEM characterization of samples was performed to investigate the formation and structuring of the composite materials. Nanocomposite materials with higher than 2% graphene content were found to consist of a quite inhomogeneous bulk, formed by different polymerization domains as well as regions with concentrated graphene. In addition, at high graphene concentrations, large graphene agglomerations were observed in some cases, suggesting that a limit of concentration might exist, above which graphene cannot be fully dispersed in the LDPE molten material.At graphene concentrations higher than 2.5%, the structural inhomogeneity is accentuated and a mix of compact and granular regions as well as “graphene bubbles” were observed, as can be seen in Following the SEM characterization, one can understand the effect of the graphene concentration on the composites structure and morphology. The increase of concentration seems to promote the formation of graphene agglomerates, air gaps, and inhomogeneities being present at the same time. These structural SEM observations, related to the effect of the graphene concentration on the evolution of the bulk morphology, seem to correlate with the dielectric constant measurements, as it will be shown later. Finally, it should be mentioned here that to the best of our knowledge, such detailed morphology study of LDPE-graphene composite and its evolution with graphene concentration has not yet been presented in the literature before.Structural features for the LDPE composites of different graphene concentrations were further investigated using grazing incidence X-ray diffraction (GI-XRD), where the incidence angle was kept to 0.5°. In order to assess the influence of graphene, the attention was focused in the range of 5°–40°. The results can be seen in With increasing graphene concentration, the occurrence of the characteristic diffraction peak for carbon around 26° appeared for graphene concentrations higher than 2% graphene. To get the mean crystallite size τ for LDPE and graphene composites, the Scherrer equation  was empl002 = 3.37 Å, at the highest graphene concentration.The obtained results strongly indicate the embedding of the graphene in the LDPE lattice and suggest an enhancement of its crystallinity at high graphene concentration. At the same time, the interlayer distance of the graphene employed becomes closer to the hexagonal graphene one (2H-polytype), for which dRaman spectra were also recorded, using a 532 nm diode-pumped solid-state laser  with a power of 10 mW. A 20× objective and a 25 μm slit aperture were used in order to obtain quite detailed spectra, while a total acquisition time of 10 s (1 s exposure × 10 exposures) and 600 grooves/mm grating was chosen for each Raman spectrum. The spectrometer scanning data collection and processing were carried out using the WiTec Project Five software.−1, the 2D band is at about 2786 cm−1, while there is also evidence of the so-called disorder-induced D bands around 1345 cm−1. Literature reports similar peaks for graphene, in particular, in pristine-single layer graphene, the G band appearing at 1582 cm−1 (graphite) and a 2D band at about 2786 cm−1 [−1 and 1578 cm−1 respectively, denoted by the D’, while the I2D/IG ratio increases [−1, while peaks near 1420 cm−1 (the CH2 bending mode) are also present and are indicators of crystalline and amorphous PE phases. Literature reports similar results for polyethylene, both low and high density, with Sato et al. reporting four (4) peaks at 1465, 1374, 1174 and 897 cm−1 [−1 respectively [−1 and 2848 cm−1.Examples of Raman spectra collected on melt-fabricated samples corresponding to various concentrations of graphene are presented in 786 cm−1 ,20. Morencreases . Regardi897 cm−1 . Moreoveectively . It can Regarding local homogeneity of the composite material, an example is presented in Detailed characterization of the obtained nanocomposites showed that all different batches, fabricated under similar conditions, resulted in more or less similar inhomogeneous materials exhibiting similar properties. These results indicated that the mechanical mix of GnPs in melted LDPE may not be the best method for the preparation of homogeneous GnPs/LDPE nanocomposites, at least for the case of small quantities, as those used in our work. Another approach considered for obtaining GnPs/LDPE nanocomposites formulations in small quantities could be dry mixing of the LDPE polymer pellets with graphene but, as tested, it was almost not possible, since they did not really mix. Similarly, we found out that extruding the two phases resulted in even worst composite material. For proper mixing of the specific materials hot roll milling would be more proper method but it is not easily available for laboratory small scale trials. Since GnPs are not a monodisperse material, functionalization of GnPs would not be a realistic approach to solve the dispersion problems at the mixing in the mold. In any case, more homogeneous materials are expected in larger scale production, since, in this cases mixing can be done more effectively.TGA was performed to study the mass loss if a thermal event involves loss of a volatile component. To study the phase transitions of the GnPs/LDPE nanocomposites DSC analysis was performed. Two typical DSC analyses of the 2.5% and 5% graphene containing samples compared with pure LDPE are presented in The melting temperature Tm (°C) for pure LDPE was 80 °C while the crystallization temperature Tc—98 °C. All the composite materials show Tm smaller than the pure material and Tc larger. Tm increases as the GnPs increase and Tc slightly increase when GnPs concentration increased.Finally, the dielectric behavior of all fabricated samples was investigated using BDS and its dependence on the graphene content within the LDPE matrix was analyzed. As expected, the dielectric constant significantly depends on the graphene concentration and the structural characteristics of the material. In particular, the dielectric constant can be strongly influenced by the morphology of the composite, especially if this contains air gaps and agglomerations, as was found out in the SEM results. However, at this point, a quantitative correlation between the structural/morphologic parameters and the dielectric parameters variations is not possible due to the non-quantifiable structural evolution of the composite bulk structuring with increased graphene concentration.Compared to existing literature results, Gaska et al. ,24 studiGnP/LDPE composite materials with various graphene concentrations were fabricated using the melting method and were characterized using SEM, XRD and Raman spectroscopy and microscopy in order to understand the formation of the composites and the evolution of structural and morphological characteristics with increasing graphene content. SEM characterization showed that graphene concentrations up to 2.5% lead to more compact structuring, while larger concentrations promote the random formation of a kind of “graphene bubbles”. XRD characterization showed that mean crystallite size increases as the graphene concentration increases, with a value of 26.1 nm at a concentration of 2%, this becoming 40.4 nm at 5%. Moreover, the increasing of the graphene concentration within the polymer matrix indicate an expansion of crystal interplanar distance of the embedded graphene. Regarding Raman characteristic signals of the composite regions, these appeared to increase in intensity as the graphene concentration increases, revealing the formation of a composite material and not a simple component mixing. The dielectric constants of the nanocomposites presented an increased trend, proportional with graphene increased concentration; however, it was strongly affected by the morphology evolution of the material. The random formation of “graphene bubbles” could induce the formation of conductive tunnels within the samples, this phenomenon being encountered at specimens with concentration of 4.5% and 5%, a behavior requiring further studies.


Recent advances in the field of two-dimensional (2D) materials have led to new electronic and photonic devices enabled by their unique properties at atomic thickness. Structuring 2D materials into desired patterns on substrates is often an essential and foremost step for the optimum performance of the functional devices. In this regard, optical patterning of 2D materials has received enormous interest due to its advantages of high-throughput, site-specific, and on-demand fabrication. Recent years have witnessed scientific reports of a variety of optical techniques applicable to patterning 2D materials. In this minireview, we present the state-of-the-art optical patterning of 2D materials, including laser thinning, doping, phase transition, oxidation, and ablation. Several applications based on optically patterned 2D materials will be discussed as well. With further developments, optical patterning is expected to hold the key in pushing the frontiers of manufacturing and applications of 2D materials. Nanomaterials, with at least one geometric dimension at the nanometer scale, have immense applications in miniaturization of existing technology while rendering the intense exploration of fundamental phenomena at nanoscale. 2D materials are a subset of the nanomaterials where geometric length of only one dimension reaches the nanometer regime . Since iFor application-oriented utilization of 2D materials, one often starts with their fabrication or patterning, which might involve control of either thickness, carrier density, or phase of 2D materials to precisely tune the optical, electrical, and thermal characteristics of 2D materials. The patterned 2D materials have led to applications in electrochemistry , biosensin situ patterning of 2D materials through precise digital control of laser beams. The localized nature of high-intensity laser beams offers unique opportunities to develop or pattern 2D materials based on regionally induced physical or chemical modifications. For many micro/nanodevice applications of 2D materials based on their patterned features at micro/nanoscale, the timescales of optical patterning range from a few seconds to minutes. Moreover, laser intensity can be tuned with ease to peel a desired number of atomic layers from the 2D materials [Laser-based fabrication methods provide a promising route to high-throughput, versatile, and aterials  and to caterials . Recent The physical properties of 2D materials are closely dependent on their thickness. For example, it has been reported that 2D materials exhibit thickness-dependent photoluminescence (PL) where PL intensity increases with decreasing thickness . Upon re2. The patterned GO layer was then reduced to graphene via thermal annealing or hydrazine. However, this mechanism usually takes over six hours and must be carried out in a humid atmosphere for water to intercalate between graphene layers. This thinning method could only reach down to three-layer graphene due to the high in-plane thermal conductivity of graphene that reduced the temperature in the perpendicular direction below 500°C.Initial attempts to control the thickness of graphene using high-power lasers are based on oxidative burning and subsequent reduction of graphene oxide films . The irr2 substrate that served as a heat sink for the bottom layer of the graphite adjacent to the substrate, thereby reducing the maximum temperature at the bottom layer while the top layers could reach ~1450°C to enable oxidative etching. −1 corresponds to monolayer graphene [Han et al. proposed that laser thinning of graphite to graphene could be better achieved by similar laser irradiation through substrate engineering . In one graphene . The capgraphene .in situ through four-wave mixing signals. Although throughput is compromised due to low-power multiple scanning, precise quantification of number of graphene layers has been achieved through in situ fast imaging [Despite the prominence of monolayer graphene, multilayered graphene exhibits advantages in certain applications. For instance, hydrogenation of multilayered graphene is more feasible than that of monolayer graphene, which is helpful in tuning physical and chemical properties of graphene for sensing devices . Consequ imaging .2 with patterned feature sizes of 200 nm via laser thinning [2 due to its low sublimation temperature of ~450°C [2 flake before and after laser scanning indicates the thinning of the flake  and NIR detectors. Recently, Nagareddy et al. have proposed a humidity-controlled laser thinning technique where layer-by-layer laser thinning of MoTe2 was achieved for the sample that was maintained at a relative humidity of more than 65-70% [2 result in an uncontrolled bond-breaking at relatively higher laser powers. As relative humidity increases, the water molecules adsorb on all available sites offered by the top layer of MoTe2  has also demonstrated great potential in applications requiring high carrier mobility . HoweverBesides the laser-induced thickness-control procedures in 2 and the optical patterning was imaged through upconversion luminescence [μm [Preliminary work on 2D material ablation was performed on graphene, where a high-powered femtosecond laser was utilized to convert the carbon to CO and COnescence . With a nescence . The ablence [μm . While tence [μm .2 substrate (left panel), and the scanning electron micrographs of transferred graphene nanoribbons with variable widths ranging from 100 nm–9 μm on Si/SiO2 substrates (middle and right panels). Despite its capability of achieving the multiscale and high-resolution graphene patterns on an arbitrary substrate, the ablation-and-then-transfer method has been limited using multiple wet chemical processes, which may cause graphene contamination and compromised pattern alignment.Park et al. demonstrated a procedure that involved laser ablation-based graphene patterning and subsequent transfer of the patterned graphene to an arbitrary substrate . As illuthin layer is peeled off, leaving the patterned graphene/PMMA thin layer on the substrate.To simplify the procedure, Yoo et al. have developed a single-step processing method that enables laser ablation-induced graphene patterning and simultaneous transferring to any substrate . In thisAlthough femtosecond laser ablation has been primarily demonstrated on graphene, it can potentially be employed to pattern many other 2D materials . To achiμm2, which enables localized ablation of 2D materials at the laser spot. The authors examined the feature size of patterned 2D materials under varying laser powers and exposure times  were fabricated, which enabled a photodetector with enhanced performance .  Figurencreases , 76. By hickness . In addihickness  for applhickness , topologhickness , and pulhickness .p-type or n-type doping with high site selectivity and high stability. This can be achieved by laser-induced doping of the 2D materials. As shown in 2 bilayer exhibits an increased source-drain current as a function of back-gate voltage by four orders [p-type characteristics of the WSe2. As another example, p-n junctions in graphene, which are fabricated via direct-laser-writing-induced spatially modulated doping in graphene covered by a pentacene thin film [p-type graphene to n-type at the laser-irradiated sites, which enabled the creation of p-n junctions on graphene with excellent photoresponses.Many 2D applications in electronic and optoelectronic devices such as photodetectors and FETs require the capability of tuning charge carrier type and density of the 2D materials to achieve r orders . The inchin film . Photoox2 to solve the issue of Schottky contact between the semiconducting 2D channel materials and the metallic electrodes. Briefly, a laser beam is imposed on the surface of mechanically exfoliated flake of multilayer MoTe2 in 2H phase to induce phase transition from semiconducting 2H phase to metallic 1T′ phase in the desired area where metal electrode will be located  shifting plates have been utilized to overcome this limitation; the resolution of optically patterned 2D materials is still of the same order , 92. SpaOther methods such as tip-based scanning probe lithography techniques , 99 and in situ measurement techniques based on optical imaging and spectroscopy can be used as a feedback mechanism to control the working laser parameters, as already used in colloidal manipulation and assembly works [So far, optical patterning technologies have mainly been focused on homogeneous 2D materials. Recent reports show the immense potential of van der Waals heterostructures of 2D materials in energy storage , memory ly works –112. Spaly works , 113 andly works .Optical patterning techniques have played a vital role in the tremendous growth of scientific research and applications based on 2D materials. Such techniques enable various physical and chemical changes induced precisely at the required sites of the 2D materials to create functional nanostructures and devices. With their advantages of tunable laser intensity, tunable laser wavelength, and site specificity, optical patterning holds the key to attain versatile high-throughput fabrication of 2D materials with submicron features. Further studies in this field would enable optimized, on-demand and universal fabrication of heterogenous 2D materials with feature sizes down to atomic scale, marking a significant advance in nanoscience and nanoscale industrial revolution.


Celiac disease (CD) is a common intestinal inflammatory disease involving both a genetic background and environmental triggers. The ingestion of gluten, a proteic component of several cereals, represents the main hexogen factor implied in CD onset that involves concomitant innate and adaptive immune responses to gluten. Immunogenicity of some gluten sequences are strongly enhanced as the consequence of the deamidation of specific glutamine residues by type 2 transglutaminase (TG2), a ubiquitous enzyme whose expression is up-regulated in the intestine of CD patients. A short gluten sequence resistant to intestinal proteases, the α-gliadin peptide 31-43, seems to modulate TG2 function in the gut; on the other hand, the enzyme can affect the biological activity of this peptide. In addition, an intense auto-immune response towards TG2 is a hallmark of CD. Auto-antibodies exert a range of biological effects on several cells, effects that in part overlap with those induced by peptide 31-43. In this review, we delineate a scenario in which TG2, anti-TG2 antibodies and peptide 31-43 closely relate to each other, thus synergistically participating in CD starting and progression. The enzyme type 2 transglutaminase (TG2) plays a key role in the pathogenesis of celiac disease (CD), primarily for its enzymatic activity that transforms common food proteins, i.e., gluten proteins contained in cereals, in unhealthy molecules for genetic predisposed individuals . HoweverCeliac disease (CD) is a complex inflammatory and auto-immune disorder triggered by the ingestion of gluten, a proteic component of several cereals, such as wheat, barley and rice . Gluten Gluten is a heterogeneous mixture of seed-storage proteins present in cereals such as wheat, barley, rye and oats . From a 2+-dependent manner, of an isopeptide bond between lateral chains of glutamine and lysine residues present into proteins. Due to this transamidating activity, TG2 acts as a stabilizing enzyme of protein networks, such as those formed by ECM proteins [KB activation; TG2 may also cross-link beclin-1, thus inhibiting the autophagic process [2+ ions. TG2 is inactive at the very low Ca2+ concentration in the cytosol, but is rapidly activated after perturbation of Ca2+ homeostasis. Actually, the modulation of TG2 activity is very complex and involves conformational changes between an open active conformation, bound to Ca2+, and a closed inactive conformation, bound to GTP, a negative regulator of the transamidating activity [The enzymatic deamidation, which is of crucial importance in enhancing gliadin immunogenicity, is only one of several catalytic activities displayed by TG2. This protein belongs to a wide family of cross-linking enzymes with members in all reigns of the living . Well-knproteins ,31. In t process . In the  process . When av process . In the  process . All theactivity ,36. Moreactivity ,38. TG2 activity , a proteactivity , and an activity . In addiactivity ,42.It has been reported that TG2 expression is increased in the inflamed intestinal mucosa of CD patients ,44,45. I2+ mobilization [An increasing interest on possible biological functions of auto-antibodies to TG2 into the organism has been registered in the last years. Since antibodies are not able to over cross the plasmatic membrane, they have to act on the cell surface, where they may interact with membrane-bound TG2 and eventually they can be internalized together with TG2, during the normal turnover of this protein. Studies on the ability of anti-TG2 antibodies to modulate TG2 transamidating activity have given quite contrasting results, thus it remains unclear whether auto-antibodies interfere in vivo with the repairing function of TG2 in ECM or may block those reactions necessary to the breakdown of tolerance to gluten  . In an elization ,57. In alization ,59. In alization . The genlization . It is alization . AuthorsAmong peptides obtained by the in vitro digestion in a multi-compartment system, P31-43 and the longer peptide 31-49 have been detected . Thus, iFrom biophysical studies investigating the secondary structure of P31-43 emerges that this peptide is able to self-organize in solution as a polyproline II conformation, in equilibrium with β-sheets-like structures ,66. AnotSeveral biological/toxic effects of P31-43 are very rapid and some of them may be induced sudden the peptide comes in contact with cell surface. What happens after this contact and how P31-43 enters cells are still debated issues. Experiments performed to elucidate these topics often compare the behavior of P31-43 and of peptide 57-68 (P57-68), the most immunogenic known peptide. Even if these two peptides are of similar length and composition  they are handled by cells in a very different way. In Caco-2 cells, P31-43 is internalized by endocytosis, mainly clathrin-dependent, as methyl-β-cyclodextrin reduces its entrance. P31-43 uptake is unaffected by filipin, indicating that lipid raft/caveolae-mediated endocytosis is not involved ,70. On tIn this scenario, how can anti-TG2 antibodies reduce P31-43 uptake by cells? Maybe antibodies regulate the endocytosis of TG2 itself and of other TG2 membrane partners; P31-43 could merely have undergone a reduced uptake as a consequence of its interaction with membranes near TG2. Another explanation could be that antibodies trigger a signaling that at the end is able to partially block the uptake of the peptide. Further investigations are needed to clarify these issues. In any case, by reducing the uptake of P31-43, anti-TG2 antibodies also attenuate P31-43-induced effects. This observation suggests that P31-43 needs to be internalized by cells to exert its effects and that a protective role towards negative effects induced by P31-43 could be attributed to the auto-immune response to TG2. However, as described in one of the next paragraph, in CD cells this protective role of auto-antibodies seems to fail.2+ ions from intracellular stores and to elicit an endoplasmic reticulum (ER)-stress response in Caco-2 cells [One of the first demonstrations that P31-43 is the main actor in inducing an innate immune response has been reported in the work of Maiuri et al. in 2003 . They fo-2 cells . Finally-2 cells .It is important to underline that several effects due to P31-43 treatment were observed in enterocytes of duodenal biopsies or other cells from CD subjects but not from control subjects ,81,82. FIt has assumed that TG2 participates to CD onset by enhancing gluten immunogenicity, through the deamidation of specific glutamine residues, and causes the auto-immune response toward itself and also towards other self proteins, through its transamidating activity. A less defined role of TG2 in CD has emerging in correlation with the interplay between TG2 and P31-43. In general, P31-43 seems to modulate TG2 activity and expression, even if with important differences in celiac and non-celiac cells; on the other hand, inhibitors of TG2 activity can attenuate some effects induced by P31-43; finally, in some cases, P31-43 has been described as a TG2 substrate.2+ mobilization from intracellular deposits [Regarding the influence of P31-43 on TG2, it has been demonstrated that in CD normalized duodenal biopsies (but not in non-celiac specimens) P31-43 causes an up-regulation of TG2 activity and expression . A possideposits . Prolongdeposits . A recendeposits . TG2 appdeposits . Moreovedeposits . These dNot only P31-43 upregulates TG2 in CD cells, but also TG2 itself seems to affect the biological activity of P31-43. In works investigating how TG2 modulates P31-43 effects , enzymatic inhibitors of TG2 have often been employed . Competitive aminic TG2 substrates  or non specific inhibitors that binds to the TG2 catalytic site, such as cystamine, have often been used ,91. In aIn CD duodenal biopsies, the use of the TG2 inhibitor R283 reduced the T-cells activation induced by the treatment with P31-43, followed by the sequential treatment with P57-68, whereas the clone CUB 7402 exerted a very low protective effect . Similar2+ ions, was able to activate cell surface TG2, probably present in a closed conformation. As a consequence, the hypothesis of a direct interaction between P31-43 and cell surface TG2 was proposed as a way to induce a conformational change able to activate the enzyme [In recent work, TG2 inhibitors have been used to evaluate whether agglutination of K562 cells induced by PT-gliadin was related to the activity of cell surface TG2 . Authorse enzyme . In the e enzyme , thus suA role for P31-43 and TG2 has also been proposed in the context of neurological manifestations of CD, in particular epilepsy . In miceFinally, in a very recent in vivo model, consisting of transgenic DQ8 mice over-expressing IL15 , an elegThus, the use of TG2 sensible and specific inhibitors represents the best tool to investigate TG2 role in mediating the effects of P31-43, or more in general of PT-gliadin, particularly if these compounds are able to inhibit specifically extracellular or intracellular TG2.2+ intracellular chelator BAPTA-AM [P31-43 appears as a good TG2 substrate in both in vitro and in situ transamidating reactions ,74 even BAPTA-AM . Given t2+ ions from the same deposits engaged by P31-43, i.e., ER and mitochondria, and consequently both antibodies and P31-43 activate the intracellular TG2 [The idea of a connection between anti-TG2 antibodies and P31-43 in CD has an old origin. In their work, Picarelli et al. (1999) demonstrated that the exposition to P31-43 was sufficient to induce an auto-immune response: in duodenal biopsies of patients on a gluten-free diet, challenged with P31-43, an accumulation of anti-endomysial antibodies (EMA) was detected, whereas no EMA were observed in cultures of control specimens . More reular TG2 ,89. Finaular TG2  and by Pular TG2 ,107. Affular TG2 ,63. Whenular TG2 , whereasular TG2 . Howeverular TG2 . For a dWhich are the biological consequences into the cell of the simultaneous presence of anti-TG2 antibodies and P31-43 have still been poorly investigated. When Caco-2 cells were treated with both antibodies to TG2 and P31-43, no additive effect was observed in cell proliferation . HoweverFinally, it has to be considered that anti-TG2 antibodies reach all tissues and organs by blood circulation, thus potentially participating in CD extra-intestinal manifestations ,110. IntIt has been well established that TG2 has two main roles in CD onset. First, TG2 enhances the adaptive immune response to gluten, through the reaction of deamidation of specific glutamine residues. Second, the transamidating activity of TG2 causes the generation of a strong auto-immune response to TG2 itself and to other self proteins. Other emerging roles could be related to the different subcellular distribution of TG2 in cells from CD or control subjects and to the influence that P31-43 could have on TG2 function and viceversa, in the context of CD. In addition, anti-TG2 auto-antibodies have the potential to influence the biology of CD cells by interacting with cell surface TG2. Antibodies seem to act on the same pathways engaged by P31-43, thus, antibodies and P31-43 could produce a synergic negative effect in celiac mucosa. In the future, investigations on CD-derived samples (such as biopsy cultures or isolated cells) with the concomitant presence of anti-TG2 antibodies and P31-43 could contribute to clarifying the role of the interplay between these three important “players”, i.e., TG2, anti-TG2 antibodies and P31-43, in CD trigger and progression.


Bacillus licheniformis. Firstly, our results implied that SAM exogenous addition benefited bacitracin production, which yield was increased by 12.13% under the condition of 40 mg/L SAM addition. Then, SAM synthetases and Methionine (Met) synthetases from B. licheniformis, Corynebacterium glutamicum, and Saccharomyces cerevisiae were screened and overexpressed to improve SAM accumulation, and the combination of SAM synthetase from S. cerevisiae and Met synthetase from B. licheniformis showed the best performance, and 70.12% increase of intracellular SAM concentration (31.54 mg/L) and 13.08% increase of bacitraicn yield (839.54 U/mL) were achieved in resultant strain DW2-KE. Furthermore, Met transporters MetN and MetP were, respectively, identified as Met exporter and importer, and bacitracin yield was further increased by 5.94% to 889.42 U/mL via deleting metN and overexpressing metP in DW2-KE, attaining strain DW2-KENP. Finally, SAM nucleosidase gene mtnN and SAM decarboxylase gene speD were deleted to block SAM degradation pathways, and bacitracin yield of resultant strain DW2-KENPND reached 957.53 U/mL, increased by 28.97% compared to DW2. Collectively, this study demonstrated that SAM supply served as the critical role in bacitracin synthesis, and a promising strain B. licheniformis DW2-KENPND was attained for industrial production of bacitracin.Bacitracin is a broad-spectrum veterinary antibiotic that widely used in the fields of veterinary drug and feed additive. S-Adenosylmethionine (SAM) is a critical factor involved in many biochemical reactions, especially antibiotic production. However, whether SAM affects bacitracin synthesis is still unknown. Here, we want to analyze the relationship between SAM supply and bacitracin synthesis, and then metabolic engineering of SAM synthetic pathway for bacitracin production in  B. licheniformis and Bacillus subtilis. Bacitracin biosynthetase gene cluster in B. licheniformis spans over 45 kd of DNA, which containing four genes, bacT, bacA, bacB and back. Gene bacT encodes thioesterase, and the relationship between bacitracin synthesis with bacT is unclear. BacA is responsible for activating and polymerizing five kinds of amino acids  at bacitracin tail, BacB and BacC are responsible for activating and polymerizing seven kinds of amino acids in heptapeptide loop -Ori was applied for gene deletion, promoter replacement and gene integration in B. licheniformis, pHY300PLK was applied for constructing gene expression vector. All primers used in this research were provided in The strains and plasmids used in this research were listed in mbinants , and Esc4)2SO4, 0.6% CaCO3, natural pH), and then cultivated at 37°C, 230 r/min for 48 h. In order to analyze the function roles of Met transporters MetN and MetP, ME medium  was applied (Luria-Bertani (LB) medium was served as the basic medium for strain cultivation, and corresponding antibiotics  were added when necessary. The seed culture was inoculated in a 250 mL flask containing 20 mL LB medium for 6 h, and transferred (1 mL) into bacitracin production medium -Ori at restriction sites SacI/XbaI, diagnostic PCR and DNA sequence confirmed that gene deletion vector was constructed successfully, named T2-metN. Then, T2-metN was transferred into B. licheniformis DW2 via electroporation, and the positive transformants were cultivated in LB medium with 20 mg/L kanamycin at 45°C, and sub-cultured for three generations. Then, transferred into LB medium and sub-cultured for six generations at 37°C. The gene metN deletion strain was attained via homologous double crossover, diagnostic PCR and DNA sequence confirmed that metN deletion strain was constructed successfully, named as DW2ΔmetN.The method for gene deletion in research , and MetSaccharomyces cerevisiae, gene SAM2 mediated by P43 promoter was integrated into the chromosome of B. licheniformis DW2, following to previously reported protocol , equipped with C18 column (ZORBAX SB-C18). Cell biomass was determined by dilution coating method . SAM conlization .P < 0.05 and P < 0.01, and a t-test was applied to compare the mean values using the software package Statistica 6.0, and bars represented the standard deviations, ∗P < 0.05; and ∗∗P < 0.01 indicated the significance levels between recombinant strains and control , and our results in bacT, bacA, bacB and bacC were measured under optimal condition, and our results implied that SAM addition improved transcription of bacitracin synthetase, increased by 1. 43-, 1. 34-, 1. 31-, and 1.29-fold, respectively . In addition, cell biomasses of these strains showed no significant differences, and bacitracin produced by per cell were 1.95∗10–8 U/CFU, 2.00∗10–8 U/CFU and 2.05 U∗10–8/CFU, 7.97%, 10.46 and 13.43% higher than that of DW2/pHY300 (1.81∗10–8 U/CFU), respectively. Furthermore, SAM2 expression cassette was inserted into chromosome of DW2, attaining SAM2 integrated overexpression strain DW2-K. Based on the results of ∗10–8 U/CFU, increased by 6.30% (1.84∗10–8 U/CFU). The concentration of intracellular SAM (36 h) in DW2-K was 26.32 mg/L, 41.96% higher than that of DW2 (18.54 mg/L). Additionally, transcriptional levels of genes bacT, bacA, bacB and bacC were also increased significantly , C. glutamicum (MetH), S. cerevisiae (Met6) were strengthened in DW2-K, attaining strains DW2-K/pHY-MetHBl, DW2-K/pHY-MetHCg and DW2-K/pHY-Met6, respectively. Our results implied that overexpression of MetH from B. licheniformis benefited bacitracin synthesis, however, strengthening Met synthases from C. glutamicum and S. cerevisiae has no positive effect on bacitracin production. After that, the promoter of metH of DW2-K was replaced by bacitracin synthetase cluster promoter PbacA, a proven strong promoter that has been confirmed in our previous research . The bacitracin produced by per cell was 2.23∗10–8 U/CFU, 21.42% higher than that of DW2 (1.84∗10–8U/CFU).Furthermore, the fermentation curves of Bacillus, and low production efficiency limits its promotion and application. SAM is an important cofactor in cell metabolism, which serves as the critical roles in syntheses of multiple antibiotics (Bacillus, especially for bacitracin, is still unknown. In this study, our results confirmed that strengthening intracellular SAM accumulation was beneficial for bacitracin production, and a metabolic engineered strain B. licheniformis DW2-KENPND with bacitracin yield of 957.53 U/mL was attained, via rewiring SAM synthetic and Met transportation pathways.Bacitracin is a widely used veterinary antibiotic that mainly produced by ibiotics . HoweverB. licheniformis (Streptomyces, moreover, our results implied that strengthening SAM supply increased the transcription of bacitracin synthetase gene cluster, and transcriptional levels of genes bacT, bacA, bacB, and bacC were enhanced all under the condition of SAM addition. In addition, the transcriptional level of regulator gene abrB was decreased by 32.54% (bacTABC (Acting as a critical methyl donor, SAM serves as the important role in the biosynthesis of multiple antibiotics , in addiniformis , differey 32.54% . Since r(bacTABC , our resB. licheniformis, C. glutamicum and S. cerevisiae were tested, and SAM2 from S. cerevisiae showed the best performances on SAM accumulation and bacitracin synthesis, consistent with the previous results of our group (E. coli, Streptomyces coelicolor etc., (B. licheniformis DW2, although it has not been identified or annotated. In addition, SAM decarboxylase gene speD was deleted to block synthetic pathway of byproduct, meantime, based on our results, two other harmful by-products, cadaverine and putrescine (Based on the previous researches, Met yield produced by microbial is generally at a high level , while Sur group . Along wur group , couplinur group , increasur group , etc. Inor etc., , also, ttrescine , were altrescine , and thitrescine . Furthertrescine , the ATPC. glutamicum (Bacillus has not been identified until now, although much work has been done on bacitracin microbial breeding and regulation (Engineering transporter has been proven as an efficiency tactic for enhancement production of metabolite. Overexpression of ABC transporter AvtAB benefited avermectin secretion, and further led to the 50% increase of avermectin production . Lys trautamicum . Howevergulation . Here, tgulation , overexpgulation , indicatgulation , LysE, Lgulation , TcyP angulation . The synB. licheniformis DW2-KENPND was attained via rewiring SAM synthetic and degradation, Met transportation pathways. Based on our results, SAM synthetase SAM2 from S. cerevisiae and Met synthetase MetH from B. licheniformis showed the best performance on SAM accumulation and bacitracin syntheses, and Met transporters MetN and MetP were identified as Met exporter and importer in B. licheniformis DW2, respectively. Finally, The concentration of intracellular SAM of DW2-KENPND was 47.53 U/mL, increased by 1.56-fold compared to DW2, and bacitracin yield reached 957.53 U/mL, increased by 28.97%. Taken together, this research demonstrated that SAM served as the critical role in bacitraicn synthesis, and a promising strain B. licheniformis DW2-KENPND was attained for industrial production of bacitracin.S-Adenosylmethionine served as the critical role in antibiotic production, however, whether it affects bacitracin synthesis is still unknown. Here, our results confirmed that exogenous SAM addition benefited bacitracin production, and a metabolic engineered strain The datasets generated for this study are available on request to the corresponding author.DC and SC designed the study. DC, BZ, JZ, and HX carried out the molecular biology studies and construction of engineering strains. DC, BZ, JZ, and PL carried out the fermentation studies. DC, ZW, JL, ZY, XM, and SC analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.JL was employed by Lifecome Biochemistry Co., Ltd., in China. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.


For some decades, the scientific community has been looking for alternatives to the use of fossil fuels that allow for the planet’s sustainable and environmentally-friendly development. To do this, attempts have been made to mimic some processes that occur in nature, among which the photosystem-II stands out, which allows water splitting operating with different steps to generate oxygen and hydrogen. This research presents promising results using synthetic catalysts, which try to simulate some natural processes, and which are based on Au@ZnO–graphene compounds. These catalysts were prepared by incorporating different amounts of gold nanoparticles  and graphene (1 wt.%) on the surface of synthesized zinc oxide nanowires (ZnO NWs), and zinc oxide nanoparticles (ZnO NPs), along with a commercial form  for comparison purposes. The highest amount of hydrogen (1127 μmol/hg) was reported by ZnO NWs with a gold and graphene loadings of 10 wt.% and 1 wt.%, respectively, under irradiation at 400 nm. Quantities of 759 μmol/hg and 709 μmol/hg were obtained with catalysts based on ZnO NPs and commercial ZnO, respectively. The photocatalytic activity of all composites increased with respect to the bare semiconductors, being 2.5 times higher in ZnO NWs, 8.8 times higher for ZnO NPs, and 7.5 times higher for commercial ZnO. The high photocatalytic activity of the catalysts is attributed, mainly, to the synergism between the different amount of gold and graphene incorporated, and the surface area of the composites. If you were asking to identify one of the many challenges that our world is facing right now, you would probably say climate change, energy production, or sustainability. Most of these problems are directly related to the continuous growth of the world population and the use of fossil fuels as our primary energy source .x) or sulfur oxides (SOx) are release to the atmosphere ..2− ions C groups ,42, and Under irradiation at 320 nm, the hydrogen production of ZnO NWs (365 μmol/hg) decreased when compared to that obtained at 280 nm, but then increased again (427 μmol/hg) at 400 nm. This was not expected either since the wide band gap energy of ZnO (3.37 eV for wurtzite) does not favor the production of hydrogen under visible light. Different studies ,43,44 ha2 [2–/SO32–), and the electrons are excited to the conduction band. Electrons transferred from the conduction band of the semiconductor are injected into the graphene because graphene has a slightly lower redox potential than the semiconductor conduction band [+ ions for H2 evolution [Both ZnO NPs and the commercial ZnO obtained similar results in all the wavelengths that were evaluated. At 500 nm, the hydrogen production of the unmodified catalysts was almost zero, with the exception of ZnO NWs that obtained a high value of 350 μmol/hg, showing high catalytic activity. Incorporation of gold and graphene greatly increased hydrogen production in both the UV and visible regions of all the composites. The presence of co-catalysts such as Au and graphene improve the charge separation and suppresses the recombination of excited photogenerated carriers, resulting in a better evolution of H2 . Differe2 ,46,47 ha2 . This ac2 . Further2 ,48. The ion band ,48. Grapvolution ,48.+ ions to form H2. Thus, the synergetic effect between both co-catalysts, plasmonic Au nanoparticles and graphene, can effectively suppress photogenerated charge recombination, enlarge the active adsorption sites and reaction space, and consequently enhance the photocatalytic activity for H2 evolution [2–graphene composites had significantly increased the visible light absorption and enhanced the photocatalytic H2 production activity compared to the Au@TiO2. Luo et al. [2-P25, the hydrogen production via water splitting increased nine times more than bare TiO2-P25.According to other authors ,49, somevolution ,49. In to et al.  found thIn this research, the highest hydrogen production of the Au@commercial ZnO–graphene catalysts was 709 μmol/hg and was obtained by 10%Au@ZnO commercial graphene under irradiation at 400 nm. This enhancement represents a difference of 615 μmol/hg when compared to the highest amount obtained by the unmodified commercial ZnO catalyst (94 μmol/hg), and the fact that the maximum production of the commercial catalyst was obtained at 400 nm (visible light) is an indication that the Au NPs are allowing the use of visible light ,30,37. OIn the case of Au@ZnO NPs–graphene catalysts, the highest hydrogen production measured was 759 μmol/hg, representing a difference of 673 μmol/hg when compared to the maximum hydrogen production of the unmodified ZnO NPs catalyst (86 μmol/hg), and was obtained with the 10%Au@ZnO NPs–graphene catalyst at 400 nm. The highest hydrogen production for 1%Au@ZnO NPs–graphene, 3%Au@ZnO NPs–graphene, and 5%Au@ZnO NPs–graphene catalysts was 537 μmol/hg, 622 μmol/hg, and 728 μmol/hg, respectively. These results confirm once again that the presence of Au NPs allows the use of visible light to produce hydrogen.Au@ZnO NWs–graphene catalysts showed the highest hydrogen production (1127 μmol/hg) with a gold loading of 10 wt.% at 400 nm, representing a difference of 685 μmol/hg when compared to the unmodified ZnO NWs catalyst (442 μmol/hg). The highest amount of hydrogen produced at 400 nm with the 1%Au@ZnO NWs–graphene, 3%Au@ZnO NWs–graphene, and 5%Au@ZnO NWs–graphene catalysts was 701 μmol/hg, 828 μmol/hg, and 944 μmol/hg, respectively.Under irradiation at 500 nm d, the ma2 g−1 for 10%Au@ commercial ZnO–graphene; 117 m2 g−1 for 10%Au@ZnO NPs–graphene; 247 m2 g−1 for 10%Au@ZnO NWs–graphene). Materials with high surface areas can be attained either by fabricating small particles or clusters where the surface-to-volume ratio of each particle is high, or by creating materials where the void surface area (pores) is high compared to the amount of bulk support material [In all cases, the highest amounts of hydrogen reported in this investigation were obtained with the catalysts with the highest surface area  leads to the generation of electrons in the conduction band, and electron holes in the valence band of the semiconductor [2 conduction band leading to the generation of holes in the Au NPs and electrons in the TiO2 conduction band [2 are injected into the reduced graphene in a graphene/TiO2 system because the graphene/graphene redox potential is slightly lower than the CB of TiO2 [2 likely transfer directly to the Au NPs deposited on the surface of the semiconductor by ohmic interconnection or to carbon atoms on the graphene sheets, and the electrons then react with the adsorbed H+ ions to form H2 [In the case of Au/graphene–TiOting see . One of ting see A, a direonductor . The eleonductor . When irion band ,52. Thenion band . The ele of TiO2 . In addi form H2 ,26. This form H2 ,25,26. W form H2 . The wat form H2 ,19,20 ha form H2 . Thus, t form H2 ,19.2 using catalysts based on ZnO and graphene–gold. The materials studied in this research will have to be contrasted by other researchers to establish a much deeper knowledge that allows us to know the complex mechanism of hydrogen production with ternary compounds based on ZnO.Graphene and different amounts of gold nanoparticles were incorporated on the surface of synthesized ZnO supports , and on the commercial form . These catalysts were fully characterized by different techniques, and their photocatalytic activity was determined by measuring the hydrogen produced by water splitting under UV–vis irradiation.The highest amount of the unmodified ZnO support was 442 μmol/hg and was obtained by the ZnO NWs catalyst under irradiation at 280 nm. This unexpectedly high hydrogen production may be attributed to the morphology (nanowires) and possible defects in the crystalline structure. The maximum hydrogen production for the ZnO NPs and commercial ZnO catalysts was 94 μmol/hg and 86 μmol/hg, respectively, at 280 nm.The maximum hydrogen production obtained with the commercial ZnO composites containing gold and graphene was 709 μmol/hg at 400 nm and was obtained with a gold loading of 10 wt.%. The enhancement in the hydrogen production was 7.5 times higher than that reported by the commercial ZnO.The higher hydrogen production for the Au@ZnO NPs–graphene catalysts was 759 μmol/hg at 400 nm and was obtained with 10%Au@ZnO NPs–graphene. The enhancement in the hydrogen production was 8.8 times higher than that reported by the ZnO NPs catalyst.In the case of the Au@ZnO NWs–graphene composites, the higher hydrogen production (1127 μmol/hg) was obtained with the 10%Au@ZnO NWs–graphene under irradiation at 400 nm.The catalysts did not show a reduction in the surface area nor in the hydrogen production with the increment in gold loadings and incorporation of graphene. These results suggest that the best graphene and gold loading for the Au@ZnO–graphene catalysts could be higher than 1 and 10 wt.%, respectively.