The permeability, selectivity and diffusivity of different gases as a function of graphene loading, from 0.3 to 15 wt %, was measured at 35 and 65 °C. Samples with small loadings of graphene show a higher permeability and He/CO2 selectivity than pure PPO, due to a favorable effect of the nanofillers on the polymer morphology. Higher amounts of graphene lower the permeability of the polymer, due to the prevailing effect of increased tortuosity of the gas molecules in the membrane. Graphene also allows dramatically reducing the increase of permeability with temperature, acting as a “stabilizer” for the polymer matrix. Such effect reduces the temperature-induced loss of size-selectivity for He/N2 and CO2/N2, and enhances the temperature-induced increase of selectivity for He/CO2. The study confirms that, as observed in the case of other graphene-based mixed matrix glassy membranes, the optimal concentration of graphene in the polymer is below 1 wt %. Below such threshold, the morphology of the nanoscopic filler added in solution affects positively the glassy chains packing, enhancing permeability and selectivity, and improving the selectivity of the membrane at increasing temperatures. These results suggest that small additions of graphene to polymers can enhance their permselectivity and stabilize their properties.We fabricated novel composite (mixed matrix) membranes based on a permeable glassy polymer, Poly (PPO), and variable loadings of few-layer graphene, to test their potential in gas separation and CO Most of those processes are carried out at high temperature, and a high-temperature purification process would allow exploiting the thermal level of the gas stream [Membrane separations are considered suitable, and environmentally friendly, technologies to capture COfrom H2) ,4,5,6. MIn membrane processes, the main way to optimize the separation is to use highly permeable and highly selective membrane materials. However, as pointed out in the literature, it is often not possible to simultaneously increase the permeability and the selectivity, which are governed by a tradeoff mechanism .2-selective behavior, when exposed to mixtures of H2 and CO2, i.e., it exhibits a size-sieving ability, because hydrogen has a smaller kinetic diameter than CO2 (0.29 vs. 0.33 nm) [2/CO2 selectivity value for PPO is 1.5 at room temperature, while an economically feasible membrane separation process would require higher values [2/CO2 membrane separation performances are known to be significantly increased by increasing temperature [Poly (PPO) is a glassy polymeric material suitable for membrane-based gas separation, due to a high permeability and moderate selectivity . In partA further strategy to enhance the selective behavior of the polymer can be based on the addition of nanosized fillers to the polymer matrix: in particular, nanofillers can modify the chain packing of the polymer, increasing its selectivity. It was reported by many authors that separation performance of polymeric materials can be improved by the addition of nanosized particles of different shapes, such as nanospheres ,14,15,16Graphene is a nanosized material that has been just recently applied to the field of gas separation membranes, as initially it was mostly considered for improving the barrier effect ,26,27,282 permeability of PIM-1 is 0.1 wt %, and molecular simulations indicate that the presence of graphene nanolayers modifies the polymer distribution [Some other works evaluated the combination of graphene, obtained by direct exfoliation of graphite in polymeric solution, to a polymer of intrinsic microporosity, PIM-1. The studies indicate that the optimal concentration of graphene to maximize the COribution ,33,34.2/He and CH4/He [2/He and CH4/N2, CH4/He. The addition of this filler mainly lowers the CO2 diffusivity, leaving the gas solubility unaltered, while the solubility and diffusivity are both enhanced by addition of GO . Multiple layer graphene  lowers the permeability of PTMSP to a significant extent (up to 30%), with a weight fraction of just 1 wt % [The addition of 1 wt % of monolayers of GO slightly enhances the gas permeability of poly(trimethyl silyl propyne) (PTMSP), and the selectivity for the couple COd CH4/He . The addt 1 wt % .2, CH4 and CO2 permeability. Graphene platelets, due to their high aspect ratio, act as physical barriers to the rearrangement of polymer chains, and the diffusion of free volume domains, which cause the ageing. Such explanation is supported by the fact that ageing is mostly reduced by the fillers that have the higher aspect ratio [Both GO and few-layer graphene allow to impair the aging process of PTMSP, as tracked with He, Nct ratio .In view of the previous findings, in this work, we studied whether the separation performance of PPO with respect to some gas mixtures, can be improved by addition of graphene nanoplatelets. Furthermore, in the aim of assessing the effect of temperature on permselectivity, we tested the membranes performance to temperatures as high as 65 °C.Tg (213 °C), that makes it suitable for use in high temperature separations. The melting point is 268 °C and the density at 25 °C is 1.06 g/cm3. The permeability to hydrogen is rather high, and ranges between 87 and 112 Barrer at 25 °C [2 for safety reasons: literature results indicate that the permeability of He is moderately lower than that of H2 in this polymer, its permeability ranging between 56 and 75 at 25 °C [2 leads to conservative estimates for the H2 permeability and H2/CO2 selectivity.The solid polymer PPO was purchased by Sigma Aldrich, and chloroform  was used as solvent. It is an amorphous material with a rather high at 25 °C ,37; suchat 25 °C . TherefoTwo commercial grades of graphene in powder, produced by Graphene XT, were used: Graphene XT6, with a lateral dimension of 5 m and a thickness between 6.0 and 8.0 nm, and Graphene XT7, with a nominal lateral size of 20 m and a thickness of 2 nm.The procedure to prepare membranes was optimized considering also a previous work .The polymer was dissolved in chloroform and then graphene powder was added. The resulting suspension was sonicated for at least 15 min, and stirred for one day. In the case of Graphene XT7, the sonication time was increased to 60 min to improve the disaggregation of nanoplatelets, as this materials has a higher aspect ratio than the other filler used. Once the dispersion was complete, the solution was poured on a Petri dish and placed in a clean hood, where the temperature was kept at 50 °C, to ensure fast evaporation of the solvent. The solid film was then removed from the Petri dish, and treated in an oven under vacuum for 1 day at 200 °C. Such treatment not only removes traces of solvent, but also stabilizes the properties of the glassy PPO, with respect to ageing, for a sufficiently long time. It must be noticed, however, that PPO membranes treated at such temperatures show a somewhat lower permeability than untreated ones. 2 , CO2  and He .The gases used were NdV is the calibrated downstream volume, R is the universal gas constant, T is the system temperature, A is the membrane area, l is the thickness of the sample and The permeability was measured with a variable pressure apparatus described in previous works , by appl2 and CO2.The apparatus is placed in a thermostatic chamber which can reach temperatures in the order of 80 °C. Tests were conducted at 35 °C; and for most samples we also carried out tests at 65 °C to investigate temperature effect on permeability of the different gases investigated, i.e., He, NThe permeability value is also affected by the uncertainty on the membrane thickness, which depends on the sample considered. The error on ideal selectivity of a single sample however is negligible, because such value is unaffected by downstream volume, permeation area and membrane thickness values as it is clear from Equation (1).Ideal selectivity for the different gas couple of interest is calculated using Equation (2), which is valid in the case of negligible downstream pressure, as in the tests considered here:ectivity .tL, that is related to the gas diffusivity in the membrane by the following relation [In many cases, we were able also to determine the characteristic time of permeation, i.e., the time required to reach a stable flux across the membrane, the so-called time-lag, relation :(3)D=16iS, a straight forward relation holds true between the gas permeability, and its diffusivity and solubility coefficient in the polymer:The time lag is actually estimated as the intercept, on the time axis, of the asymptotic straight line representing the downstream gas pressure versus time. It can also be noticed that, due to the phase equilibrium between the gas and the polymer phase, according to which the gas is absorbed onto the polymer surface proportionally to its solubility coefficient According to which, the selectivity can be decomposed into a diffusivity-based, and a solubility-based contribution:2 is concerned. For this reason, not all polymeric membranes have a H2/CO2 selectivity higher than 1, but some of them, due to a very high CO2 solubility, exhibit a CO2-selective behavior [Usually, for a same gas couple, the two contributions are, respectively, lower than unity and higher than unity. In general, the diffusivity-selectivity prevails, so that smaller gases are generally more permeable than larger ones in most polymeric membranes. In some cases, the solubility prevails, as happens for instance when CObehavior .PE and DE are the permeation and diffusion activation energies, respectively, which are usually positive for gases in glassy polymers, and ΔSH is the sorption enthalpy, which is usually negative. Accordingly, the permeability and diffusivity increase with temperature, while the solubility decreases with it.The temperature dependence of permeability, diffusivity and solubility is governed by Arrhenius-like laws, as follows:Due to Equations (4)–(8), the permeation activation energy has a diffusivity and solubility contribution as follows:2/CO2 and He/CO2 in PPO form an exception, because H2 and He are more permeable at room temperature than CO2, but also have a higher activation energy of permeation. The activation energy of CO2 is low, due to a high contribution of sorption, an exothermic process, on the permeation. Therefore, for H2-selective membranes, as the ones of the present paper, increasing the temperature enhances the H2 permeability and H2/CO2 selectivity.For the selectivity dependence on temperature is concerned, one can combine Equations (2) and (6) to obtain:First, neat graphene in powder was characterized. The Graphene XT7 was chosen for this analysis and two images are reported in Pictures of the cross section of PPO + graphene membranes are reported in In 2 seems unaffected by the presence of graphene up to a loading of 1 wt %. The permeability of N2, on the other hand, follows a same qualitative behavior as that of helium, increasing by about 20% for graphene loadings lower than 1 wt %, and decreasing for higher values. The permeability enhancement observed could be due to various factors, such as the formation of additional free volume at the interphase between polymer and filler, and the permeation of the gas through graphene layers.It can be seen that, while the addition of small amounts (below 1 wt %) of both types of graphene enhance the He permeability, by about 10%, increasing the filler loading to higher amounts (5 and 15 wt %) reduces the permeability, for all gases considered. This is because the wide graphene platelets, in this range of concentration, significantly increase the tortuosity of gases diffusive path in the membrane. The permeability of COAt 65 °C, no increase of permeability could be detected after addition of graphene: we believe that, at this temperature, the transition between the regime where the free volume increase prevails and the one in which the tortuosity increase dominates occurs at very small graphene loadings, or the first regime does not occur at all. At higher temperatures, indeed, the mobility of both diffusing gases and polymeric chains is higher, and the diffusion and permeation process relies less on the presence of intrinsic free volume in the solid matrix.2, He/N2 and CO2/N2. The first separation (He vs. CO2) is favored by high temperatures, i.e., both permeability and selectivity increase with increasing temperature, due to higher activation energy of permeation for He than for CO2. The other separations considered, namely He/N2 and CO2/N2 are, in terms of selectivity, not favored by temperature, as the selectivity of polymers for such couples decreases with temperature, due to the unfavorable difference between the permeation activation energies of the two gases (see Equation (10)).The ideal selectivity values calculated with Equation (2) at 35 °C are reported in 2 selectivity of the polymer at 35 °C, of about 10%. For the He/N2 separation, the selectivity values decrease monotonically with increasing graphene content at 35 °C, going from 26.0 to 21.1 at the largest loading. For the CO2/N2 mixture, the selectivity also decreases monotonically with increasing graphene loading, from 20.3 to 15.0. Such trends are due to the gas-dependent variations of permeability induced by addition of graphene. The gas that experiences the highest increase of permeability after addition of small loadings of graphene is the largest one, namely nitrogen. For samples containing larger amounts of graphene, the smallest reduction of permeability recorded is again that of nitrogen. Thus, both He/N2 and CO2/N2 selectivity decrease. Such trend is attributable mostly to the kinetic term of permeability, the diffusivity, which is strongly affected by molecule size, as will be seen in the following. When the He/CO2 selectivity is considered, on the other hand, the size of the molecule plays a less important role, as CO2 permeability is also strongly affected by solubility, which depends on the molecule condensability rather than on its size. Therefore, for this couple of gases, the size-sieving capacity of the membrane slightly increases with the addition of graphene, rather than decreasing. When the temperature is increased to 65 °C, the situation slightly changes, in particular for the case of the He/N2 mixture, for which graphene addition positively affects the selectivity. The incorporation of filler is also beneficial for the He/CO2 separation, similar to the lower temperature case. The CO2/N2 selectivity, on the other hand, decreases with graphene addition also at 65 °C. The different trends of selectivity at different temperatures are due to the effect that graphene has on the thermal dependence of the gas permeability in the polymer, which we will discuss below.The addition of graphene in all proportions produces an increase of He/COThe diffusivity is a kinetic quantity that contributes to the permeability, according to Equation (4). In particular, diffusivity is strongly affected by variations of the polymer free volume, and by the chain packing of the membrane. Therefore, it is expected to be the quantity that is most strongly affected by the introduction of filler with a peculiar morphology, such as the high aspect ratio graphene platelets with one nanometric dimension. Furthermore, in size-selective polymers such as the one that we are considering, diffusivity dictates the selective behavior, rather than solubility, as happens for instance in the case of higher free volume glassy polymers such as PTMSP and PIM-1. In D/D0 close to 1 for the larger molecule, namely N2, and much smaller than unity for the smallest molecule, Helium.It is often seen, in mixed matrix membranes comprising nanometric fillers, that the variations of permeability induced by filler are gas size-dependent ,16. UsuaP/P0, to the ones observed for diffusivity, D/D0, in 2 and, less markedly, for N2, the permeability trend is strongly associated to the diffusivity one, confirming that diffusion plays a strong role in such membranes, for He, the behavior is different. For this gas, the permeability of composite membranes is reduced less than the diffusivity by addition of graphene. Although the error associated to the evaluation of the diffusivity from the time lag measurement is larger in the case of Helium, which can cause some significant random scattering, such trend is systematic, i.e., all the data fall above the bisector. If the deviations were due merely to scattering associated to experimental error, they should have lied both above and below the bisector. According to the solution–diffusion model expressed by Equation (4), one should have:P/P0 = D/D0, as it approximately happens in the case of N2 and CO2, one concludes that S/S0 = 1, i.e., that the solubility of such gases is not affected by the presence of graphene. On the other hand, the same equation applied to the case of helium indicates that S/S0 > 1, i.e., that graphene enhances the solubility of helium in the membrane. Although the solubility of helium in solids is generally small in absolute value, we are considering relative variations here, which can be significant. Furthermore, the helium molecule, due to its small size, could be adsorbed onto the graphene layers interface but also between the layers of graphene platelets. This solubility behavior counterbalances the strong reduction of diffusivity observed in the case of helium.To effectively correlate the observed variations in permeability to variations of diffusivity, we plotted a parity curve, reporting the observed variations in permeability after addition of graphene, It is very interesting to analyze the effect of temperature on those composite membranes. Normally, the transport properties of polymers, especially the permeability, are strongly dependent on temperature, and, to our knowledge, this is the first analysis of the temperature effect on transport properties of polymer/graphene composites for separation.2. These data appear for the first time and indicate that graphene nanoplatelets hinder the polymer mobility and flexibility. Indeed, it is known that the strong increase of gas diffusivity and permeability observed in polymers at increasing temperature is mostly due to the thermally-enhanced polymer chain flexibility and mobility, which makes the diffusive jumps of gas molecules more frequent. When increasing amounts of graphene are added, up to 5 wt %, to PPO, the thermal activation of diffusivity and permeability are strongly inhibited. In particular, adding 5 wt % of graphene to PPO reduces the relative increase of permeability (corresponding to a 30 °C increase) from 0.7 to 0.2 for N2, and from 0.47 to 0.36 for He. The relative dependence of CO2 permeability in pure PPO is much weaker, for the reasons discussed above, and numerically is expressed by a value of +0.13, which becomes −0.17 when the sample contains 5 wt % graphene. At graphene loadings higher than 5 wt %, the effect discussed above is partially lost, possibly because the graphene nanoplatelets start to form aggregates and lose part of their ability to immobilize the polymer.2 selectivity, favored by temperature, graphene further enhances this trend. For CO2/N2 separations, unfavored by temperature, the negative effect of a temperature increase seems mitigated by graphene addition. For He/N2 the selectivity starts to increase, rather than decrease, with temperature, after graphene addition. Such trends are a natural consequence of the permeability trend previously discussed, and indicate that graphene can have a marked, and beneficial, effect on the polymer mobility, which reduces the negative effects of temperature on selectivity. Indeed, at higher temperatures, the polymer becomes more flexible and loses part of its size selectivity. The addition of graphene in small amounts seems to mitigate such trend, due to the peculiar, thin and long, shape of graphene platelets which hinder polymer mobility and the loss of discriminating ability. Such effect is totally consistent with what observed when adding graphene to other glassy polymers prone to ageing: it was shown that graphene reduces ageing via the same mechanism, i.e., by acting as a physical constraint, or stabilizer, of the polymer matrix [In r matrix .2 separation is concerned, are CO2-selective, rather than He-selective: this is due to their high free volume, and to a strong impact of the solubility on the separation. In 2, in the PPO of this work, and in PIM-1. We can notice that, in the case of helium, no significant differences between the two polymers are observed.The permeability variations observed with graphene have comparable order of magnitude as the ones obtained by adding 1 wt % of graphene into other glassy polymers, namely PTMSP  and PIM-2 (and similar results are noticed for N2), that is a slightly larger molecule, the situation changes strongly: while in PPO the CO2 permeability is not much affected by graphene, in the loading range inspected, marked increases of permeability are observed at very low loadings (0.1 wt %) in PIM-1. One can say that such difference can be due to the, probably, smaller aspect ratio of the graphene used in the paper by Althumayri et al., [2 selectivity higher than 1, while PIM-1 has a He/CO2 selectivity much smaller than unity.When it comes to CO et al., . FurtherThe comparison with other polymers case indicates that, as far as graphene addition to gas separation polymers is concerned, it seems that is advisable to work in the low loading range to retain the beneficial effects. The quantitative enhancement obtained are still limited, but there is large room for improvement by working on the morphological aspects, i.e., the aspect ratio of graphene, which could be optimized by varying the preparation method. Furthermore, the effect of a surface chemistry modification of graphene platelets on the final composite properties has not been studied yet: chemistry affects the solubility contribution to permeability, but also the adhesion and morphology of the composite material. Both aspects will be investigated in future works.2 and CO2 at 35 and 65 °C.We fabricated mixed matrix membranes based on PPO and increasing amounts of few layer graphene, from 0.3 to 15 wt %, and tested them for the permeability of He, N2 and He/N2 is also positively affected by addition of graphene, and the effect is observed at both 35 and 65 °C. Furthermore, the graphene addition allows adjusting the thermal dependence of permeability and selectivity, generally improving it, by acting as a physical constraint to the relaxation of polymer chains which compromises size selectivity at high temperatures.In general, the best effect of graphene addition is observed when the loadings are small, below 1%, and this was attributed to the fact that, at high filler loading, the effect of increasing the tortuosity prevails on the effect of enhancing the polymer chain distribution. Such aspect is in agreement with previous findings on other glassy polymer filled with graphene. The selectivity for He/COThe permeability trend follows closely the diffusivity one, confirming the validity of the solution-diffusion mechanism, and that solubility of gases is less strongly affected than diffusivity by graphene addition, for all gases except helium, which might be adsorbed onto graphene insertions.These preliminary results suggest that small additions of graphene to PPO can enhance the permselectivity, as it does in other glassy polymers. The effect could be quantitatively improved by optimizing the aspect ratio of particles, and testing chemical modification of graphene.


Atomic‐scale heterojunction‐type photocatalysts are regarded as a promising and effective strategy to overcome the drawbacks of traditional photocatalysts for higher photoenergy conversion efficiencies. Herein, an atomic‐scale heterojunction composed of a boron nitride monolayer and graphene (h‐BN‐C/G) is constructed to significantly shorten the charge transfer path to promote the activation of molecular oxygen for artificial photosynthesis (exemplified with oxidative coupling of amines to imines). As the thinnest heterojunction, h‐BN‐C/G gives the highest conversion, which is eightfold higher than that of the mechanical mixture of graphene and boron nitride monolayers. h‐BN‐C/G exhibits a high turnover frequency value (4.0 mmol benzylamine g Nevertheless, most metal‐free photocatalysts are still suffering from the poor mobility, as the intrinsic property of semiconductor‐based photocatalysts even engineered into nanostructures, and thus low activity for specific photocatalytic reactions, especially in the realm of artificial photosynthesis. As the thinnest heterojunction, the dyadic bilayers of graphene‐like 2D metal‐free materials can significantly shorten the charge transfer path to promote its activity for artificial photosynthesis, even though the synthesis of atomic‐scale heterojunction‐based photocatalyst still remain a great challenge for material scientists and chemists.Typical strategies to promote the activity of these metal‐free photocatalysts are mainly focused on elevating the crystallinity,Thus, we turned toward the atomic‐scale heterojunction based on graphene and hexagonal boron nitride (h‐BN) monolayers for the construction of photocatalysts for highly efficient artificial photosynthesis. We here establish that highly selective and oxidative coupling reactions of amines, as a model reaction, using molecular oxygen as the green oxidant are efficiently catalyzed by the metal‐free photocatalyst of the atomic‐scale Mott–Schottky heterojunctions of boron nitride and graphene (h‐BN‐C/G). Albeit h‐BN with a wide bandgap is nearly inactive for visible‐light photocatalytic reactions, a little amount of carbon dopants introduced into the framework of h‐BN can reduce its bandgap largely and make it responsive to visible light. More importantly, the ultrathin structure of h‐BN‐C/G can principally enlarge the active surface area for possible catalytic reactions and also make the nanoheterojunction transparent for maximizing the utilization ratio of the light irradiation in a heterogeneous catalytic system.Figure3N4) acts as the lamellar templates for further growth of h‐BN and graphene inside their interlayers spaces. A high temperature (>750 °C) can lead to the thorough removal of g‐C3N4, releasing the confined BN and Gr layers simultaneously. The synthetic process was depicted in Figure x heterojunction composites were obtained by changing the weight percentage of glucose in boric acid  with fixed amounts of urea . The X‐ray photoemission spectroscopy (XPS) analysis results  demonstrates the chemical structure of N atoms mainly in the form of “pyridinic” N atoms and N—B bonds. The Fourier transform infrared spectroscopy (FTIR)  with slightly more B atoms. Typical FTIR signal of cubic carbon nitride centered around 1200 cm−1 was not observed for all h‐BN‐C/G‐x samples. Indeed, the introduction of a slight amount of C dopants into the lattice of the h‐BN was well reflected by the obvious redshift in the optical absorption edge of h‐BN‐C/G‐1.6 from 245 nm for pristine BN as revealed by the UV–visible absorption spectra . This observation directly demonstrated the formation of graphite domains in the samples and thus a possible Mott–Schottky heterojunction structure of BN (semiconductor) and graphene .Large‐area scanning electron microscopy (SEM)  and transmission electron microscopy (TEM)  and mesoporous carbon nitride (mpg‐C3N4). h‐BN‐C/G‐x materials and control samples showed excellent selectivity to the imines , hydroxyl radical (•OH), hydrogen peroxide (H2O2), and singlet oxygen (1O2), respectively. The photocatalytic oxidation of benzylamine is obviously suppressed by the catalase, while negligible effects are observed when using other scavengers.A further set of control reactions were tested to demonstrate the real role of h‐BN‐C/G‐15 as a photocatalyst in triggering the reaction. The high chemical stability of the h‐BN‐C/G‐15 catalyst was well reflected by the excellent reusability under standard conditions with nearly the same conversions in the following four runs  and also unchanged structure . Also, the final turnover number of the h‐BN‐C/G‐15 catalyst was estimated to be 1.82, calculated on the basis of conversions of the five successive recycling reactions, rather speaking for the role of the h‐BN‐C/G‐15 sample as a heterogeneous catalyst here. The dependence of conversions of benzylamine on the wavelength of light source matched well with the optical absorption of the h‐BN‐C/G‐15 material and thus demonstrated a photocatalytic process depending on its light absorption Figure3a. A tx dyad with graphene as metal  counterpart and boron nitride as semiconductor, optimizing the weight ratio of graphene and h‐BN was thus a direct way to modulate the interfacial Shottky barrier and thus the valance band position of the semiconductor part in the graphene/h‐BN dyad. The fixed photoluminescence peaks  were finally observed in the Raman spectrum of the h‐BN‐C/G‐40 sample after the fluorescence was completely quenched by the graphene components, again demonstrating the rectify contact between the h‐BN‐C and graphene components. Most importantly, the valance and conduction band position  of the boron nitride part of h‐BN‐C/G‐15 sample were lowered obviously as compared with those of the h‐BN‐C/G‐1.6 sample presumably due to the band bending at the interface of Mott–Schottky heterojunction. This well explains the much higher catalytic activity of the h‐BN‐C/G‐15 catalyst with optimized weight ratio of graphene and h‐BN, as directly indicated by its remarkably high yield of the target product (95%), even though types of functional groups of all h‐BN‐C/G‐x catalysts are similar , the gradual decrease in the Brunauer–Emmett–Teller (BET) surface areas of BN, h‐BN‐C/G‐1.6, h‐BN‐C/G‐15, and h‐BN‐C/G‐20 samples was mainly induced by the formation of multilayer heterojunctions.The preadsorption and photocatalytic activation of benzylamine and oxygen molecules on the surface of graphene/h‐BN dyad were mainly induced by the electron relocalization and electron exchange under light irradiation, respectively, between the reactants and photocatalysts, largely depending on the band structure of the nanoheterojunctions. Considering the heterojunction structure of the h‐BN‐C/G‐ks Figure4a of t−1 h−1) of the h‐BN‐C/G‐15 photocatalyst in this work  and electron‐donating  groups. Heterocyclic ring amines with sulfur or nitrogen heteroatom were also converted into the corresponding imines with high selectivity. The excellent and functional‐group tolerance of the Mott–Schottky photocatalyst was again due to its intrinsic activation mechanism by enhancing the electron relocalization of the aromatic rings via the charge‐transfer interaction with conjugated framework of graphene or boron nitride. Note that metal‐based catalysts were usually selective to absorb and activate specific functional groups to accelerate such an oxidative coupling process but were easily poisoned by S or N‐contained functional groups.The synergetic effect of the Mott–Schottky photocatalyst on the basis of graphene and h‐BN dyad is general to promote the photocatalytic activation of oxygen molecules and various substituted benzylamines for possible oxidative coupling reactions under visible light irradiation. As shown in Table S5 (Supporting Information), h‐BN‐C/G‐15 photocatalyst gave similar conversions of In summary, we introduce the construction of Mott–Schottky photocatalysts on the basis of atomic‐scale heterojunctions of boron nitride and graphene (h‐BN‐C/G) for highly efficient artificial photosynthesis. Such an atomic‐scale Mott–Schottky photocatalyst was prepared from the direct copolymerization of glucose, boric acid, and urea with controllable contents of graphene and boron nitride layers. The interfacial Schottky barrier of the ultrathin nanoheterojunction can largely promote the separation of the photogenerated charges carriers for direct activation of the substrate and oxygen molecules. Long‐distance charge transfer path, which could result in obvious combination of separated electrons, was not involved in this atomic‐scale dyadic system. The lifetime of the photogenerated electrons in the h‐BN‐C/G Mott Schottky photocatalysts was thus largely lengthened. All these merits enhanced the photocatalytic activation of oxygen gas for oxidative coupling of amines into imines over the h‐BN‐C/G dyads with high selectivity and reusability under mild conditions.Materials: All chemical reagents used in the present experiments were of analytical grade. All the reagents  were purchased from Aladdin Industrial Corporation without purification.Preparation of h‐BN‐C/G and Control Samples: Anhydrous urea and boric acid, with a mass ratio remaining at 40:1, were roughly blended with different amounts of glucose by an agate mortar. After grinding, the mixed precursor was transferred into a covered corundum crucible and heated in a muffle furnace at 1000 °C for 7 h at a heating rate of 2.4 °C min−1 under the protection of nitrogen flow. After cooling naturally to room temperature, the as‐obtained samples were directly used for further examination and characterizations. The control sample BN was synthesized without the addition of glucose.Oxidative Coupling of Amines into Imines: 10 mg of the catalysts (h‐BN‐C/G‐15), 5 mL of acetonitrile and benzylamine (0.1 mmol) were added into a 10 mL round‐bottom flask. After the flask was purged with oxygen gas for three times, the mixture was stirred under 1 bar of oxygen at room temperature for 6 h under the visible light irradiation. A 150 W Xe lamp with a water filter was used as the light source for catalytic reactions. The wavelength of the incident light was controlled by using a cutoff filter. The temperature of the reactant solution was maintained at 288 ± 5 K by a water bath during the reaction. The liquid sample for further examination (GC with a flame ionization detector (FID) and/or for GC‐MS analysis) was separated by suction filtration after the reaction.Recycling Reaction of Oxidative Coupling of Amines into Imines: The catalyst was collected by suction filtration and washed with acetonitrile after every reaction was finished. The obtained catalyst was reused for the next catalytic reaction.Confirmation of Possible Active Oxygen Species: Different amounts of scavengers  were added to identify the active oxygen species on the basis of the standard conditions of oxidative coupling of amines into imines.Characterization: Powder X‐ray diffraction patterns were recorded on a Bruker D8 Advance X‐ray diffractometer with Cu‐Kα radiation  operating at an acceleration voltage of 200 kV. The XPS measurements were conducted on a Kratos Axis Ultra DLD spectrometer using a monochromated Al Kα radiation. Nitrogen sorption experiments (BET) were operated with a Quadrasorb at 77 K, and data analysis was performed with Quantachrome software. Samples were degassed at 150 °C for 16 h before measurements. The AFM measurements were performed on a Multimode NanoscopeIIIa. Raman spectra were acquired using an in Via‐reflexmicro‐Raman spectrometer  with a 532 nm wavelength incident laser. UV–vis absorption spectra were measured on a Hitachi U‐4100 instrument. The GC analysis was performed on Shimadzu GC‐2014gas chromatograph. For TPV measurement, the sample chamber consisted of a glass substrate covered with FTO as top electrode, the same as bottom electrode, and a 10 µm thick mica spacer as electron isolator. The samples were excited with a laser radiation pulse  from a third harmonic Nd:YAG laser . Intensity of the pulse was regulated with a neutral gray filter and determined by an EM500 single‐channel joule meter . The illuminated area was 0.1 cm2. The TPV signals were registered by a 500 MHz digital phosphor oscilloscope  with a preamplifier.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


Introduction: Hepatocellular carcinoma (HCC) typically develops in cirrhotic livers, with increased programed death ligand 1 (PD-L1) and transforming growth factor beta (TGF-β) activity implicated in immunosuppression.Methods: In an observational study of HCC liver samples, we determined the incidence of PD-L1 and immune cell (IC) infiltrates, and signs of TGF-β activity. HCCs were characterized by the incidence and distribution of PD-L1+ cells, and CD8+, CD68+, and FoxP3+ infiltrating ICs in HCC and surrounding liver. Gene expression signatures (GESs) associated with TGF-β activity and ICs were evaluated by RNAseq.Results: In non-neoplastic cirrhotic and non-cirrhotic liver, PD-L1 occurred on sinusoidal lining cells (mostly Kupffer cells), endothelial cells and ICs. In HCC, PD-L1+ tumor cells were rare. Most PD-L1+ cells were identified as ICs. CD8+, CD68+, and FoxP3+ ICs were associated with HCC, particularly in the invasive margin. CD8+ cell incidence correlated with PD-L1+ cells, consistent with PD-L1 being upregulated in response to pre-existing cytotoxic T-lymphocyte activity. TGFB1 mRNA levels and TGF-β activation GES correlated with the strength of the tumor-associated macrophage GES.Conclusion: Inhibition of PD-L1+ ICs and TGF-β activity and their respective immunomodulatory pathways may contribute to antitumor effects in HCC. Cancers develop through the acquisition of genetic and epigenetic changes that incidentally lead to the expression of novel antigens. These neoantigens are recognized in the course of immunosurveillance, enabling the elimination of many nascent tumors . To deveCompared with most other cancer types, hepatocellular carcinomas (HCCs) develop in an environment that is rather peculiar. The liver environment is intrinsically immunosuppressive, an adaptation that prevents the development of inflammatory liver damage secondary to routine exposure to foreign antigens derived from the gut . In addi+ T cells, regulatory CD4+ T cells (Tregs), natural killer (NK) cells, macrophages, dendritic cells (DCs), and myeloid-derived suppressor cell (Important molecular components of the immunosuppressive network implicated in HCC include the checkpoint inhibitor programed death ligand 1 (PD-L1) and the immunosuppressive cytokine transforming growth factor beta (TGF-β) –9. Togetsor cell , 10.+ T cells and some other ICs, such as macrophages. PD-L1 binding to PD-1 inhibits cytotoxic T cell activity and promotes other immuno-inhibitory effects , and double labeling was performed to characterize PD-L1-expressing cell types. Tumor-infiltrating ICs were evaluated by quantitative IHC, while TGF-β activity was assessed from gene expression signatures (GESs) determined from RNAseq data.To gain further insight into the potential immunosuppressive mechanisms present in HCC and the liver, we evaluated PD-L1, TGF-β, and infiltrating ICs in an observational study of a series of HCCs. The prevalence and distribution of PD-L1The study set consisted of 68 formalin-fixed, paraffin-embedded (FFPE) HCC resection specimens, from 68 patients, procured from Indivumed GmbH  and Agilent . All patients who provided samples to Indivumed and Dako/Agilent gave written informed consent in accordance with the Declaration of Helsinki.+ (Dako) secondary reagents were used. For double labeling, the LabVision™ Multivision Polymer Detection System (ThermoScientific) was used according to the manufacturer's directions.To determine PD-L1 expression in HCC resection samples, serial 4–5 μm sections were stained with hematoxylin and eosin for histopathological assessment and were used for IHC. The diagnosis of HCC and the histologic subtype were confirmed independently by two board-certified pathologists (LMT and CI). Histologic grading and classification of HCCs were performed according to the World Health Organization (WHO) guidelines and the Edmondson & Steiner grading system . IHC wasPD-L1 staining of non-neoplastic liver tissue was assessed qualitatively to evaluate PD-L1 expression in adjacent non-HCC parenchyma. PD-L1 staining of tumor cells was assessed qualitatively and semi-quantitatively by two board-certified pathologists (LMT and CI). Discrepant scores were discussed during joint examination and a consensus score agreed. The proportion of viable tumor cells with membrane staining of any intensity was determined by manual evaluation of the entire slide. The case was scored positive if ≥1% of the tumor cells were stained. The proportion of tumor area and contiguous peritumoral stroma occupied by IC with any degree of PD-L1 staining was also determined by semi-quantitative scoring.+ (T cells), CD68+ (macrophages), and FoxP3+ (regulatory T cells) ICs were assessed quantitatively in the following tumor regions identified by two board-certified pathologists : the whole tumor region (ALL), the area occupied by tumor; CT, the center of the tumor; and IM, the invasive margin . The frequency (% IHC+) of CD8+, CD68+, and FoxP3+ cells in each region was determined at a contract research organization  using a computerized image analysis system. The incidence of ICs was defined as low (≤ median) or high (>median).To evaluate immune cell infiltration in HCC, the numbers of CD8GESs associated with TGF-β activity and ICs were evaluated by RNAseq. RNAseq was conducted at a CRO  on 50 samples, of which two failed quality control and were removed, leaving 48 samples in the final analysis. For each sample, the tumor was annotated by a board-certified pathologist (CI) and RNA was extracted from three whole-slide scrapes of 4–5 μm-thick sections with a tumor content >50%, using Recoverall Total Nucleic Acid Isolation Kit for FFPE (ThermoFisher Scientific). 200 ng of total RNA, quantified using RiboGreen® RNA reagent (Life Technologies), was depleted of ribosomal RNA using the Ribo-Zero Gold rRNA Removal Kit (Illumina). Strand-specific libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit (NEB) and sequenced on an HiSeq2500 (Illumina) using 2 × 50 base-pair paired-end sequencing. Approximately 50 million reads per sample were obtained.Sequencing reads were aligned against the hg19 reference genome using Bowtie2 version 2.2.3 . Gene ex+ T cells . Double labeling of PD-L1 and CD68 showed that PD-L1+ cells along the sinus were mainly Kupffer cells . The incidence of ICs positive for CD8, CD68, and FoxP3 was highly variable between different cases . The area occupied by PD-L1-positive ICs was highly variable (median 2%), and in most (41/43) cases it was <25%, indicating that only a small number of HCCs have a high content of PD-L1+ ICs (data not shown).Cytoplasmic and/or membrane PD-L1 staining in tumor cells and/or ICs was present within HCC in all 68 cases . Levels une cell . Indeed,nt cases . ICs posere seen , inset. + cells correlated with CD8A and CD8B mRNA levels .TGF-β gene expression and activity were evaluated in HCC samples. TGF-β activity via TGF-β mRNA levels of TGF-β genes in 48 HCC samples were determined from RNAseq data. All isoforms were detectable, with abundant . Moleculabundant  showed tabundant . The TGFabundant . The strabundant . The TGFabundant . NotablyCD274) mRNA levels correlated with the incidence of CD8+ cells across the HCC samples  in HCC tissue samples. Importantly, TGF-β expression and activation shows a strong correlation with the strength of TAM activity GES. In addition, PD-L1 expression is largely restricted to IC. Thus, PD-L1+ ICs and TGF-β activity are features of HCC that likely play a role in immune evasion.The primary objectives of this observational study were to investigate the expression of PD-L1 in HCC and adjacent non-tumor liver, the frequency and identity of ICs in HCC, and the expression/activity of TGF-β1 in HCC. The purpose was to gain a greater understanding of the immunogenic environment associated with HCC, including the TME and infiltrating ICs, building on previously published research , 13. To + tumor cells was overestimated. CD8+, CD68+, and FoxP3+ cells were found to occur with variable incidences in HCC, with the highest numbers at the invasive margin, suggesting greatest immunological activity in this region. However, as shown by semiquantitative IHC, the content of PD-L1+ immune cells in HCC is generally low. The expression of PD-L1 in HCC primarily on infiltrating ICs rather than tumor cells suggests that tumors may evade immune surveillance primarily by manipulation of PD-L1+ ICs, e.g., cytotoxic T lymphocytes. Additionally, PD-L1 expression by endothelial cells in small blood vessels, central veins, and intratumoral microvessels may have a role in immune regulation. Liver sinusoidal endothelial cells (and hepatocytes) have previously been shown to act as unconventional antigen presenting cells (APCs) in the liver, so when expressing PD-L1 they may anergize infiltrating ICs (+ endothelial cells may also block the transmigration of PD-1+ ICs into the liver (PD-L1 is implicated in immune suppression in HCC by its presence in tumors and adjacent tissue, and high PD-L1 expression in HCC has been positively correlated with liver cirrhosis, poor Barcelona Clinical Liver Cancer stage, portal vein invasion, and reduced overall survival . We founting ICs . Speculahe liver .+ cells correlated with higher levels of mRNAs for perforin, granzyme A, and granzyme H. We also found a correlation between the incidence of PD-L1+ cells and the incidence of CD8+ cells and the strength of a CD8-associated GES, suggesting that PD-L1 expression may increase as an adaptive anti-inflammatory response to pre-existing CTL activity (We found that the incidence of CD8activity , 35.TGFB1, TGFB2, and TGFB3 transcripts in HCCs using RNAseq data. However, we considered that expression levels of individual isoforms of TGFB mRNA may be poor indicators of TGF-β activity, because activity is also dependent on availability of mature and free TGF-β ligands, turnover, and cellular context. We therefore additionally used a previously described TGF-β1-associated GES as a measure of TGF-β activity in HCC. Our results demonstrated that TGF-β1 mRNA is detectable in HCC.IHC is an established method for detecting clinically relevant expression of PD-L1 in HCC , 36 and The observed correlation between TGF-β1- and EMT-associated GESs is consistent with the established role of TGF-β in the induction of EMT in HCC , and indOnly one section per patient was available for analysis in this study, as evidenced by the relatively low proportion of patients with adjacent non-neoplastic liver parenchyma. Since heterogeneity in this setting is an issue, this is acknowledged as a limitation to this study. In addition, as this study was executed on procured tissue samples, and clinical characteristics were not available, it was not possible to correlate tissue histology with clinical characteristics, such as cirrhosis and clinical behavior.Our study contributes to the hypothesis that inhibition of PD-L1 and TGF-β and their respective immunomodulatory pathways may contribute to antitumor effects in HCC. Our observations are consistent with those of others who have identified roles for PD-L1, TGF-β1, and infiltrating ICs in HCC , 36, 41.Our results suggest that inhibiting both PD-L1 and TGF-β may increase antitumor immunity beyond that achievable by inhibiting either in isolation. Therefore, there may be an opportunity to combine PD-1/PD-L1 inhibitors with TGF-β-inhibiting compounds to improve the activity of immuno-oncology treatment approaches.Merck KGaA operates a responsible Data Sharing Policy and requests for access to the datasets for this study can be made directly to Merck in line with this policy.CI, YZ, ID, LT, and BN contributed to the concept and design of the study. PR and EF-K contributed to experiments and procedures. All authors contributed to the writing of the manuscript.CI and EF-K are employees of Merck KGaA. BN, YZ, PR, and ID are employees of EMD Serono. LT is a consultant pathologist for Merck KGaA.


Graphene and graphene-based materials exhibit exceptional optical and electrical properties with great promise for novel applications in light detection. However, several challenges prevent the full exploitation of these properties in commercial devices. Such challenges include the limited linear dynamic range (LDR) of graphene-based photodetectors, the lack of efficient generation and extraction of photoexcited charges, the smearing of photoactive junctions due to hot-carriers effects, large-scale fabrication and ultimately the environmental stability of the constituent materials. In order to overcome the aforementioned limits, different approaches to tune the properties of graphene have been explored. A new class of graphene-based devices has emerged where chemical functionalisation, hybridisation with light-sensitising materials and the formation of heterostructures with other 2D materials have led to improved performance, stability or versatility. For example, intercalation of graphene with FeCl The discovery of graphene  and morePhotodetectors are used in nearly every electronic device which interfaces with the external world or with other devices. Several industrial sectors make use of light sensors to include telecommunications, food production, transport, defence and healthcare. Although the miniaturisation of electronic devices such as transistors has allowed higher computational speeds and smaller, portable devices, the miniaturisation of light sensors did not proceed at the same rate, as several physical factors limit the scaling of such devices. In particular, the realisation of ultra-thin and flexible photodetectors is particularly challenging with conventional semiconductor technologies, due to the brittle nature of the materials used and low absorption at nano-scale thickness. Graphene-based light sensors have shown some exceptional performances, spanning from high speed  to largeGraphene can be obtained via different methods. The first and most direct approach is micro-mechanical cleavage of bulk graphite . This meChemical functionalisation refers to the use of chemical species to modify the properties of a material and in graphene it can take different forms , which a3 , (2) the3 , or (3) graphene . The fungraphene . Each tygraphene ,22,23 tographene . A functgraphene ,25,26 sulectrode ,28.A multitude of techniques can be employed in order to form hybrids and heterostructures between graphene and other materials. These include coating techniques such as spin/spray/dip/cast/bar coating, printing techniques such as inkjet and contact printing and deposition methods such as thermal evaporation, CVD, electrochemical deposition, etc. However, the majority of these methods result in a certain degree of defects being introduced in the graphene material. In this review, we focus our attention onto non-destructive techniques which allow the realisation of heterostructures which preserve the high-mobility of graphene field-effect transistors (FETs) and enhance the light absorption of the device. For this reason, we will consider the following two techniques: spin-coating  of nanoparticles or quantum dots (QDs) directly on the surface of graphene and stacking different 2D materials in a van der Waals (vdW) assembly , as showcontacts  , photovoltaic (PV) and photogating (PG) effects. These three mechanism are schematically shown in Other three mechanisms responsible for photodetection in graphene devices are the bolometric effect , the DyaThe basic characterisation techniques rely on shining light onto the device whilst recording its electrical response. Light can impinge on the whole surface of the device, known as flood illumination, or it can be delivered with a focused laser onto a specific area to allow for a spatially-resolved photo-response, such as in scanning photocurrent mapping (SPCM) . Both teA is the area of the device. Jones (cmE: The responsivity (cmHz/W) . The lin(cmHz/W) . It is dq is the electron charge and A junction between two materials with different Seebeck coefficients  voltage . In grap1−S2 see c, causesrelation ,43:(2)S=∼0.1mV/K . In geneThe exponent in Equation  is commoIt has been shown that the PTE is responsible for the light-sensing ability of pristine graphene and that the photo-active areas are confined to the junctions between two different materials such as the graphene/metal ,45,46, munctions .W and L are the width and length of the device, The term “photovoltaic effect” has been commonly used by the solar cell research community to describe a broad variety of mechanisms by which the absorption of photons, generation of excitons, separation into free charge carriers and collection of free charge carriers at electrodes sequentially takes place. This definition is quite broad and does not necessarily require the presence of an electric field. However, within the research field of atomically thin materials the PV effect refers to the process of separation of photo-generated carriers by a built-in electric field ,45,48,57To increase the absorption of graphene-based photodetectors, a semiconducting material is placed in close proximity to the channel of a graphene field-effect transistor (FET). Upon illumination, a photoexcited charge carrier is transferred from the semiconductor to graphene. This changes the carrier density in the graphene FET, which manifests itself in electrical measurements as a shift in the charge neutrality point (D is described by ,852/Si sRobin et al.  have engAside from QDs, perovskites have shown promise in the next generation of hybrid graphene photodetectors with ideal properties such as direct band gap, large absorption and relatively high mobility ,92,93,94Recently, the first image sensors based on graphene photodetectors have been reported ,127. TheOrganics have also been investigated as light-sensitising elements in graphene FETs. They are of particular interest owing to their intrinsic affinity to biological systems as well as the ability to tailor their spectral selectivity through chemical functionalisation. A number of different materials have been investigated including chlorophyll , rutheni0.6%  was achi0.6% . The lonThe family of 2D materials extends beyond graphene to include insulators (hBN), semiconductors , and materials exhibiting more exotic properties such as superconductivity and magnetism . SimilarThe first demonstration of a graphene-TMD phototransistor was found in the work of Roy et al. . Here, g2) see a. Electrectively b. In a fy 90meV ,103, wit, wit2 (sications d. To extg∼0.3eV)  and MoTeg∼1.1eV) . HoweverThe slow temporal response of these heterostructure photodetectors limits their use to steady state imaging applications as the presence of long-lived charge traps that provide the extreme photo-sensitivity comes at the expense of operational speed. Recently, Mehew et al. fabricated graphene–tungsten disulphide  compared to pristine graphene, chemical functionalisation allows photodetection from UV to THz wavelengths. Furthermore, quenching of PTE results in an increase of the LDR of such detectors. Chemical functionalisation also allows the creation of solution-processed materials, such as GO, with clear advantages in scalability.Improvements in responsivity and operating bandwidth have been achieved by combining graphene with other materials to form hybrid photodetectors. Photodetectors based on hybrid interfaces of graphene with QDs, semiconductors to include atomically thin TMDs, perovskites and organic crystals offer improvements in responsivity and high gain owing to the photogating effect, which enables charge multiplication in the graphene channel (charge recirculation). The majority of these devices have a limited LDR due to the charge relaxation time, which quickly saturates the available states for photoexcitation, leading to a drop in responsivity with incident optical power. In some architectures, however, this effect is compensated by the low NEP, giving both high LDR and high responsivity. Therefore, a thorough investigation of charge trapping mechanisms is necessary to design a high performance PD. The speed of these devices is limited by the charge trapping times with reported operating bandwidths between Future developments of atomically thin PDs will focus on the optimization of the responsivity and bandwidth for a given application. For instance, as quantum technologies become more important, the ability to manipulate single quanta of light is a priority and single-photon detectors will have to be integrated in optical communications systems and computation circuits at room temperature. Current state-of-the-art commercial single photon detectors have a dark count of ng 30MHz . This co∼ 100 K) .Although high responsivity is an important parameter, in applications where high levels of illumination are present, it is more important to have a large LDR, in order to avoid saturating a detector undergoing abrupt changes in radiation intensity. Such scenarios include surveillance or monitoring in harsh environments such as space or inside a nuclear reactor. For instance, recent efforts in nuclear fusion have been focussing on igniting a plasma using high-power lasers. In this case, monitoring the power of the laser and the corresponding plasma is vital and small-footprint photodetectors could significantly facilitate the operation of such reactors. FeCl0MW/cm2)  and in h0MW/cm2) . The larnt power .Healthcare applications will also benefit from ultra-thin, flexible photodetectors able to operate across a wide range of wavelengths . UV radiIn summary, graphene-based photodetectors, whilst offering a small footprint promising for next-generation flexible and wearable electronics, are also more energy-efficient and could provide features that are not available in bulk semiconductors, such as polarisation sensitivity  and stra


Aberrant expression, dysfunction and particularly aggregation of a group of RNA-binding proteins, including TDP-43, FUS and RBM45, are associated with neurological disorders. These three disease-linked RNA-binding proteins all contain at least one RNA recognition motif (RRM). However, it is not clear if these RRMs contribute to their aggregation-prone character. Here, we compare the biophysical and fibril formation properties of five RRMs from disease-linked RNA-binding proteins and five RRMs from non-disease-associated proteins to determine if disease-linked RRMs share specific features making them prone to self-assembly. We found that most of the disease-linked RRMs exhibit reversible thermal unfolding and refolding, and have a slightly lower average thermal melting point compared to that of normal RRMs. The full domain of TDP-43 RRM1 and FUS RRM, as well as the β-peptides from these two RRMs, could self-assemble into fibril-like aggregates which are amyloids of parallel β-sheets as verified by X-ray diffraction and FT-IR spectroscopy. Our results suggest that some disease-linked RRMs indeed play important roles in amyloid formation and shed light on why RNA-binding proteins with RRMs are frequently identified in the cellular inclusions of neurodegenerative diseases. In particular, aggregation of RNA-binding proteins is considered to be one of the major hallmarks of neurodegenerative diseases, based on characterization of cellular inclusions containing TAR DNA-binding protein 43 (TDP-43) and Fused in Sarcoma (FUS) in the diseased neurons of patients suffering amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar dementia (FTLD)7. RNA-binding proteins usually contain one or more RNA recognition motifs (RRMs), raising the intriguing question as to whether RRMs can mediate protein aggregation.RNA-binding proteins play primary roles in RNA metabolism, coordinating networks of RNA-protein and protein-protein interactions and regulating many important events such as RNA splicing, maturation, translation, transport and turnover. Aberrant expression, dysfunction, and aggregation of RNA-binding proteins have been identified in several major classes of human diseases, including neurological disorders, muscular atrophies and cancer8. TDP-43 contains an N-terminal domain (NTD), two RRMs (RRM1 and RRM2), and an unstructured C-terminal domain (CTD)9. Most ALS- and FTLD-linked mutations are located within the CTD, so its unstructured Glycine-rich region has been extensively studied for its involvement in protein aggregation12. A prion-like region has been identified in the CTD, and small peptides in the Glycine-rich region form twisted fibrils in vitro20. However, the major components in the cytoplasmic inclusions of diseased neurons in ALS/FTLD patients are 35-kD and 25-kD C-terminal fragments of TDP-43, comprising not only the CTD but also RRM1 and RRM223. Some studies have demonstrated that RRMs or parts thereof could be involved in TDP-43 aggregation and cytotoxicity. For example, expression of a series of TDP-43 truncation fragments showed that the CTD alone is not efficient at inducing protein aggregation and that RRMs are required for robust protein aggregation in the cytoplasm of mammalian26 and yeast cells27. Overexpression of RRM1-deleted TDP-43 in a transgenic Drosophila model also demonstrated that RRM1 deletion mitigated the degeneration of motor neurons compared to the effect of full-length TDP-4328. Moreover, our previous study revealed that two small peptides in the β strand region of TDP-43 RRM2—β3 (residues 227–233) and β5 (residues 253–259)—as well as a truncated RRM2 (residues 208–265), can form fibril-like aggregates in vitro26. Recently, it has also been reported that RRMs from TDP-43 have amyloid-like properties30 and that various peptides from the TDP-43 RRM2 domain can form amyloid fibrils in vitro32, further supporting the role of RRMs in promoting protein aggregation and amyloid formation.TDP-43 is the key component of toxic cytosolic inclusions in ALS and FTLD34. As for TDP-43, mislocalization and inclusions of FUS are not limited to ALS and FTLD but have also been detected in other neurodegenerative diseases, including Huntington disease37. FUS contains a QGSY-rich prion-like domain, a Glycine-rich region, a C-terminal low complexity domain and a single RRM, with this RRM necessary for manifesting FUS cytotoxicity38 and being capable of self-assembling into amyloid fibrils in vitro39. In contrast, RBM45 only comprises three RRMs (RRM1-3), thus lacking any other structural domain, but the role of RRMs in RBM45 aggregation is not clear. RBM45 forms homo-oligomers and physically associates with TDP-43 and FUS in the nucleus40, suggesting a close link between RBM45, TDP-43 and FUS in aberrant RNA metabolism and protein aggregation in ALS.Similar to TDP-43, the RNA-binding proteins FUS and RNA-binding motif 45 (RBM45) have also been identified in the cytoplasmic inclusions of ALS and FTLD patients41. RRMs are folded, with two α-helices packed against a β-sheet consisting of four or five antiparallel β-strands42. The crystal and NMR structures of RRM1 and RRM2 of TDP-43 reveal how these two RRMs bind nucleic acids45. Here, we compared the biophysical and fibril-formation properties of five RRMs from the disease-linked proteins, TDP-43, FUS and RBM45, as well as five RRMs from RNA-binding proteins not linked to disease, i.e., U2AF47, UP148, and PABP49. We observed that most of the disease-linked RRMs display reversible thermal unfolding-refolding properties and have slightly lower thermal melting points compared to the non-disease-associated protein RRMs. The full domain of TDP-43 RRM1 and FUS RRM, as well as three β2 peptides from these two RRMs, are prone to fibril formation in vitro, and all these fibrils are amyloids in nature. Our results thus confirm, for the first time, by X-ray diffraction and FT-IR analysis that the full domain of RRMs could form amyloids, and contribute to the aggregation-prone properties of TDP-43 and FUS, and could possibly drive protein self-assembly. Thus, our results provide an important basis for further studies of RRMs and RRM-containing proteins in terms of their roles in protein aggregation and neurodegeneration.In fact, RRMs are the only domains shared by these three aggregation-prone RNA-binding proteins, TDP-43, FUS and RBM45 see Fig. . RRMs arE. coli, including 5 RRMs from disease-linked proteins , and 5 RRMs from non-disease-associated proteins   Fig. . These tata Fig. .47 RRM1, UP1 RRM1, and UP1 RRM2—did not revert to the original CD profile upon cooling, and the refolded CD profiles of PABP RRM2 and PABP RRM4 only partially matched their original low-temperature profiles . Together with our CD data, these results show that RRMs from disease-linked proteins mostly exhibit reversible thermal unfolding and refolding, and have slightly lower melting points compared to non-disease-associated RRMs.We next performed differential scanning fluorimetry to measure the thermal melting points of all ten RRMs. We used SYPRO Orange dye as the fluorophore, which binds to unfolded hydrophobic protein surfaces to produce fluorescence signals. We found that RRMs from the aggregation-prone proteins, including TDP-43 RRM2, FUS RRM, RMB45 RRM1 and RBM45 RRM2, shared similar differential scanning fluorimetry profiles, characterized by a sudden decrease in fluorescence signal upon protein melting by raising the temperature   and TDP-43 RRM1 β2b (128GEVLMVQVKK137). A segment in the β1 region of TDP-43 RRM1 also presented high predictive ability to form amyloid fibrils, so we also designed two peptides in this region, namely β1a (104SDLIVL109) and β1b (102KTSDLIVLG110). Three β-strand regions in FUS RRM were also predicted by ZipperDB to be prone to forming amyloids , β2 (321MINLYT326) and β4 (364IKVSFA369). Three of these seven peptides formed needle-like fibrils, including TDP-43 RRM1 β2a, TDP-43 RRM1 β2b and FUS RRM β2, which were visualized by negative-stain TEM , suggesting that these fibril-like aggregates exhibit amyloid-like characteristics  that preferentially binds to amyloids and gives a strong fluorescence signal at ~485 nmics Fig. . However55. Not only did TDP-43 RRM1 and FUS RRM fibrillar aggregates present the two characteristic diffraction rings—one at 4.7 Å of inter-strand spacing and one at 10 Å of inter-sheet spacing—so did the three β2 peptide fibrils  from TDP-43 RRM1 could form fibrils in vitro, but these fibrils only diffracted X-rays to produce a ring at 9.2 Å, but not the ring at 4.7 Å31. Thus, our X-ray diffraction data confirm for the first time that the fibrils formed by TDP-43 RRM1 and FUS RRM (and their respective β2 peptides) are amyloids in nature.To further confirm the amyloid nature of RRM fibrillar aggregates, we used X-ray diffraction to establish if these fibrils could produce the characteristic cross-beta diffraction pattern of amyloids59. We acquired ATR-FTIR spectra for the fresh proteins, fresh peptides, and all fibrils. The ATR-FTIR spectra of the fresh β2 peptides gave the highest absorbance intensities (mainly at 1630–1640 cm−1), revealing a characteristic β-strand structure, whereas the spectra for the fresh FUS RRM and TDP-43 RRM1 were broad with a wide frequency range reflecting different secondary structure components  spectroscopy; a widely used technique to study the fibrillar conformational changes of β-sheet-rich amyloids60. In doing so, we observed troughs that clearly shifted from 1630–1640 cm−1 for fresh proteins/peptides to 1620–1630 cm−1 for the aggregates/fibrils  when compared to the spectra of fresh RRM proteins and peptides (black lines). This type of transition is a characteristic representation of amyloid assembly, indicating that antiparallel β sheets are being transformed into parallel β sheets in proteins and peptide fibrils59. Previous study has also revealed that TDP-43 RRM2 peptides form amyloids of parallel β-sheets32, which is in agreement with the parallel amyloid structures observed here for TDP-43 RRM1, FUS RRM and their peptides. In summary, all of our results, including from ThT fluorescence assays, X-ray diffraction and ATR-FTIR spectroscopy, confirm that TDP-43 RRM1, FUS RRM and their β2 peptides can self-assemble into fibril-like amyloids consisting of parallel β-sheets.To more thoroughly investigate hidden local peaks in the overall spectrum, we calculated second derivatives of each spectrum23. In this study, we compare the biophysical properties of ten RRMs of disease-related and non-disease-associated RNA-binding proteins to reveal if the disease-linked RRMs share any particular features making them prone to self-assembly. Unexpectedly, we found that the disease-linked RRMs are not resistant to thermal denaturation as suggested previously44, and their tertiary structures could be disrupted by raising the temperature to ~56 °C, as revealed by near-UV CD. This thermal unfolding process is reversible for most of the disease-linked RRMs (except TDP-43 RRM1), as the unfolded RRMs could be refolded upon cooling. In contrast, thermal denaturation of most of the non-disease-associated RRMs is not reversible and these RRMs are mostly precipitated upon heating. We noted that TDP-43 and FUS have previously been shown to undergo liquid-liquid phase separation (LLPS) and form reversible dynamic assemblies mediated by their low complexity C-terminal domains63. The unique property of reversible thermal unfolding and refolding of disease-linked RRMs supports their conformational flexibility and reversibility, which may induce the intrinsically unfolded C-terminal domains to undergo phase transition or to drive pathological aggregation.Previous studies revealed that TDP-43 RRMs are present in the pathological aggregates in ALS brains by mass spectrometry, suggesting that RRM may contribute to protein aggregation65. TDP-43 aggregates expressed in yeast cells are not amyloid-like27, and TDP-43 purified from inclusion bodies in bacterial cells were structurally amorphous and also non-amyloid66. In contrast, fine structural analysis of neuronal inclusions using high-resolution immuno-gold labelling with electron microscopy provided evidence that amyloid-like fibrils are formed by TDP-43 in the affected neurons of patients with neurodegenerative disease68. Several studies have further reported that the fibrils formed by different regions of TDP-43 are amyloids69. Recently, it has also been shown by Thioflavin S-staining that TDP-43 forms amyloid-positive aggregates in ALS patients70, and the cytoplasmic inclusions in most cases of ALS/FTLD were found to be Thioflavin S-positive upon modifying the staining protocol to remove lipid autofluorescence background71. These studies provide strong evidence that protein aggregates in the cytoplasmic inclusions of ALS/FTLD have amyloid-like properties. Our results herein also support that protein aggregates formed by TDP-43 RRM1 and FUS RRMs are amyloids in nature.In this study, we also show by X-ray diffraction and ATR-FTIR spectroscopy that TDP-43 RRM1, FUS RRM and their β2 peptides form fibrils and fibril-like aggregates that are amyloids in nature  RIPL cells, and then incubated at 37 °C overnight in LB media supplemented with 100 μg/mL ampicillin or 40 μg/mL kanamycin. A single bacterial colony was used to inoculate a small 25 ml culture, which was grown overnight. An aliquot (10 ml) was added to 1 L of LB media with antibiotic and this was grown at 37 °C for 3–4 hours. When the optical density of culture at 600 nm (OD600) reached 0.6, it was cooled down and induced by adding 0.8 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG). The induced bacterial culture was grown at 18 °C for 16–18 hours for protein overexpression. The cells were harvested by pelleting down the culture and were resuspended in 50 mM phosphate buffer (pH 7.5), 500 mM NaCl and 10 mM β-mercaptoethanol (βME) containing a tablet of cOmplete EDTA-free protease inhibitor cocktail (Roche). The cells were lysed by a microfluidizer (Microfluidics M-110P). The cell lysate was pelleted down by high-speed centrifugation and the supernatant was applied to a HisTrap FF column . The His-tagged recombinant proteins were eluted by using an imidazole gradient from 0 to 500 mM. The eluted proteins were further purified by HiTrap Heparin HP columns  and loaded into Superdex 75 10/300 GL columns  for a final round of purification. Purified protein samples were eluted in a buffer containing 50 mM phosphate buffer (pH 7.5), 150 mM NaCl and 10 mM βME for further experiments. The proteins assessed were: TDP-43 RRM1 (residues 101–191), TDP-43 RRM2 (residues 192–265), FUS RRM (residues 281–375), RBM45 RRM1 (residues 24–109), RBM45 RRM2 (residues 116–197), U2AF47 RRM1 (residues 148–237), UP1 RRM1 (residues 7–92), UP1 RRM2 (residues 99–182), PABP RRM2 (residues 95–178), and PABP RRM4 (residues 290–373).The cDNA of each RRM from the human cDNA library was amplified using 2x PfuUltra II Hotstart PCR Master Mix (Agilent Technologies) and cloned into bacterial expression vector PQE30 or pET28a to generate N-terminal His-tagged recombinant protein-expressing clones. The expression plasmids were transformed into The near-UV CD spectrum of each protein sample was measured using a Chirascan-plus CD spectrometer (Applied Photophysics). To record the near-UV CD signal, each RRM was assessed at a concentration of approximately 2 mg/ml in a buffer of 50 mM phosphate (pH 7.5), 150 mM NaCl and 0.5 mM Tris(2-carboxyethyl)phosphine (TCEP). The spectra were recorded in the near-UV range (from 260 nm to 310 nm) in a 10-mm path length quartz cuvette at temperatures increasing from 20 °C to 90 °C at 10-degree intervals with an equilibration time of 3 minutes. The samples were then slowly cooled down to 20 °C within 30 minutes and the spectra were recorded again to monitor the structures upon re-cooling.® Protein Melting Analysis.The thermal melting curve of each RRM was measured in triplicate by differential scanning fluorimetry using a LightCycler 480 system (Roche). A final protein concentration of 10 μM and 15X SYPRO Orange dye (Invitrogen) was mixed in each well in a LightCycler Multi-well Plate 96 White (Roche). The temperature was raised from 20 °C to 85 °C at a rate of 0.06 °C/second with 10 acquisitions per degree. An excitation filter of 465 nm and an emission filter of 580 nm were used for SYPRO Orange detection. Melting temperatures (Tm) were calculated by LightCyclerg for 10 minutes before being filtered through a 0.22 µm Millex-GV filter (Millipore) to remove any insoluble material or aggregates. All β2-peptides (synthesized by Mission Biotech) of TDP-43 RRM1 and FUS RRM were dissolved in water, and both RRM and peptide samples were agitated at room temperature for two days to promote fibril formation. The freshly-formed fibrillar solutions (5 μL) were placed on 200-square-mesh carbon-coated, glow-discharged grids (Electron Microscopy Science). Each grid was washed with water and stained by 1% uranyl acetate for 2 minutes at room temperature and then air-dried. Fibrils on the grids were examined by a Tecnai G2 Spirit TWIN transmission electron microscope (FEI Company).The purified sample of each RRM was diluted into a buffer containing 10 mM phosphate (pH7.5) and 50 mM NaCl to a final concentration of 50 μM. RRM samples were then centrifuged at 20,000 Thioflavin T  was dissolved in water to make a stock solution (1 mM), which was filtered through a 0.22 μm filter and stored at −20 °C with protection from light. This ThT stock was diluted in 10 mM phosphate buffer to a concentration of 25 μM. Fresh protein or fibrillar solutions (100 μL) were mixed with 400 μL ThT dye  and incubated for 5 minutes in the dark. The samples were excited at 442 nm, and the fluorescence emission signal was recorded from 460 to 600 nm using a Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent) in a 400-μL fluorescence micro cell (Varian).The freshly made fibrillar solution of peptides/RRMs were centrifuged at 20,000 g for 60 minutes and washed with Milli-Q water. The dried fibrils were placed on the CrystalCap CryoLoop. X-ray diffraction images were recorded at room temperature using Rigaku Ultimate HomeLab protein crystallography system equipped with Saturn 944+ CCD detector and FR-E+ SuperBright microfocus rotating anode generator.2O water to remove any residual proteins and were then re-suspended in a small amount of D2O. The samples were evenly spread on an internal reflection element (IRE) crystal using a micropipette tip. The buffer and D2O spectra were used as background for protein and fibril samples, respectively. The data were collected as an average of 128 scans at 1 cm−1 resolution. PeakFit (Systat Software Inc.) was used for spectral processing and data analysis. We plotted the amide I band at 1600–1700 cm−1 to record specific spectral features in amyloids.The structural transition from proteins/peptides to fibrils was studied by attenuated total reflection FTIR (ATR-FTIR). The ATR-FTIR spectra were recorded on a Tensor 27 FTIR spectrophotometer (Bruker) in conjunction with OPUS data collection software. We used high concentrations (~12 mg/ml) of fresh protein samples for FTIR analysis. The freshly prepared fibrils were washed three times with DSupplementary Information


The maximum value of the parameter of ordering is η by the relation η →1, the electrical conductance of graphene In the one-band model of strong coupling, the influence of substitutional impurity atoms on the energy spectrum and electrical conductance of graphene is studied. It is established that the ordering of substitutional impurity atoms on nodes of the crystal lattice causes the appearance of a gap in the energy spectrum of graphene with width  In recent times, a special attention has been paid to the possibility of a targeted modification of graphene with the help of purposely introduced impurities, formed defects, and atoms or chemical functional groups deposited on a surface. In this case, wide possibilities to change the physical properties of graphene are opened, due to the controlled introduction of impurities by the method of ion implantation. In such a way, graphene becomes a basic system that generates a new class of functional materials. Sometimes, such materials find unexpected applications: from nanoelectromechanical systems to hydrogen-accumulating systems. Of course, the main hopes are set upon graphene, because it has all possibilities to become, in the nearest future, a substitute of silicon in electronic devices, which will allow one to significantly enhance the level of their miniaturization and to increase the working frequencies. The quasirelativistic spectrum of charge carriers determines the unique properties of graphene and, simultaneously, hampers the use of graphene in field-effect transistors due to the absence of a gap in its spectrum. It is known that the impurities can lead to the appearance of such a gap, whose width depends on the type of impurities and their concentration.1 which is realized with softwares WASP and GAUSSIAN1. The numerical calculations made within the method have demonstrated the appearance of a gap in the energy spectrum of graphene caused by the presence of an impurity. However, in order to clarify the nature of this effect, it is necessary to supplement the mentioned numerical calculations by analytic studies of the influence of impurities on the energy spectrum and properties of graphene.The recent investigations of the energy spectrum of graphene are based on the density functional theory. It is worth to note the advantages of this theory related to the self-consistent meta-generalized gradient approximation within the projector-augmented-wave method2. The energy band gap in the bilayer graphene/hexagonal boron nitride material then increases, due to symmetry breaking in the curvature graphene component and due to the charge transport at the toroidal border of the hole compared with a similar graphene mesh2. Also, in earlier work, the complex structure based on the graphene monolayer and the twisted BN monolayer was investigated3. The changes in the electronic structure during the hydrogen adsorption at low concentration were considered. It was found that the presence of the BN layer did not impact significantly on the electronic structure and that only the H-atom adsorption on the boron atoms could open the band gap in the layered structuré. An investigation of the dependence of the band gap on the hydrogen concentration on the Moire surface was made. Upon increasing the hydrogen concentration, the value of the band gap was increased up to 0.5 eV3.Recently, new 2D-material nanomeshes based on Moiré graphene/hexagonal boron nitride bilayers were predicted, on the basis of first-principle calculations within the framework of density functional theory, with standard norm-conserving pseudo-potentials, flexible numerical LCAO double zeta + polarization orbital basis sets4. There, the effects of the opening of a gap in the energy spectrum, which are connected with the breaking of a symmetry of sublattices due to the appearance of internal mechanical stresses, were analyzed. The influence of a magnetic field on the energy spectrum of a heterostructure was studied as well4.The effects generated by the Moiré superlattice of a van-der-Waals heterostructure of graphene grown on ultrathin layers of hexagonal boron nitride (h-BN) were studied in review5 in the frame of the density functional theory with the use of the method of pseudopotential, the electron structures of an isolated monolayer of graphene, two and three layers of graphene, and graphene grown on ultrathin layers of hexagonal boron nitride (h-BN) were calculated. It was shown that, for graphene grown on an h-BN monolayer, the energy gap 57 meV in width arises. By an analogous method, graphene with aluminum, silicon, phosphorus, and sulfur impurities was studied in work6. It was shown6 that graphene with a 3-% phosphorus impurity has a gap 0.67 eV in width.In work7 demonstrated the possibility to open a gap in the energy spectrum of graphene at the introduction of impurity atoms of boron and nitrogen (gap 0.49 eV in width), as well as impurity atoms of boron and atoms of lithium adsorbed on the surface (gap 0.166 eV in width).By using the software QUANTUM-ESPRESSO, the authors of work10 present the theory of reconstruction of the spectrum of graphene, which occurs at a growth in the concentration of point-like impurities, and foresee the possibility of the metal–dielectric transition in such a system. The course of the reconstruction of the spectrum, which was predicted on the basis of the analytic calculations, was confirmed with the help of a numerical experiment. The latter showed the existence of a quasigap filled with localized states and demonstrated the dominating role of the scattering by pairs and triples of impurity centers in the localization.Works16, the influence of impurity atoms or atoms adsorbed on the surface on the electron structure and electrical conductance of graphene was studied. The numerical calculations were carried out in the frame of the quantum-mechanical formalism. In work17, the influence of the ordering of atoms on the energy spectrum and the electrical conductance of an alloy was studied analytically in the above-mentioned one-band model. It was found17 that, at the long-range ordering of the alloy, a gap appears in the energy spectrum of electrons. Its width is equal to the difference of the scattering potentials of the components of the alloy. The appearance of the metal–dielectric transition was established in the case where the Fermi level enters into the indicated gap at the long-range ordering of atoms in the alloy. However, no explanation about the nature of the influence of the ordering of impurities on the appearance of a gap in the energy spectrum of graphene is available until now.In works19. In particular, it was established in work18 that the presence of impurities can cause a significant decrease in the electrical conductance of graphene.It is worth to note that, at the appearance of a gap in the energy spectrum of graphene in the case where the Fermi level enters into the indicated gap, the velocity of an electron on the Fermi level can decrease. This causes a decrease in the mobility and electrical conductance of electrons, which can deteriorate the functional characteristics of graphene, which would be used as a material for field-effect transistors instead of the traditional materials based on silicon and germanium. The influence of disordered impurities on the electrical conductance of graphene was investigated in works5 and graphene with an impurity adsorbed on the surface and with a substitutional impurity10. In the present work, we will demonstrate one more cause for the appearance of a gap in the energy spectrum of graphene that is related to the ordering of a substitutional impurity. We will study the influence of the ordering of a substitutional impurity on the conductivity of graphene.The authors of the above-indicated works studied the effects related to the appearance of a gap in the energy spectrum of a heterostructure of graphene grown on ultrathin layers of hexagonal boron nitride (h-BN)In order to clarify the influence of the ordering of substitutional impurities on the energy spectrum and electrical conductance of graphene, we use the Lifshitz simple one-band model.17The Hamiltonian describing the one-electron states of graphene with substitutional impurity atoms can be presented in the formA or B) is located at the node in; i is the number of a sublattice, and n is the number of a node of the sublattice.where In formula , we now In Eq. , \documez in the upper half-plane, readsThe retarded Green function of graphene, which is an analytic function of the complex energy The Green function satisfies the Dyson equationwhereT-matrix of the scattering by a random potential is determined by the relation17is the Green function for the effective Hamiltonian entclass1pt{minimaand satisfies the equationwhich follows from formulas  and 6)..6).T takes the formThe scattering matrix T-matrix as an infinite series17:Substituting relation  for the formulas  in 7), , \documeHere,I is the identity operator. The terms of series is scattering operator on the same site, and 17, we present the density of one-electron states of graphene in the formNeglecting the contribution of the processes of scattering by the clusters of three and more atoms, which are small in some parameterwhere 20 and neglecting the contribution of the processes of scattering by clusters composed of at least three atoms, we get the static electrical conductance of graphene in the form (T = 0)17:Using the Kubo—Greenwood formulai of the crystal lattice y is the concentration of impurity atoms, and η is the parameter of atomic ordering.In formulas  and 13)13), \doclj by an atom of the sort 0i .In formulas  and 13)13), \doc17:The coherent potential is determined from the condition Setting elations , we get1is the difference of the scattering potentials of components of graphene.In the analytic description of the energy spectrum and electrical conductance of graphene, we consider only the first terms in relations  and 13)13), whicThe mentioned components can be presented in the form:The operator of the  formula  is:20\dowhere The wave vector in formulas  and 19)19) varieWe calculated In the used model, the main contribution to the energy spectrum of electrons in the middle of the zone is given by the values of the wave vector 21 and the distance between the nearest neighbors, respectively.Substituting formulas  and 22)22) in 1 and passwhere Let us consider the influence of the ordering of atoms on the energy spectrum of electrons of graphene with a substitutional impurity in the limiting case of weak scattering where In this case, the solution of the system of equations , 23) is is23) iswhere where In Eqs –27, \docThe analysis of formulas  and 25 sRelation  implies formulas  and  and and22) awhere It is worth to note that the factor  formula  can be oThus, the above-presented results imply that the appearance of a gap in the energy spectrum of graphene is related to the ordering of substitutional impurity atoms.We have shown that, at the ordering of impurity atoms, a gap If the Fermi level falls in the region of such a gap, then the electrical conductance If the Fermi level is located outside the gap, then, as is seen from formula , the eleAt the concentration