The development of ultrathin barrier films is vital to the advanced semiconductor industry. Graphene appears to hold promise as a protective coating; however, the polycrystalline and defective nature of engineered graphene hinders its practical applications. Here, we investigate the oxidation behavior of graphene-coated Cu foils at intrinsic graphene defects of different origins. Macro-scale information regarding the spatial distribution and oxidation resistance of various graphene defects is readily obtained using optical and electron microscopies after the hot-plate annealing. The controlled oxidation experiments reveal that the degree of structural deficiency is strongly dependent on the origins of the structural defects, the crystallographic orientations of the underlying Cu grains, the growth conditions of graphene, and the kinetics of the graphene growth. The obtained experimental and theoretical results show that oxygen radicals, decomposed from water molecules in ambient air, are effectively inverted at Stone–Wales defects into the graphene/Cu interface with the assistance of facilitators. Graphene holds promise as a protective coating; however, lattice defects may hinder its practical applicability. Here, the authors investigate the oxidation behavior of graphene-coated copper foils and unveil the interplay between structural defects and oxygen radicals from water molecules in ambient air. However, the replacement of Al by Cu in interconnections required substantial advancements in fabrication technologies, including Cu patterning methods and the introduction of barrier materials to isolate the Si from potentially damaging Cu atoms. The inability to etch Cu using plasma has led to a new approach of patterning Cu, i.e., the -Damascene process3, which is currently in common use in the semiconductor industry; however, intensive effort is still underway to develop a controllable physical barrier material for Cu in the form of films and coatings 4–7. Furthermore, Cu exhibits high reactivity toward oxygen, resulting in the formation of a surface Cu oxide layer, even at room temperature8. This layer causes substantial degradation in the interconnection capabilities during processing and/or device operation because of the formation of trap states at the Cu/Cu oxide interface11. Therefore, the barrier material must completely surround all of the Cu interconnections with a designated thickness, yet possess minimum influence on the physical and electrical properties of the underlying Cu. Many approaches, including coating with metals and alloys such as Ta/TaN and Ru/Ti layers5 and CuMn and CoW alloy films7, were concentrically pursued to protect the Cu surface; however, with the scaling down of the feature size to atomic thickness, a stack comprising the aforementioned barrier metal films and a Cu conductor exhibits a greater total resistance than the Al interconnects, eliminating any benefit.Si-based integrated circuits that use Cu instead of Al for interconnections have improved the system performance and power dissipation in ultra-large-scale integration (ULSI) devices, because Cu-based chips have smaller metal components and require less energy to pass electricity because of the low resistivity and high electromigration resistance of Cu compared to those of Al12. Thus far, graphene (Gr), the thinnest two-dimensional (2D) material known, appears to be the best candidate to satisfy this requirement because of its unique combination of properties that include ultrathin thickness, high carrier mobility, high chemical and thermal stabilities, and impermeability to all atoms, ions, and molecules19. Furthermore, Gr/Cu composites have the advantages of conducting a high current density with an enhanced thermal reliability compared to that of bare Cu20; in addition, ultrathin graphene films can be directly formed on Cu surfaces with various shapes using a chemical vapor deposition (CVD) process, thereby minimizing any adhesion issues at the interface of graphene and Cu.According to the literature, the thickness of the barrier material of Cu should be reduced to < 2 nm to satisfy the requirement of sub−22 nm interconnect technology21. Statistical observations suggest that various topological imperfections in graphene substantially degrade its properties24; however, the study of the nature of intrinsic graphene defects and their influence on the physical and barrier properties of graphene are still in its infancy. Recently, several pioneering reports have demonstrated that engineered, polycrystalline graphene films can serve as effective diffusion29 and oxidation34 barriers for Cu. However, the literature contains extensive contradictory results, especially with respect to the effectiveness of graphene coatings as an oxidation barrier for Cu; the detailed mechanism by which Cu is oxidized through a polycrystalline graphene barrier remains a subject of debate36.However, practically engineered materials are nearly always polycrystalline, and the presence of a certain amount of disorder in crystalline materials is inevitable according to the second law of thermodynamics. In fact, typical films of large-scale graphene produced by CVD are polycrystalline and contain a high density of intrinsic structural defects such as vacancies, adatoms, and line defectsHere, we use optical and electron microscopies to systemically investigate selective oxygen transport through various intrinsic graphene defects in Gr-coated Cu foils. Using an air oxidation process of Gr/Cu composites on a hot plate at ~200 °C, we develop a facile, scalable characterization technique to observe various microscopic topological defects with different origins, such as 0D and 1D defects at nucleation points, intra- and inter-granular grain boundaries (GBs) of graphene grains, multilayer graphene flakes and folded wrinkles in large-area sheets. This technique can be used to correlate the presence and density of intrinsic defects with the properties of macro-scale graphene. The oxidation behavior of Cu through various microscopic graphene defects suggests that the degree of structural deficiencies in as-synthesized graphene can vary depending on the origins of the structural defects, the crystallographic orientations of the underlying Cu grains, the growth conditions of graphene, and kinetics of graphene growth in the CVD reactor. From combined experimental and theoretical studies, we observe that oxygen radicals decomposed from the water molecules in ambient air are the direct cause of the oxidation of Cu and that selective oxygen transport occurs at Stone–Wales (SW) defects through the interface between graphene and Cu with the assistance of facilitators, leaving carboxyl (O = C–OH) groups on graphene as by-products. Finally, we propose a selective oxidation mechanism of Cu through an atomic-scale engineered polycrystalline graphene barrier.α and Cu Lβ, respectively  at 533.3 eV (4.7 at% of O atoms), carbonyl (C = O) at 531.4 eV (67.6 at% of O atoms), Cu2O/CuO phase at 530.3 eV (8.1 at% of O atoms), and carboxyl (O = C–OH) at 529.5 eV (19.6 at% of O atoms)  paired with Arms) Fig. ; however41; thereby, the long-axis length of the line features decreased to ~3.1 ± 0.6 μm at a methane flow rate of 50 sccm  grains can be fully revealed throughout the whole surface by increasing the annealing time up to 240 min  grain was the most favorable component of both the as-received and annealed Cu foils with volume fractions of ~91% and ~74%, respectively. Figure 47, it is well known that the Cu oxides formed after oxidation at low temperatures below 300 °C generally have fine crystals composed of three regions: a thin CuO layer, a thick Cu2O layer, and an oxygen-containing Cu region, and the volume change associated with the solid-state transformation at the CuO/Cu2O interface produces compressive stresses during the oxide growth, which stimulate the formation of fine oxide particles to accompany the interface reaction.We halted growth before the graphene islands merged with each other to form a continuous graphene film. Figure 2/Si(100) substrates for Raman spectroscopy. The intensities of the characteristic graphene Raman peaks, D (~1351 cm−1), G (~1588 cm−1) and 2D (~2685 cm−1), were extracted50; their spatial dependences are plotted in Fig. ID/IG intensity ratio was negligibly small (<0.05) over most of the area within the graphene islands, with the notable exception of the central area displaying a relatively large ID/IG related to the rhombus-shaped (ID/IG ≈ 0.3) and cross-shaped (ID/IG ≈ 0.2) dot assemblies. We mapped numerous (>30) graphene islands and observed that every island contains two such overlapping features in the center, regardless of the size of the graphene islands grown under various growth conditions , G ~158 cm−1 anFigure In the next observation, the cross-shaped oxidation line features centered at the nucleation point were distinctively characteristic of the graphene grown on the Cu(100) grains, which were different from the intra-granular GBs presented between lobes  of molecular oxygen (O2) between graphitic layers52, these observations strongly suggest that the oxygen molecules present in air were not a major cause of the oxidation of the Cu surface. Similarly, careful observations revealed that the white line features in the fully grown, annealed Gr/Cu samples always exhibited a kink when they met the folded wrinkles in graphene, as shown in Fig. Another key finding is the disappearance of the rhombus-shaped white dot assemblies at the center of some graphene islands grown on Cu(100) grains, as shown in Fig. P ≈ 10−3 Torr, Fig. 2 atmosphere  itself was not a direct cause for the oxidation phenomenon of Cu and that earlier observations in our study were irrelevant to the segregation of oxygen species that intrinsically resided in the bulk of Cu during the annealing process37. Previous theoretical and experimental studies on the dissociative adsorption of O2 and H2O on graphene with or without various defects54, have suggested that O2 has relatively high dissociative reaction barriers of ~1.78 and ~2.80 eV on a pristine graphitic lattice and at the GBs of graphene grains, respectively, except for the di-vacancy region (~0.42 eV); by contrast, H2O molecules on a defective graphitic lattice such as edge (~0.06 eV), mono-vacancy (~0.04 eV) or di-vacancy (~0.35 eV) regions can be easily spilt into hydrogen and hydroxyl radicals, even at room temperature. We therefore performed oxidation experiments in a wet O2 atmosphere (relative humidity (RH) ≈ 80%, Fig. 2O and observed that the oxidation line features using wet O2 were significantly thick and clearly visible compared to those using ambient air  graphene grains with an aberration-corrected HR-TEM; however, detecting the presence of vacancies or pores in our graphitic frameworks was very difficult. Instead, we dominantly observed the graphene GBs consisting of alternating pentagon and heptagon carbon rings, i.e., a chain of SW-like bond rotations . We obtained an inversion energy barrier of ~2.46 eV for an epoxide (C-O-C) at the SW defect, and this energy barrier was dramatically diminished when H, OH, or O atoms were attached to sites around the epoxide, as shown in Table G) after oxygen inversion process through the SW defect is around 0.1 eV, indicating that the inversed epoxide is thermodynamically stable in the Gr/Cu system. Furthermore, considering the fact that a bond breaking energy of the carbon dioxide on Cu is ~1.0 eV59, the inversed epoxide is expected to be more easily dissociated on Cu in our experimental conditions because the epoxide has a single bond (C-O), resulting in the selective oxidation of the underlying Cu through graphene GBs.So, a key question is how the underlying Cu is oxidized through the graphene GBs, which do not involve any removed atoms. To answer this question, we performed spin-polarized density functional theory (DFT) calculations2O molecules at GBs, but it may be hardly possible because the energy barrier for the dissociation (~2.71 eV) is too high54. Instead, we have studied the possibility that the dissociation takes place at other defected sites on graphene. Previous experimental and theoretical studies have indicated that intrinsic defects are always present and stabilized in typical films of large-area graphene produced by CVD and that they can catalyze the dissociation of H2O molecules in ambient air; this dissociation can even occur at room temperature given the very low dissociation energy barriers of the H2O molecule on various intrinsic defects such as edge (~0.06 eV), mono-vacancy (MV) (~0.04 eV) and di-vacancy (~0.35 eV) regions54. For instance, we have performed spin-polarized DFT calculations on the configuration of the H2O molecule on a graphene MV, as shown in Supplementary Figs. 2O molecule is dissociated to one O and two H atoms on a graphene MV and that both the dissociated O and H atoms can diffuse laterally with the energy barriers of ~1.9 eV. Although the energy barriers for O and H atoms for lateral diffusion from the MV are still high, they are significantly lower than the dissociation energy barrier of H2O at GBs (~2.71 eV)54. Once the O and H atoms dissociated at the graphene MV diffuse laterally to non-defected sites (to outward positions), the graphene MV can return to its original configuration, implying that the graphene defects/vacancies will produce O radicals repetitively in a catalytic manner. Importantly, we found that the presence of the underlying Cu greatly reduces the activation barriers of both the dissociated O and H atoms into neighboring C rings. We do not rule out the possibility of the dissociation of H2O at graphene GBs, but it is reasonable to expect that O radicals, which penetrate the graphene through the SW defects, come from the defected sites such as vacancies or edges after the dissociation of H2O molecules. In addition, the resulting atomic oxygen, which escapes from the defected sites, diffuses on perfect graphene with relatively low barriers (mostly < 1 eV), although its actual diffusion rate is different depending on the local environment61. Furthermore, the atomic oxygen can readily diffuse on graphene with Cu substrate compared with a pristine graphene surface without Cu substrate because of the spontaneous electron doping of graphene by Cu63. Finally, upon reaching the GBs, the O atoms accumulate because the high barrier (~1.4 eV)61 for reverse displacement should ensure their stability at low temperatures. From all this we may conclude that the oxygen radicals are generated more frequently by the dissociation of H2O molecules at defected sites of graphene rather than at graphene GBs. And, they diffuse and accumulate on graphene GBs and then penetrate the graphene through SW defects with the assistance of facilitators.Here, it is worthwhile discussing on where oxygen radicals, which penetrate graphene through the SW defects on GBs and finally oxidize the Cu surface, are generated. One possibility is the direct dissociation of H54. In Step 2, the dissociated H and O atoms diffuse laterally from the MV defect site to the six-ring configuration of graphene with the energy barrier of ~1.9 eV each. The H atom is bonded at the top of the C atom and the O atom is stabilized in the middle of the C atoms. In Step 3, the H and O atoms continue their lateral diffusion from one stable six-ring to a neighboring six-ring configuration with the energy barriers of ~0.3 eV64 and ~0.75 eV60, respectively. In Step 4, the accumulation of O atoms at SW defects of graphene is relatively preferred by the diffusion of O atom from perfect hexagon to graphene GBs with the forward and backward energy barriers of ~0.3 and ~0.8 eV, repectively61. Finally, in Step 5, the inversion of the O atom through the SW defects on GBs takes place with the assistance of additional O atoms, with the energy barrier of ~0.70 eV. We note that the suggested oxidation mechanism clearly shows a catalytic cycle of H2O splitting at the graphene defect sites.In Fig. 65, and therefore we can expect that the final state of the carboxyl group returns to the same configuration in which a carbonyl group and two H atoms exist, which results from the direct dissociation of a water molecule at MVs. This means that forming a carboxyl group at a graphene MV site is energetically stable, but it can transform to a carbonyl group and thus can further contribute to oxidation processes. Thus, we may conclude that the formation of a carboxyl group does not halt the oxidation process. In other words, the oxidation process is either hardly affected by the formation of carboxyl group or is affected within an allowable margin. Similarly, we found that the stable hydroxyl group at graphene MV can only contribute to the oxidation process after it has transformed to a carbonyl group and a single H atom. It is noticeable that the possibility that the formation of hydroxyl groups decelerates the oxidation process was higher than that in the case of carboxyl groups because of a relatively high diffusion barrier (~1.23 eV) of the H atom  and carboxyl (O = C–OH) groups on graphene are expected to be present at defective regions in graphene, e.g., the under-coordinated C atoms in graphene GBs since they possess dangling bonds68. It is interesting to note that the O/C ratio of the Gr/Cu samples after air oxidation at ~200 °C for 120 min corresponded approximately to the estimated fraction of intrinsic defects in graphene  group present on graphene in the oxygen transport, we carried out the controlled oxidation experiments of the Gr/Cu samples at ~200 °C as a function of annealing time. The average width of the oxidation line features shows a strong dependence on the annealing time, increasing linearly from ~0.25 to ~1.40 μm with increasing the annealing time from 2 to 48 h, as shown in Fig. We further conducted NEB and DFT calculations to examine the stability of carboxyl groups at defects other than MVs and the possibility that they penetrate at SW defects. For the stability of carboxyl groups, we considered the disconnection between C atoms, so-called unzipping, instead of vacancies for two cases: perfect six-ring and SW defect structures of graphene, as shown in Supplementary Fig. The suggested selective oxidation mechanism of Cu with a polycrystalline graphene barrier is schematically illustrated in Fig. -μm-thick Cu foils  that had been previously cut with razorblade into 4 × 4 cm2 strips. These strips were electrochemically polished in phosphoric acid for 15 min to clean the bare Cu surface and were subsequently rinsed with distilled water followed by isopropyl alcohol (IPA). Immediately after the cleaning process, the Cu strips were loaded into a 4 in. quartz tube followed by evacuation of the chamber to ~3 mTorr; the temperature was then increased to 1000–1050 °C under an H2 atmosphere (H2 flow rate of 5 sccm). After the Cu strips were annealed for 10 min, the graphene was synthesized by introducing methane gas (CH4 flow rate of 10 sccm) and then cooling the Cu strips under the same conditions. Following the growth, the graphene layers were transferred onto SiO2/Si for further investigation using a conventional wet-transfer method assisted by a poly(methyl methacrylate) (PMMA) supporting layer and an aqueous solution of 1M ammonium persulfate.A homemade hot-walled CVD system was used to grow graphene on 25x–y translation stage and a 532 nm excitation source; the laser spot had a diameter of ~640 nm for a ×50 objective lens with NA= 0.5. During the measurements, the laser power was ~1 mW at the sample to avoid laser-induced thermal effects or damage. The Raman spectra and mapping images were analyzed using the WiTec Project software.The Raman spectroscopy and mapping were carried out on a WiTec Alpha 300R M-Raman system equipped with a computer-controlled −9 mbar. To obtain the O composition, we have carried out the curve fitting of the HR O 1s line-scan XPS spectrum using a Gaussian-Lorentzian peak shape after performing a Shirley background correction37.The XPS studies were conducted on a K-alpha spectrometer (Thermo Fisher) using a non-monochromatic aluminum Kα X-ray excitation source operated at a power of 72 W; the diameter of the analysis area was ~0.4 mm and the pass energy for the electron analysis was 50 eV. The base pressure of the analysis chamber was less than ~1 × 10We performed EBSD measurements using a FEI Quanta 3D FEG SEM equipped with AMETEK EBSD system to determine the crystallographic orientation of the Cu foils before and after graphene growth. During the measurements, the probe current, the accelerating voltage, and the incident angle of the beam were maintained at 16 nA, 15 kV, and 70°, respectively. The EBSD data were collected with a 0.25 μm step size and were analyzed using the TSL OIM Analysis 6 software for digitized inversed pole figures.69, we transferred the graphene layer onto Au Quantifoil TEM grids. HR-TEM images and the corresponding selective area electron diffraction (SAED) patterns were taken by an FEI Titan cube G2 60-300 equipped with an image-aberration corrector and a monochromator. It was operated at an acceleration voltage of 80 kV to decrease the beam damage to the graphene layer, especially for detecting the graphene GBs.Using a direct transfer method58. We used ultra-soft pseudo-potential, and local density approximation of Ceperley and Alder type71. The electrons of wavefunctions were extended up to 500 eV of cutoff energy in a plane-wave basis set. The calculations were relaxed until forces on ions became less than 0.01 eV Å−1. For sampling Brillouin zone, the Monkhorst-Pack k-point grid was 5 × 5 × 172. For the penetration calculations, we employed a 6 × 6 structure consisting of 72 carbon atoms for a free-standing graphene and a 4 × 4 graphene sheet (32 carbon atoms) and 3 layers of Cu(111) slab (48 Cu atoms) for a graphene on a Cu substrate. For the diffusion simulations, we employed the same structures but one carbon atom is missing to model the MV. The minimum energy pathway for the penetration and diffusion was examined by the nudged elastic band method (NEB)73.We have performed DFT calculations on the penetration energy of single oxygen atom through a graphene sheet and on the lateral diffusion of O and H atoms from unsaturated C atoms. In this work, all calculations were performed using Vienna Ab-initio Simulation Package (VASP) based on the spin-polarized density functional theoryThe data that support the findings of this study are available from the corresponding authors upon request.Supplementary Information


Chlamydia trachomatis are higher in new arrivals to a community and in travelers who leave for extended periods, suggesting they are sources of re-infection. This community-randomized, clinical trial was designed to determine if a surveillance program that targeted newcomers and travelers, identified weekly, would result in more communities achieving levels of infection of ≤1%.Trachoma is targeted for global elimination. Infection rates with 52 communities were randomly allocated 1:1 to the control  or intervention arm . In each community, surveys were completed every six months on a random sample of 100 children ages 1–9 years for trachoma and infection. The primary outcome was the proportion of communities in the intervention arm, compared to the control arm, which had a prevalence of infection at ≤1% by 24 months. Registered: clinicaltrials.gov(NCT01767506).Intervention communities experienced an average of 110 surveillance events per month. At 24 months, 7 (27%) of 26 intervention communities achieved a prevalence of infection ≤1% compared to 4 (15%) of the 26 control communities . At 24 months, the average infection prevalence in the intervention communities was 4·8, compared to 6·9 in the control communities (p = ·06).Despite surveillance programs for community newcomers and travelers, the proportion of intervention communities with a level of infection ≤1% was lower than expected and not significantly different from control communities. Chlamydia trachomatis, and remains the leading infectious cause of blindness world-wide.  In InC. trathers.[,  The commWhile we believe that the significant effort put into supervision of the surveillance program succeeded, one possible limitation is the design of the program itself. We did not plan to detect newcomers who were solely children, as we reasoned it would be unlikely they would arrive without a guardian to an existing house. We know of instances when grandmothers picked up children from elsewhere to take care of them, but these were not strictly new child and guardians, so these children were not tracked. Movement of children alone within a village was not of interest, as they would not have brought in infection from outside. Also, we defined traveling families as those whose members all left, and were gone for a continuous eight weeks. Some members who travel, or those who left for less than eight weeks, would not be tracked. This definition was deliberate, to avoid having to track the considerable number travel for many for a short period of time to a farm, where it is unlikely the family would encounter anyone else. These short trips would have placed a significant burden on a surveillance program. We may have missed infections brought in by those who did not meet our criteria for surveillance, although we felt this was minimal. Moreover, the effort and cost of mounting a surveillance program that intensive without more evidence it would be effective may not be justified.Despite our earlier finding of higher rates of infections in neighborhoods where newcomers settled, we cannWe must also interpret the data in light of the significant numbers of study communities who did not have MDA at baseline and one year, because as communities they had reached a prevalence of trachoma <5%. All these communities came into this trial having had three rounds of MDA with high coverage from a previous trial a year prior. At baseline, 17 of them did not have MDA and of these, 7 (41%) had to re-start MDA based on infection re-emerging to 6% (our pre-specified rule). These data suggest that, in a district that had not yet reached <5% trachoma prevalence, stopping MDA in individual communities or even sub-districts who have reached that prevalence is not advisable as the risk of re-emergence of infection appears to be higher than anticipated.In summary, despite a surveillance and treatment program for community newcomers and travelers to prevent reemergence of infection, the proportion of intervention communities who achieved a level of infection ≤1% was lower than expected and not significantly different from control communities. Targeting potential incoming sources of infection alone may not be justified.S1 File(DOC)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


The speed of solid-state electronic devices, determined by the temporal dynamics of charge carriers, could potentially reach unprecedented petahertz frequencies through direct manipulation by optical fields, consisting in a million-fold increase from state-of-the-art technology. In graphene, charge carrier manipulation is facilitated by exceptionally strong coupling to optical fields, from which stems an important back-action of photoexcited carriers. Here we investigate the instantaneous response of graphene to ultrafast optical fields, elucidating the role of hot carriers on sub-100 fs timescales. The measured nonlinear response and its dependence on interaction time and field polarization reveal the back-action of hot carriers over timescales commensurate with the optical field. An intuitive picture is given for the carrier trajectories in response to the optical-field polarization state. We note that the peculiar interplay between optical fields and charge carriers in graphene may also apply to surface states in topological insulators with similar Dirac cone dispersion relations. Graphene possesses a nonlinear optical response arising from its electronic dispersion. Here, the authors measure the response of graphene to an ultrafast optical field and provide an explanation of the quantum dynamics of Dirac carriers mediating the material’s nonlinear response. In particular, its electron transport properties9, which are described by a two-dimensional gas of massless Dirac fermions (MDFs) moving at a velocity of 1 nm fs−1, are of interest to develop ultrafast optoelectronic devices10. Its room-temperature carrier mobility of up to 1.5×106 cm2 V−1 s−1 is unusually high2, while the third-order susceptibility of a single-atom-thick graphene layer is 108 times stronger than in a dielectric material11. The large nonlinear response of graphene14 is attributed to its linear electronic energy dispersion17, which not only provides resonant interband transitions for a continuous range of low photon energies, but in the presence of intense optical fields leads to square-wave oscillatory motion of Dirac fermions . Third-harmonic generation16 and four-wave mixing were observed early on, while an extreme nonlinear effect has been recently reported, consisting of transient plasmons enabled by strong optical pumping17. High-harmonic generation (HHG) up to the 5th order has been also observed at THz frequencies18, and more recently the light polarization dependence has been investigated for high harmonics19. Additionally, the introduction of a slight bandgap in the graphene dispersion effectively enhances the nonlinear response compared to that of gapless graphene21. Consequently, the nontrivial interactions among optical fields and Dirac fermions in graphene, even at low-field strengths, demands an investigation of the non-equilibrium response of the material’s charge carriers as a first step toward the implementation of ultrafast optoelectronic control23.Graphene is a remarkable material19. Due to the low-field regime of our investigation. In particular, we observe a blue-shifted nonlinear response that is not unique to graphene and is known to occur in semiconductors , as well as in gas-filled photonic crystal fibers undergoing ionization26. Our findings reveal that remarkably, despite the negligible propagation path length within the two-dimensional medium, the blue-shift signature is measurable for the generated harmonics, thus providing insight into the ultrafast dynamics of MDFs in graphene, matched by excellent agreement with simulations.Here, we focus on the ultrafast nonlinear response of graphene arising from the photo-generated free carriers at moderate pump intensity, describing their effect on HHG along with the dependence on elliptical polarization, which we find quite different from ref. 29. Finally, electron–phonon coupling leads to electron relaxation and electron-hole recombination, thereby gradually reducing the electron temperature over a characteristic picosecond time scale. The described non-equilibrium carrier dynamics of graphene have been experimentally investigated, in particular through measurements in the THz regime30 and also using ca. 10-fs-duration pulses in the visible29. Such measurements were instrumental to elucidate the dynamics of charge carriers in this material, but an implementation of ultrafast optoelectronics requires additional knowledge on how charge carriers influence the optical fields themselves.Figure π electrons are treated as MDFs; and complementary time-domain simulations combining an atomistic tight-binding model for the electrons with the random-phase approximation formalism (TB-RPA). We find that these two approaches yield results in excellent agreement for the experimentally relevant conditions.Here, we investigate the direct back-action of the carrier dynamics on the optical field at the petahertz scale through harmonic generation. Indeed, spectral reshaping of the fundamental optical field is amplified by the harmonic order, and the response at the generated frequencies is disentangled from that of the fundamental excitation pulse itself. We employ two different theoretical models to describe this interaction : one based on a non-perturbative continuum picture in which −2. The absence of a bandgap, in combination with the linear energy dispersion of graphene, leads to an instantaneous photo-generation of free carriers by the optical field and a delayed recombination with a metal-like instantaneous response and time-dependent plasma frequency. This behavior is markedly different compared to systems with a large bandgap, such as free atoms and insulators32. To illustrate the difference, we show the temporal evolution of the ionization yield for an atomic medium (orange curve), where the well-known step-like response of the emitted electrons is clearly discernable. Depending on the strength of the optical field, the medium may be fully ionized already at the leading edge of the pulse, in such a way that the trailing edge experiences a constant carrier density. The scenario is distinctly different in graphene because free carriers are instantaneously generated whenever an optical field is present. In addition, efficient and rapid electron–electron scattering occurs due to the high mobility of free carriers, thus affecting the optical field in a transient manner, which is markedly different from the abrupt temporal variation of the atomic medium.Figure Consequently, in graphene the generated free carriers oscillate synchronously with the optical field at early times (leading edge of the pulse), but modify the optical response for later times during the optical field (trailing part of the pulse). The result is an induced asymmetry on the leading and trailing parts of the optical field Fig. , and a sThe back-action of charge carrier motion to the optical field is revealed by the time-energy plot of the resulting nonlinear light emission in Fig. 33. We focused the pulses at normal incidence onto a 5-monolayer graphene sample that was transferred onto a 0.4-mm thick CaF2 substrate  FWHM pulses with a central wavelength of 3.1 μm (0.4 eV photon energy) from a mid-IR optical parametric chirped pulse amplifier (OPCPA)−2. We observed the generation of harmonic frequencies up to fifth order and with proportional blue-shifts compared to the nominal third and fifth harmonic (dashed curves). This first observation of the fifth harmonic is commensurate with predictions35 and contradicts earlier measurements in multilayer graphene at terahertz frequencies in which the absence of optical harmonics was attributed to fast carrier scattering36, which at THz frequencies is heavily affected by hot thermal carriers. To further investigate the influence of the charge carriers, we measured the scaling of the blue-shift with peak intensity and pulse duration. Longer pulses, up to 340 fs duration, at constant peak intensity, were generated by spectrally narrowing the laser output and increasing the pulse energy proportionally with the increase of pulse duration. We resorted to spectral narrowing rather than simple frequency chirping to exclude additional dynamical effects associated with pulse propagation in graphene. Figure A characteristic measurement for linear polarization is shown in Fig. k-space and their effect on the nonlinear response, we repeated our measurements with varying polarization for the incident mid-IR field. Figure ∈ and that the resulting third harmonic was two orders of magnitude weaker for a circularly polarized field (ε = 1) compared with a linearly polarized field (ε = 0). Of particular interest is the dependence of the blue-shift on ellipticity37. Fig. To gain further insight into carrier dynamics in 38 with circular polarization are well-known phenomena, the scenario here investigated is markedly different due to the availability of carriers, the ballistic transport of Dirac fermions, and the band topology in graphene. Our simulations clearly confirm a reduction of anharmonic response, and hence a diminished harmonic generation that reproduces the measured reduction in blue-shift with increasing ellipticity. The origin of this behavior becomes clear from a trajectory analysis of field-driven Dirac fermions, as shown in Fig. π phase in k-space during each optical cycle. The availability of carriers across k-space ensures that a matching trajectory always exists that is π out of phase, so that any anharmonic response is effectively canceled for circular driving fields. It is worth emphasizing that the observed ellipticity dependence of harmonic generation is not unique to graphene because it inherently arises from crystal symmetry. Three-fold rotational symmetry (which is present in graphene) generally forbids THG under circularly polarized illumination39. Thus, in a simple picture, the quenching of blue-shift for circularly polarized light can be readily understood from the vanishing of the harmonic generation process.Our simulations qualitatively reproduce the experimental findings and are indicated by the dashed blue curves superimposed on the experimental data presented in Fig. We identify both the dynamics of graphene Dirac carriers driven by optical petahertz fields and the back-action of these dynamics on the driving field as revealed by blue-shifts in the generation of third and fifth harmonics. Theoretical modeling explains the results and allows us to track down carrier trajectories in the specific topology of Dirac cones, which readily explains the disappearance of the blue-shifts when the incident light is circularly polarized. These investigations of the dynamics of Dirac carriers in graphene provide important insight for the design of future graphene-based ultrafast optoelectronic devices, while they open an avenue toward the exploration of topology-dependent carrier dynamics in other two-dimensional systems, such as surface states of topological insulators and other two-dimensional materials.2) graphene monolayers grown by chemical vapor deposition on a sputtered Cu film (Aixtron) and transferred on top of one another onto a 0.4-mm thick CaF2 substrate. Each graphene layer is transferred by spin-coating polymethyl methacrylate (PMMA) on top of the graphene/Cu surface and etching the Cu copper using an oxidizing solution of 0.1 M ammonium persulfate. The floating PMMA/graphene layer is transferred to DI water and scooped using the CaF2 substrate. Once the sample is dry, PMMA is removed with acetone and the sample is rinsed in isopropanol. We performed electrical measurements on similar graphene samples and typically found an intrinsic hole doping of ~5 x 1012 cm−2, with a room-temperature hole mobility of ~500 cm2 V−1 s−1.The graphene sample consists of a 5 large . The electric field components along the major and minor axes of the polarization ellipse were separated with a polarizing beam splitter cube  placed after the sample. The generated third harmonic signal was separated from the fundamental and simultaneously focused into a fiber-coupled spectrometer  with transmissive BK7 optics.In the experiment, the mid-IR output of an OPCPA system is used as fundamental pump beam to investigate the optical response of graphene. We used output pulses at a center wavelength of 3.1 μm, with pulse energies of 1.3 μJ and pulse durations of 80 fs at a pulse repetition rate of 160 kHz. In the experimental setup, the mid-IR driving pulses are focused with a 30-cm-focal-length CaFπ electrons are treated as MDFs; and (ii) an atomistic tight-binding model for the electrons combined with the TB-RPA. These two approaches yield results in excellent mutual agreement under the conditions of the present experiment. (i) The MDF model (see Supplementary Note k + (e/c)A, where k is the electron momentum, whereas E. Electrons are treated independently and their dynamics described by the Dirac equation for massless fermions, which can be recast as in the form of Bloch equations, involving the population inversion nk, the interband coherence ρk, and a phenomenological relaxation time τ. We numerically integrate these equations to extract the k-resolved induced current and is integral over k to yield the total current. A self-contained presentation of this method is given in Supplementary Information  and ϕ is the electric potential produced by the incident light plus the Hartree interaction. We express the density matrix HTB with complex-number expansion coefficients τ = 100 fs in both approaches.Our simulations are based on two complementary approaches to describe the nonlinear optical response of graphene: (i) a non-perturbative continuum picture in which mentclass2pt{minimThe data that support the findings of this study are available from the corresponding author upon request.Supplementary Information


The development of electrochemical biosensors for the simultaneous detection of ascorbic acid (AA), dopamine (DA), uric acid (UA), tryptophan (Trp), and nitrite ( Human body fluids contain electrolytes, including a wide variety of electroactive materials, including biological, organic, and inorganic electrolytes. L-ascorbic acid (AA), dopamine (DA), uric acid (UA), tryptophan (Trp), and nitrite , reduced graphene oxide (rGO), and chemical vapor deposition (CVD) graphene. Of these, GO and rGO are the most commonly used in electrochemical sensorsBased on these above considerations, a biosensing electrode with CVD graphene as sensitive layer was here developed, and Ta wire served as the basis electrode. The present work focuses on evaluating the simultaneous detection of AA, DA, UA, Trp, and 2  of graphene/Ta wire electrode. The thickness of graphene nanosheets can be confirmed using high-resolution transmission electron microscopy (TEM)  Fig.  and atomEM) Fig.  images. −1, G band at around 1592.7 cm−1, and 2D band at approximately 2702.5 cm−1. The D band is a characteristic feature, indicating defects. It can be caused by excitation of the edges of graphene sheets and their random orientation48. The G band shows sp2 vibrations in the graphitic plane, which confirms the presence of graphene sheets. Intense, well-defined 2D bands with a narrow width of full width at half maximum is peculiar for few layer graphene. The X-ray diffraction (XRD) pattern of the graphene/Ta wire is shown in Fig. 46.The Raman spectrum of graphene layer is shown in Fig. 3[Fe(CN)6] and 5.0 mM K4[Fe(CN)6], respectively. For the TaC/Ta wire, the Nyquist plot consisted of two semicircles in high- and medium-frequency regions followed by an inclined line in low-frequency region. For the graphene/Ta wire, the Nyquist plots consisted of a semicircle in high-frequency region followed by an inclined line in a medium-low-frequency region. According to the Randles-type circuit in Fig. Rs) is created by the electrolyte. The semicircle indicates the charge-transfer resistance (Rct) at the external graphene/electrolyte interface; the interface resistance (Rint) is contributed from the bottom graphene layer. As shown, the diameter of the semicircle for the TaC/Ta wire (Rct = 200 Ω cm2) is much larger than that of the graphene/Ta wire (Rct = 37.2 Ω cm2), indicating the graphene/Ta wire electrode exhibited better charge transfer performance than the TaC/Ta wire and had excellent conductivity.Figure 49. The graphene/Ta wire for use as a sensing electrode, the oxidation potential is dominated by both the difference in energy between the Fermi level of graphene (EF ≈ −4.6 eV)50 and the highest occupied molecular orbital (HOMO) of analytes, and energy gaps between the HOMO and lowest unoccupied molecular orbital (LUMO) of the analytes. HOMO and LUMO energies approximately represent the a ability of molecules to donate and accept electrons51. H. Y. et al.49, reported that the oxidation potential increases with the increase of LUMO-HOMO energy gap. In this study, molecules and MO orbitals were prepared using GaussView 5.0 and all the calculations were performed using Gaussian V9.5. Moreover, all the molecules were fully optimized using the density functional theory (DFT)/B3LYP method with 6–31 G basis sets in water. The MO data was calculated at the B3LYP/6-311++G  level for AA and DA, while that of UA, Trp, and EF (of graphene)-HOMO  values decreases in the order UA (4.17) > 52. Dissociation constant (pKa1) values of AA, DA, UA, Trp, and I = 5.89), and 3.29. The DA molecules did not dissociate into anions in pH 7.0 PBS, which further increased the oxidation potential of DA. Corresponding to the difference in the dipole moment, the HOMO and LUMO of Trp are localized on the indole ring, the LUMO of 2ˉ > UA > Trp > AA > DA.The individual electrochemical response of these analytes needs to be studied before the sensitivity of graphene/Ta wire electrodes toward the simultaneous detection of AA, DA, UA, Trp, and 2 atoms from the six-benzene-ring graphene and the aromatic rings from to DA, UA, and Trp. Hence, high oxidation currents translate to higher sensitivity in the detection of DA, UA, and Trp than in the detection of AA and The sensing mechanism of the graphene/Ta wire is shown in Fig. res Fig. . Graphen55.Due to the availability of both large peak separations and enhanced currents, the graphene/Ta wire electrode is the best electrode for sensing the simultaneous detection of AA, DA, UA, Trp, and Because pH has a profound effect on the electrochemical response of the sensing electrodes toward the simultaneous determination of AA, DA, UA, Trp, and In order to identify the key parameters for detection of five species at graphene/Ta wire as a biosensor, the DPV curves of the mixtures of AA, DA, UA, Trp, and 2ˉ. H. Y. et al. explained the phenomenon with the change of adsorption interaction49. The interface interaction of monolayer adsorption is strong, while that of multilayer adsorption is weak; hence, the slope of the working curve changed with increase in the concentration of analytes. Further, since the DA molecule contains an aromatic ring, a well-ordered organic layer was formed between the graphene and the aromatic ring of DA through π-π stacking interactions, which resulted in strong adsorption; therefor, the slope did not change within this concentration range. Compared with the flat graphene composite electrode, the sensitivity of the graphene/Ta wire electrode reached the same level, and the detection limits of DA, UA, and Trp are very advantageous56.When the five analytes were detected simultaneously, i.e. when the concentrations of all five were changed, the calibration curves were linear and within the concentration ranges of 5–1500 μM for AA, 0.25–75 μM for DA, 0.4–120 μM for UA, 0.25–75 μM for Trp, and 2–600 μM for 59. All this evidence supports the idea that the graphene/Ta wire electrode has an excellent ability to determine AA, DA, UA, Trp, and The LOD for AA, DA, UA, Trp, and The stability of the graphene/Ta wire electrode was also studied in this work. When the electrode was cyclically swept for 100 cycles, there was a 4.8% decrease in the initial response of the graphene electrode, indicating that the graphene electrode had excellent stability Fig. . The rep2, 500 μM CuSO4, 500 μM KCl, 500 μM Mg(NO3)2, 500 μM Ni(NO3)2, 500 μM Zn(NO3)2, 500 μM Na2S, 500 μM FeSO4, 25 μM aniline, 25 μM phenol, 25 μM hydroquinone, 25 μM catechol, 25 μM resorcinol, 25 μM citric acid, 25 μM glucose, 25 μM L-alanine, 25 μM L-cysteine, 25 μM L-glycine, 25 μM L-lysine, and 25 μM L-tyrosine did not significantly influence the height of the peak currents. The tolerance limit was defined as the concentration giving an error of ≤5% in the determination of AA, DA, UA, Trp, and Several forms of inorganic, organic, and biological interference were tested to determine whether they could affect the detection of AA, DA, UA, Trp, and O2− Fig. . The res60, HPLC-CL61, GC/MS62, and UV methods63; however, the concentration of NO2ˉ is significantly higher than that measured by the spectrophotometric methods in the ref. To evaluate the application of the graphene/Ta wire electrode, an attempt was made to detect five species in the human serum samples. Each sample was diluted 5 times with 0.1 M PBS (pH 7.0) before measurement. The electrochemical responses for detection of AA, DA, UA, Trp, and Free-standing graphene nanosheets were here fabricated on Ta wire, which was used to selectively and simultaneously detect AA, DA, UA, Trp, and 2) were purchased from Tianjin Guangfu Fine Chemical Research Institute . Phosphate buffered solutions (PBS) with various pH values were prepared using 0.1 M Na2HPO4 and 0.1 M NaH2PO4. Ultra-pure water was used in all experiments.AA and DA were purchased from Aladdin Reagent Co., Ltd. . UA was purchased from Sigma Aldrich (U.S.). Trp and sodium nitrite (NaNO3)2), and drying. The main deposition parameters are shown in Table Ta wires with a diameter of 0.6 mm were cut from 5 cm length wire-like substrates. The free-standing graphene nanosheets covered Ta wires, i.e. the graphene/Ta wire electrodes were prepared using a high-power direct current (DC) arc plasma jet CVD operating at gas recycling mode. The Ta wires were cleaned and attached to catalysts as follows before being placed in the deposition chamber: ultrasonic cleaning in ethanol and ultra-pure water, drying, immersion in the catalyst precursor solution (0.6 M Ni(NOα radiation (wavelength of 0.15406 nm).The morphology of the CVD graphene obtained by scanning electron microscopy , transmission electron microscopy  and atomic force microscopy  measurements, respectively. Sample treatment for AFM measurement: The graphene/Ta wire electrode was immersed in ethanol and sonicated in an ultrasonic bath for 48 h (600 W/40 kHz). During this time, a small amount of graphene sheet was peeled off and dispersed in ethanol. The graphene/ethanol solution was then sprayed onto the Si wafer and dried. The Raman spectrum of graphene was recorded on a Renishaw Raman microscope with an excitation laser wavelength at 532 nm. X-ray diffraction (XRD) spectra were created using a Rigaku Ultima IV x-ray diffractometer with CuKThe electrochemical performance was investigated using cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) with a CHI 660E electrochemical workstation . In a three-electrode system, a graphene/Ta wire electrode/or other sample electrodes, a platinum (Pt) sheet and a saturated calomel electrode (SCE) served as the working electrode, counter electrode, and reference electrode, respectively Fig. .Supplementary Information


Fusobacterium nucleatum and Porphyromonas gingivalis were highly increased within tissues, comprising 15–40% of the total bacteria. Furthermore, biofilm formation within the tissue was observed by Alcian Blue staining and atomic force microscopy, where degradation of fibers was prominent. Taken together, bacteria formed complex biofilm communities within gingival tissues that may serve as a reservoir for persistent infection. This novel finding may instigate new research into therapeutic strategies to treat periodontitis.Periodontitis is caused by dysbiosis of subgingival plaque that results in increased bacterial invasion into gingival tissues. Although shifts in subgingival microbiota from healthy to periodontitis have been well characterized, the characteristics of bacterial communities located within gingival tissues have not been studied. To characterize microbiota within the tissues of periodontal lesions in comparison with plaque microbiota, gingival tissues and subgingival plaque were obtained from the same tooth of patients with periodontitis (n = 7). A pyrosequencing analysis of the 16S rRNA gene revealed that species richness and diversity were not significantly different between the two communities. However, inter-subject variation in intra-tissue communities was smaller than that in plaque communities. Intra-tissue communities were characterized by decreased Firmicutes and increased Fusobacteria, compared with the plaque communities. Particularly,


Flower-like MoS2 nanoflakes were successfully grown on graphene nanosheets and characterized to understand the temperature-dependent crystallization process and the electrochemical performance. The highest electrocatalytic efficiency for both the dye-sensitized solar cell and the hydrogen evolution reaction was obtained by preparing the hybrid at 180 °C, which benefits from balanced high reactivity and high conductivity. This research leads to a better understanding of temperature dependence of MoS2 crystallization and offers guidelines for better catalytic material design.This work primarily studies the crystallization condition of molybdenum disulfide (MoSThe online version of this article (doi:10.1186/s11671-017-2248-9) contains supplementary material, which is available to authorized users. Two-dimensional (2D) hybrid materials have been studied for use in photovoltaics, water splitting, sensors, batteries, and many other applications, often in the form of heterojunctions or three-dimensional (3D) frameworks –6. Benef2/graphene hybrid has been studied as one of the most promising options because of its excellent electrocatalytic activity and unique 2D structure [2 [2/graphene hybrid combines the benefits of reactivity and conductivity of the two constituent materials, thereby leading to significantly enhanced electrocatalytic performance [2 formation to improve the production rate [Compared with traditional platinum (Pt)-based catalyst materials, 2D hybrid materials offer a comparable performance and a much lower production cost, thus demonstrating their great potential for replacing Pt for commercial use. Until now, the MoStructure , 14, 15.cture [2 , 17 and cture [2 –20. The formance , 22. In ion rate –27. Beca2 and graphene through a hydrothermal reaction in organic solvents and explored the kinetics of catalytic reactions [2 onto graphene and highlighted the effect of coverage factor [2/graphene hybrids [2 could change significantly with different reaction temperatures, with amorphous MoS2 nanospheres at low temperatures (120–150 °C), flower-like MoS2 balls with a high catalytic performance at mid-range temperatures (160–240 °C), and large MoS2 nanoparticles at high temperatures (230–260 °C) [2 is not well understood, and thus further understanding of the crystallization condition is essential to optimize the material catalytic activity. In this work, we report a facile hydrothermal method to prepare MoS2 nanoflakes grown onto graphene nanosheets at different mid-range temperatures. MoS2 crystallization on graphene nanosheets can be clearly identified by various crystal characterization methods, and the effects of the crystallization on the resulting catalytic performance are studied by DSSC performance and HER reactivity.To create the hybrid, many approaches have been explored and their advantages have been compared. Dai’s group prepared the heterojunction of MoSeactions . Zhang ee factor . In rece hybrids , 29–32. –260 °C) , 34. How2/graphene hybrids were prepared by the hydrothermal method (details in the Supporting Information). First, microwave-exfoliated graphene oxide nanosheets (MEGO) were prepared from graphite oxide under an argon environment with exposure to 900 W microwaves for 90 s [Various MoSfor 90 s ; this prfor 90 s . Then, 2for 90 s . The sus2/graphene hybrid prepared at 180 °C. X-ray diffraction (XRD) was done using a Bruker D8 Discover X-ray diffractometer. Raman spectroscopy was taken with a Renishaw Raman spectrometer (Inc 1000B) with an HeNe laser (633 nm). X-ray photoelectron spectroscopy (XPS) was studied through VG ESCA 2000 with Mg, Kα as X-ray source, and peaks are calibrated with C1s peaks at 284.6 eV.The structure of prepared materials was studied with a Hitachi (S-4800) field-emission scanning electron microscope (FE-SEM). The energy-dispersive X-ray spectroscopy (EDS) mapping data were obtained using a Bruker detector on a Hitachi S-4800. A Hitachi (H 9000 NAR) system was used to take transmission electron microscope/high-resolution transmission electron microscope (TEM/HRTEM) and to study the hybrid junction of the MoS2 nanoparticle structure was formed, by doctor-blading a commercial TiO2 paste and gradually heating to 500 °C over 30 min. After the treatments, the substrates were transferred to 0.5 mM N719 ethanol solution and were soaked for 24 h. The counter electrodes were also fabricated by doctor-blading. The slurry contains 20 mg sample and 5 μL Triton ×100 in 500 μL DI water. After coating, the electrodes were annealed at 500 °C for 30 min in an argon environment. Pt-based counter electrodes were fabricated by blading 0.01 M H2PtCl6 ethanol solution with the same steps. To assemble the cell, the prepared counter electrodes and photoanodes were sealed with a commercial thermoplastic sealing film, and then a commercial electrolyte was injected into the cell.First, FTO glasses were sequentially cleaned with acetone, isopropyl alcohol, and DI water. Following earlier publications , a TiO2 2, Newport, 94021A) with a Keithley 2420 source meter. The system was calibrated with a Si-reference cell . The electrochemical impedance spectroscopy (EIS) of DSSCs was tested at a frequency from 0.1 to 10,000 Hz, under one sun illumination. The potential was set at 0.7 V, which is about the average open circuit voltage. The data was recorded by a CHI 760D electrochemical workstation.The J-V characterization was conducted under a simulated one sun illumination . To fabricate GCEs, 5 mg of material was mixed with 50 μL Nafion ethanol solution (5%) and 450 μL DI water. The mixture was well dispersed and a 5 μL suspension was dropped onto a glassy carbon electrode with a diameter of 3 mm and then fully dried.A saturated Ag/AgCl reference electrode was used in all measurements and was converted to the reversible hydrogen electrode (RHE) scale via the Nernst equation. All measurements were carried out in 0.5 M HThe linear sweep voltammetry (LSV) was tested from 0.2 to −0.8 V (vs. Ag/AgCl) at 5 mV/s; later the Tafel plot was calculated from LSV. Cyclic voltammetry (CV) was scanned between −1 V and 1 V (vs. Ag/AgCl) at 0.05 V/s. The electrochemical impedance spectroscopy was measured at a frequency ranging from 0.1 to 10,000 Hz at a constant potential 0.5 mV (vs. Ag/AgCl). The stability was evaluated for 20,000 s at a constant potential −0.5 V (vs. Ag/AgCl).2 structure grown on the graphene surface. The perpendicularly oriented, flower-like MoS2 nanoflakes were observed at all temperatures and the uniform coverage was proven by EDS  loaded on the surface of graphene (a flat gray area) was observed, as shown in Fig. 2 in 2H–crystal (002) face, and the 0.23 nm lattice spacing is close to that of the zig-zag chain gap in a single-layer graphene nanosheet [2 nanosheets crossed over to each other in the small area, representing the formation of small nanoflakes and the creation of edges and defects. The seamless stitching of the graphene nanosheet to MoS2 nanosheets, marked by the dashed circle in Fig. To gain further insights into MoSanosheet . The few2/graphene hybrids  and (105) of the 2H phase, the broadened and shifted (100) peak, and, importantly, an additional (006 + 104) peak. The rearrangement in the crystals indicates possible 1 T phase existence [2 Raman signatures, which suggest poor crystallization quality. The intensity of A1g, E2g1, and E1g peaks increased with the increasing temperature. Also, the out-of-plane Mo-S phonon mode (A1g) is preferentially excited for the edge-terminated perpendicular orientation of MoS2 nanosheets, and the high intensity of A1g shown in the MG-210 and MG-240 hybrids indicates the perpendicularly oriented structure formed on graphene nanosheets [2 Brillouin zone, which indicates improved crystallization quality at a high temperature [D/IG) of graphene with increasing temperatures, as shown in Fig. 2 nanosheets and graphene nanosheets, which enhanced the breathing mode of the hexagonal ring of graphene.To gain deeper understanding of the crystallization change at different reaction temperatures, the XRD and Raman spectra of the MoSxistence . The wea2/graphene hybrids.Additional study using XPS Fig.  also pro2 is always preferred. However, a significantly lower crystal quality leads to poorer charge transfer and lower catalytic performance [2 is known to show limited stability in ambient environment [2 formation, and the analyses are applicable here [2 crystals along the (002) face based on HRTEM results; defects are easier to form at a low temperature due to slower chemical kinetics, which exposes vacant Mo or S to the environment. Density functional theory (DFT) calculations show that reduction reactions tend to happen more along Mo-Mo grain boundaries than point defects in lattice [Previous studies reported that defects in crystals can increase the catalytic reaction rate, and a 1 T phase of MoSformance , 42. It ironment , 41, 43,ble here , 44. Fir lattice , and the2 for catalytic reactions has also been studied for pure 2D crystals. Earlier, DFT calculations suggest the 1T–MoS2 shows metallic properties and has a significantly higher catalytic reactivity compared with semiconducting 2H–MoS2 [2 nanosheets are always prepared by chemical exfoliations by alkali metal [2 [2/graphene hybrids in this work, show lower temperature requirements at 180 °C, and this can be explained by the faster seeding process with graphene as the supporting media and crystal constant alignments during crystallization. A first-principle study of MoS2/graphene heterojunction shows that the work function of graphene (4.3 eV) matches well with the conduction band (4.2 eV) of monolayer MoS2, and the calculated charge carrier density in MG hybrids are over 3 orders of magnitude higher than the intrinsic value of graphene. Furthermore, the electron-hole pairs are well separated in the structure, which promotes a higher reactivity [The importance of 1T phase of MoS 2H–MoS2 , 41, 46. 2H–MoS2 . Pure 1Tli metal , to obtametal [2 . MoS2/gractivity , 48, 49.2/graphene hybrids were first investigated in DSSCs. DSSCs have a sandwich structure with a sensitizing material-coated semiconducting layer as the photoanode, a pair of redox as the electrolyte, and a reducing catalyst as the counter electrode [2/graphene hybrid as the counter electrode in DSSCs, both the conductivity and the catalytic reactivity relevant to its electrochemical properties can be directly characterized.The electrocatalytic activity of MoSlectrode . DSSCs h2-based photoanode, I3−/I− electrolyte, and MoS2/graphene hybrid counter electrodes for DSSC measurements, as shown in Fig. VOC) at around 0.7 V, which is close to that of the Pt-based catalyst, while the short-circuit current (isc) dropped to 8.47 mA/cm2 for MG-210 and 7.71 mA/cm2 for the MG-240 hybrids. The increased fill factor (FF) for high temperature hybrids results from the lower isc and VOC. It is clear that isc is the dominating factor for the efficiency that depends on fast charge transportation in the hybrids. Comparing the MG-150 and MG-180 catalysts, the MG-180 hybrid gave a higher isc, which suggests either a better conductivity or a higher reactivity, and agrees well with the weakened charge transportation prediction by excessive defects in the MG-150 hybrid. The low performance of the MG-240 hybrid is predictable because of the over-stacking of MoS2 nanosheets, shown in SEM images of Fig. 2 crystals. A further investigation on the resistance through EIS analysis  tests indicated that MG-180 has a specific surface area of 73.5 m2/g, compared with those of MG-150 (49.5 m2/g) and MG-210 (73.4 m2/g). The result agrees well with the highly branched structures shown in the SEM images. The Tafel slope of 137 mV/decade for the MG-150 hybrid also explains its slightly lower efficiency in DSSCs. CV results  to 2H in the high-temperature region (above 210 °C), along with crystal quality improvement and reduced defect sites. The existence of the 1T phase improves the reduction reactivity and charge transfer ability of the hybrid. The controlled defect sites also improve the catalytic reaction rate. The morphology of MoS2 on graphene is essential for maintaining high catalytic performance and perpendicularly oriented structures in flower-like shape is preferred. This work provides a fundamental guideline and understanding for the rational design and construction of 2D hybrid materials for electrocatalytic applications.In summary, the crystallization condition of MoS


L1-deficient mice partially overlap with those of Reelin-deficient reeler mice. In utero electroporation of L1-80 into reeler embryos normalised the migration of cortical neurons in reeler embryos. The combined results indicate that the direct interaction between L1 and Reelin as well as the Reelin-mediated generation of L1-80 contribute to brain development at early developmental stages.The cell adhesion molecule L1 and the extracellular matrix protein Reelin play crucial roles in the developing nervous system. Reelin is known to activate signalling cascades regulating neuronal migration by binding to lipoprotein receptors. However, the interaction of Reelin with adhesion molecules, such as L1, has remained poorly explored. Here, we report that full-length Reelin and its N-terminal fragments N-R2 and N-R6 bind to L1 and that full-length Reelin and its N-terminal fragment N-R6 proteolytically cleave L1 to generate an L1 fragment with a molecular mass of 80 kDa (L1-80). Expression of N-R6 and generation of L1-80 coincide in time at early developmental stages of the cerebral cortex. Reelin-mediated generation of L1-80 is involved in neurite outgrowth and in stimulation of migration of cultured cortical and cerebellar neurons. Morphological abnormalities in layer formation of the cerebral cortex of  L1-deficient mice show severe malformations and malfunctions of the nervous system8. In humans, mutations in L1 are associated with the L1 syndrome which comprises a spectrum of mild to severe congenital X-linked developmental disorders9. The L1 syndrome is characterised by developmental and mental retardation, intellectual deficits, hydrocephalus with stenosis of the aqueduct of Sylvius, corpus callosum agenesis, adducted thumbs, shuffling gait, aphasia and spastic paraplegia9.During nervous system development, the cell adhesion molecule L112, which have been implicated in distinct L1 functions20. Cleavage of L1 within the third FN III domain by the serine proteases trypsin13, proprotein convertase PC5a10, and plasmin16 leads to promotion of neurite outgrowth and stimulates migration of neuronal cells.The protein backbone of L1 consists of a C-terminal intracellular tail, a transmembrane domain and an extracellular part composed of 6 immunoglobulin-like (Ig) and 5 fibronectin type III (FN III) domains. Full-length L1 can undergo proteolytic ectodomain shedding to release soluble fragments and to generate transmembrane fragmentsreeler27. Since Reelin was found to show serine protease activity cleaving extracellular matrix proteins28 and since Reelin-mediated cleavage of the cell surface glycoprotein Caspr had been reported to alter neurite outgrowth29, we investigated whether Reelin cleaves L1 and whether the serine protease activity of Reelin affects L1-dependent functions in the nervous system.The extracellular matrix protein Reelin controls the sequential lamination of the cerebral cortex, and the cortical layers are disorganised in the natural Reelin-deficient mutant 1283 in Reelin is required for the cleavage of L1 by Reelin. Furthermore, we show that the protease activity of Reelin is required for L1-mediated neurite outgrowth and neuronal migration, and we provide evidence that cleavage of L1 by Reelin affects early stages of brain development.Here, we show that full-length Reelin and its fragments N-R2 and N-R6 bind to L1. We also show that full-length Reelin and its fragment N-R6 proteolytically cleave L1 within amino acids 840–845, and that serinereeler mice and wild-type littermates for L1-fragments using immunoblot analysis with antibody 172, which recognises the intracellular L1 domain. In the cerebellum, cerebral cortex and hippocampus of reeler mice, the expression of a proteolytic 80 kDa L1 fragment (L1–80) was decreased when compared to its wild-type levels 28, we examined whether exchanging serine against alanine at this position would affect proteolysis of L1 by Reelin. HEK cells expressing L1 were transfected either with wild-type Reelin or Reelin with the S/A1283 mutation and cell lysates were probed by immunoblot analysis with L1 antibody 172. L1-80 was found in lysates of HEK cells expressing wild-type, but not mutated Reelin  yields two N-terminal fragments (N-R2 and N-R6), two fragments containing the central part of Reelin (R3-6 and R3-8), and one fragment containing the C-terminus (R7-8)  Fig. . Thus, w7-8 Fig. , suggestin vitro scratch injury assay using transfected HEK cells. It was not possible to perform this assay with primary cultures of neurons, since these neurons do not form uniform monolayers and do not survive upon scratching. Moreover, the low transfection efficiency of neurons precludes their usage in this experimental setup. It was plausible to use HEK cells, because they use similar signalling pathways for migration as neurons do during development, i.e. they respond to Reelin with enhanced migration when expressing L1. Widths of gaps were measured 16 h after scratching of monolayers of HEK cells which were transfected to co-express L1 and full-length Reelin or Reelin fragments N-R6, N-R3, R3-8, R3-6 or R7-8. Only the N-R6 fragment and full-length Reelin, but not the N-R2, R3-8, R3-6 and R7-8 fragments, enhanced the gap closure  and Foxp2 (deep layers). In contrast to wild-type cerebral cortices, many Brn2-positive cells were abnormally found in deep layers, and many Foxp2-positive cells were found in upper layers of L1-deficient cortices  could not be detected in these assays, implying a rapid degradation after generation of such fragment(s). Although the L1-deficient mice based on our analysis of Nissl-stained sections44. In comparison to 6-day-old wild-type littermates, L1-deficient mice did not show obvious changes in cortical lamination following Nissl staining. However, Golgi impregnation and investigation of layer-specific markers in addition to BrdU pulse-chase labelling revealed abnormalities in the positioning and morphology of cortical neurons in L1-deficient brains. These novel findings call for a re-interpretation of previous results on neuronal lamination in the L1-deficient brain44 and underscore the importance of L1 for correct brain development. The morphological abnormalities observed in cortices in L1-deficient mice partially overlap with those seen in cortices of reeler mice, suggesting that the functional interplay between L1 and Reelin and cleavage of L1 by Reelin at early developmental stages controls cerebral cortex development. Indeed, we showed that reeler neurons in the cerebral cortex normalised their movement after in utero electroporation of L1-80. These findings further support the view that Reelin-mediated L1-cleavage contributes to cortical development. Moreover, our results are consistent with L1 being important for the migration of cerebellar granule cells1 and are in agreement with observations on the deficits in L1-deficient mice, which are characterized by misorientation of neuronal processes, impaired synaptogenesis and corticospinal tract malformation46. Notably, silencing of L1 in utero has been reported to result in delayed neuronal migration47. Migration defects of cortical neurons are known to contribute to mental diseases, such as the L1-syndrome49 or lissencephaly50. Thus, our results link proteolysis of L1 by Reelin to neurodevelopmental diseases, highlighting early migratory defects in the pathogenesis of these disorders.We have previously described a normal morphology of the cerebral cortex of L1-deficient mice are also displayed by reeler mice, not all abnormalities overlap between both mutants. These findings are understandable under the premise that L1 and Reelin interact with multiple interaction partners that are distinct from each other, and thus are involved not only in overlapping functions, but also in varying pathways leading to diverse phenotypes.Although some pathological features of the 19, suggesting even more complex roles of L1 in the maintenance of cortical architecture and function. We here showed that full-length Reelin and its N-terminal fragment N-R6, but not other Reelin fragments, cleave full-length L1 and generate L1-80. Ontogenetic expression studies revealed a close correlation between the expression of N-R6 and L1-80 during the peak phase of neuronal migration, suggesting that both fragments contribute to migration.It needs to be pointed out that L1 is cleaved by other proteases at amino acids 840–845 in late developing brain structuresreeler mice by in utero electroporation partially normalises the migration of cortical neurons. Thus, our results indicate that Reelin’s N-terminus and its central part are required for Reelin’s serine protease activity to generate L1-80 and to promote L1-dependent functions via L1-80.Our results shed light on the importance of the N-terminal cleavage of Reelin by proteases, such as ADAMTS-4, to regulate Reelin’s functions. The N-terminal cleavage of Reelin abolishes the protease activity of N-R6, and the originating R3-6 and R3-8 fragments are unable to process L1. Interestingly, introduction of L1-80, but not of full-length L1, into the cerebral cortices of 51 in the terminal phase of the migratory process52. It would be also desirable to investigate whether the mechanisms of directed migration of neurons and their detachment from radial glial fibres depend solely on the L1–Reelin interaction, or whether other molecules determine the “go and stop” phenomena characteristic for neuronal migration in the cerebral cortex. It is conceivable that cytoskeletal elements at the leading edge of neurites extending along radial glial fibres are involved in regulation of migration. Moreover, cognate signal transduction mechanisms via the L1 fragment generated by Reelin could play a role in regulation of migration. Whether these downstream events would depend on the protease activity of Reelin or whether Reelin’s binding to its receptors at the cell surface would be sufficient for downstream signalling will be important to study in the future.Since cleavage of the cell surface and transmembrane adhesion molecule L1 is crucial for cerebral cortex development and for directed neuronal motility, it will be important to analyse the molecular mechanisms underlying the directed migration of neurons from the subventricular zone to the marginal zone, when migrating neurons approach the upper cortical layers and detach from radial glial fibersReeler mice21, ApoER232 or VLDLR53 single mutant mice, ApoER2 and VLDLR double mutant mice32, Dab1-deficient mice30 and L1-deficient mice7 were housed at 22 °C on a 12 h light/dark cycle with ad libitum access to food and water. All animal experiments were approved by the local authorities of the State of Hamburg  and conform to the guidelines set by the European Union.54. Aprotinin, phenylmethylsulfonyl fluoride and the Flag M2 mouse antibody (F1804) were from Sigma-Aldrich . Secondary antibodies coupled to horseradish peroxidase, Cy3 or Cy2 and human Fc were from Dianova .Mouse antibody G10 against the N-terminal part of Reelin was from Merck Millipore and mouse Reelin antibody CR50 was a kind gift of André Goffinet  and Tom Curran . Goat Brn2 antibody (sc-6029) was from Santa Cruz Biotechnologies . Mouse antibody 172 recognising the intracellular L1 domain  was from Biolegend  and the polyclonal rabbit L1 antibody Anti-L1CAM (ab123990) was from Abcam . Mouse GAPDH antibody (ab9482) and rabbit antibodies against Foxp2 (ab16046), Tbr1 (ab31940), and Cutl1 (ab140042) were from Abcam . Goat CHL1 antibody (AF2147) was from R&D Systems. Production of rabbit NCAM antibodies and of L1-Fc comprising the extracellular part of mouse L1 fused to Fc part of human IgG have been described40 and purified Reelin55 used for ELISA were kind gifts from Tom Curran and André Goffinet. HEK293T cells stably transfected with wild-type Reelin and mock-transfected HEK293T cells56 were kindly provided by Eckart Förster .CAG-IRES-GFP vector was a kind gift from Melanie Richter and Froylan Calderon de Anda . Plasmids carrying full-length Reelin and Reelin fragments N-R2, N-R6, R3-6, R3-8 and R7-82EDTA, 1 mM EGTA, 1% Nonidet P-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, and 1× cocktail of protease inhibitors ) at 4 °C. After centrifugation at 20,000 × g and 4 °C for 10 min, equal amounts of total protein from the supernatants were subjected to Western blot analysis12 with Reelin antibodies G10 , L1 antibody 172 ; CHL1, NCAM or GAPDH antibodies . Secondary antibodies coupled to horseradish peroxidase were used at a dilution of 1:10,000.Brain areas were homogenised in RIPA buffer  and collected on glass slides. Sections were deparaffinised in pure xylene several times, rehydrated in descending grades of ethanol and immunostained with Reelin antibody G10 (diluted 1:500), Brn2, Foxp2, Tbr1, or Cutl1 antibodies (diluted 1:100). Blocking solution (PBS containing 10% horse serum and 0.01% Triton X-100) was used for dilution of antibodies. Cy3- and Cy2-conjugated secondary donkey antibodies were used at a dilution of 1:500 in PBS. Sections were mounted in Fluoromount containing DAPI (Sigma-Aldrich). Images of immunostained brain sections were taken on a confocal fluorescence microscope  or a Keyence Fluorescent Microscope  and were processed by using the ImageJ software.10 were cloned into the CAG-IRES-GFP vector using the In Fusion cloning kit . For the mutation of Reelin, sequences between the SnaBI and AgeI sites were amplified using the HiFi amplification kit (TaKaRa/Clontech), the plasmid coding for full-length Reelin, the forward primer 5′-AAC CCC ACC TAC GTA CCG GGA CAG GAA TAC-3′ and the reverse primer 5′-GT TAC TGC AAA CCG GTC TCC ATC CGC CTT TCC AAA CAC CAT GGC TG-3′ carrying the mutation (underlined triplet). The plasmid coding for full-length Reelin was digested with SnaBI and AgeI and fused with the amplicon with the mutation using the In Fusion cloning kit.Wild-type and mutated L110 as template and the forward primer 5′‐GAATTC CAT ATC CAC AAA AGC CAC ATA‐3′ containing an EcoRI site and the reverse primer 5′-CTCGAG TTC TAG GGC TAC TGC AGG ATT‐3′ containing a XhoI site or the forward primer 5′‐GAAGCTTTAGCCACCATGGTC ATG GTC GTG ATG CTG CGG TAC‐3′ containing a HindIII site and the reverse primer 5′-GAATTC GAG CAG GCA GGG GCT GCA GAG‐3′ containing an EcoRI site. The amplicons were used for T/A-cloning into pGEM®-T-Easy vector . Using EcoRI and Xhol restriction sites, the L1-80 amplicon was cloned into pcDNA3 Flag-His A plasmid (Promega) in frame with the Flag-His tag. The signal sequence of L1 was cloned in frame with the L1-80 amplicon via HindIII and EcoRI restriction sites.To amplify sequences coding for L1-80 or for the L1 signal sequence, PCR were performed using murine L1-cDNA11. Serum-free supernatants from transfected HEK cells were dialysed and concentrated 5-fold using 100 kDa cut-off centrifugation columns  at 4 °C. Pull-down with L1-Fc and Fc using Protein A beads was described57.Culturing and transfection of HEK293T cells, scratch assay and assessment of gap closure have been described12) was performed as follows: 5 µg of L1-Fc and Fc, 5 µg of serum-free Reelin-containing HEK supernatants and Reelin-devoid HEK supernatants (mock) concentrated 5-fold and dialysed through 100 kDa cut-off Amicon columns, were substrate-coated in 384-well microtiter plates with high binding surface  at 4 °C overnight. After washing with PBS, blocking with 1% w/v bovine serum albumin  in PBS at ambient room temperature for 1 h, and washing with PBST (PBS with 0.05% Tween 20), 5 µg of L1-Fc or Fc were added to the substrate-coated Reelin and HEK cell supernatants, and 5 µg of purified Reelin were added to the substrate-coated L1-Fc or Fc for incubation at room temperature for 1 h. After washing with PBST, primary mouse monoclonal Reelin antibody G10 followed by an anti-mouse HRP-coupled secondary antibody (Dianova) as well as O-phenylenediamine dihydrochloride  as horseradish peroxidase (HRP) substrate were used for detection of Reelin. For detection of L1-Fc and Fc, human anti-Fc-HRP  antibody was used. The reaction was terminated by addition of 2.5 M sulphuric acid. Absorbance was measured at 490 nm with the ELISA reader .ELISA  per well for 16 h at 37 °C in a humidified atmosphere containing 5% CO2. Neurons and explants were then fixed and stained as described12. Neurite outgrowth and neuronal cell migration were analysed and quantified by measuring the total neurite length per neuron, the number of cells migrating out of the explant cores, and migration area using a Zeiss Kontron microscope with AxioVision 4.7 software .Isolation and culturing of murine cerebellar and cortical neurons and explants have been described35. Elution was performed in two steps using 140 mM and 400 mM NaCl, and eluates containing Reelin were dialysed using 100 kDa cut-off centrifugation columns (Amicon). L1 was immunopurified from mouse brains as described11. For the proteolytic assay, 10 ng of wild-type or mutated Reelin were incubated at 37 °C for 16 h in the absence or presence of 1 µM phenylmethylsulfonyl fluoride with 10 ng of L1 immunoaffinity purified from mouse brain. For the treatment of homogenates, neonatal reeler hippocampus or embryonic (E17) reeler cerebral cortex was freshly homogenized in PBS and incubated with 100 µl of HEK cell supernatants at 37 °C for 16 h.Wild-type and mutated Reelin were purified using Heparin High-Trap Sepharose columns in utero electroporation, timed-pregnant mice at E13.5 were anaesthetised, the abdominal cavity of the pregnant mice was opened and the uterine horns bearing the embryos were exposed. Thereafter, 2 µg of CAG-IRES-GFP vector coding for wild-type L1, vectors coding for L1-80, N-R2, N-R6, R3-6, R3-8 or R7-8 or empty CAG-IRES-GFP vector pre-mixed with 0.01% Fast Green were unilaterally injected into the ventricle of an embryonic brain. Five electrical square unipolar pulses  powered by a BTX electroporation apparatus  were applied to the embryos. The uterine horns were placed back into the abdominal cavity. After 60 h, pregnant mice carrying electroporated embryos were sacrificed, the embryos were isolated in ice-cold PBS and killed by decapitation. GFP-positive electroporated brains were collected under a fluorescence microscope and fixed in 4% formaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, at 4 °C overnight, followed by an overnight immersion in a solution of 15% sucrose in 0.1 M sodium cacodylate buffer, pH 7.4, at 4 °C.For Electroporated brains were embedded in 5% agarose and sectioned on a vibratome (Leica VT1000 S) at 80 µm and immersed in blocking solution , washed, and incubated with an antibody recognising flag-tag (dilution 1:500) at 4 °C overnight. Sections were washed in PBS, incubated with Cy3-conjugated secondary antibody (dilution 1:250) at 4 °C overnight, counterstained with DAPI to label nuclei, mounted in Fluorescence Mounting Medium  and subjected to fluorescence microscopy.All data generated or analysed during this study are included in this published article (and its Supplementary Information files).Supplementary Figures