Graphene is the building block of graphite, made of carbon atoms bonded into sheets of hexagonal rings just a single atom thick. Although such isolated sheets had been predicted for many decades to exist, and had been grown on other surfaces, interest in this material exploded after the discovery in 2004 that single sheets could be made easily and cheaply by separating them mechanically from graphite flakes . Although graphene is often advertised as a ‘wonder material’—electronically conducting, transparent and extremely strong and flexible—much of the interest in it is more fundamental. As a 2D conductor, graphene shows unusual electronic and magnetic properties that enable the study of quantum-mechanical effects of confinement and of correlations between electron motions—some of which might find applications in electronic devices. The excitement of this discovery was reflected in the award of the 2010 Nobel Prize in Physics to two pioneers in the field: Andre Geim and Konstantin Novoselov of the University of Manchester in the UK.This rich behavior is broadened still further when two graphene sheets are brought close enough to interact with one another. In particular, the electronic properties may then depend on the relative orientation of the sheets: how aligned the two ‘honeycomb’ lattices are. Two grids superimposed on one another may create ‘superlattices’: regularities at larger scales than the grid spacing, due to registry (commensurability) between the two at certain angles. This so-called moiré effect is sometimes evident for two closely spaced grid-like fences seen from afar. Experimentally exploring the electronic properties of such ‘twisted bilayer graphene’ requires an ability to precisely control the position and orientation of the two sheets. These phenomena are now recognized as generic to other 2D materials, such as hexagonal sheets of boron nitride. They have revealed a fertile playground for condensed-matter physics. In particular, striking electronic properties appear at certain ‘magic-angle’ twists of the layers.NSR spoke to two of the leading experts in the study of magic-angle twisted bilayer graphene (MATBG): experimentalist Pablo Jarillo-Herrero of the Massachusetts Institute of Technology and theorist Allan MacDonald of the University of Texas at Austin. NSR: How did the discovery of unusual electronic behavior in twisted bilayer graphene came about? Were such effects predicted before they were observed?PJ-H: Many theory groups started to work on twisted bilayer graphene from around 2007. In late 2009, the group of Eva Andrei reported an investigation of twisted bilayer graphene using scanning tunneling microscopy (STM) . They observed peaks in their data that they interpreted as arising from features in the electronic structure known as Van Hove singularities [where the density of electronic states diverges]—which seemed to change with the twist angle. In particular, the separation between the peaks seemed to extrapolate to zero for a twist angle of ∼1.16°. Around the same time, two theory groups worked on the theory of twisted bilayer graphene at very small angles: Eric Suárez Morell and collaborators in Chile  and Rafi Bistritzer and Allan MacDonald in Texas . Both groups predicted the existence of ‘flat’ electronic bands in twisted bilayer graphene at an angle of 1.1–1.5°. Bistritzer and MacDonald coined the term ‘magic angle’: the angle for which the velocity of electrons at the Fermi level [the energy level electrons will occupy at zero temperature] goes to zero.et al., Phys Rev Lett 2016; 117: 116804]. This is what motivated us to continue working on reducing the twist angle and to investigate devices with twist angles very close to 1.1°. In 2017, we managed to fabricate several devices with twist angles in the range of 1–1.2°, and in these devices we made two unexpected discoveries. First, MATBG can become an insulator due to correlation effects when the charge density is tuned to a certain integer number of electrons or holes per moiré unit cell . Second, if you dope the material to add a bit of extra charge density, then MATBG becomes an electrically tunable superconductor with one of the highest ratios of critical temperature to Fermi temperature . These characteristics are very reminiscent of other strongly correlated quantum materials and superconductors. The fact that we could obtain these results in such a seemingly simple system, just two layers of graphene rotated to a precise angle, and with such a degree of tunability, is one of the key reasons why our discovery attracted a lot of attention. These discoveries were totally unexpected and not predicted theoretically.My own group started to work on twisted bilayer graphene around 2010. In 2016, we showed that twisted bilayer graphene with a 1.8° twist angle already exhibited an interesting modification of the electronic structure: the velocity of the electrons at the Fermi level was substantially reduced, in agreement with theoretical calculations [Y. Cao AM: My understanding of the history—beyond what is in the publication record—comes from Eva Andrei [https://arxiv.org/pdf/2008.08129.pdf]. Eva was the first person to measure interesting changes in electronic structure, seeing features in STM density-of-states measurements on bilayer flakes that accidentally had moiré effects. Eva told me that the observation came first, and motivated the theory by Antonio Castro-Neto and João Lopes dos Santos.My interest in graphene moiré electronic structure started from a conversation I had with Ed Conrad at Georgia Tech, who showed me some data from angle-resolved photoemission spectroscopy that I did not understand. As my postdoc Rafi Bistritzer and I built up our picture, we discovered that our calculations predicted that the velocity of graphene electrons would fall to zero at a discrete set of twist angles—we called them magic angles. The largest magic angle was ∼1°. This was a complete surprise to us, and we immediately recognized that it implied a rich platform for strong correlated-electron physics. Sometime later, we noticed that a group from Chile had independently obtained at least a glimmer of the magic-angle physics. At this time, we had no idea whether or not experimentalists would be able to fabricate samples with controlled twist angles to see this physics. My colleague Emanuel Tutuc did a lot of work in this direction, some of which helped to inform Pablo's parallel efforts.NSR: What induced you to study this system? It seems now that it offers a playground for looking at correlated-electron phenomena in a controlled way—but was that the expectation, or did the discovery come as a surprise?PJ-H: Originally, my motivation to study twisted bilayer graphene was the intuition that this ‘new knob’ in condensed-matter physics, namely the possibility to change the twist angle, had to yield interesting physics. In condensed-matter physics, the systems are typically quite complex, and when one explores unchartered territory, surprises often arise. In the specific case of magic-angle graphene, my motivation was to find interesting correlated insulator states that I though may appear when one would shift the Fermi energy in graphene to the Van Hove singularities. [NSR: New electronic phases such as superconductivity have been previously observed when the Fermi energy is close to such singularities.] We did find insulators—but to our surprise they were of a different type, where the insulating behavior occurs for an integer number of electrons per moiré unit cell, rather than because of the Van Hove singularities. This was a very nice surprise. An even bigger surprise was the discovery of superconductivity, which no one had predicted.The magic-angle effect is a sort of ‘resonant’ condition … [where] it is as ‘easy’ for the electrons to go through one graphene layer as it is for them to ‘tunnel’ to the other graphene layer.—Pablo Jarillo-HerreroAM: Our original theoretical finding of magic-angle effects was not expected on the basis of earlier experiments, and we had a lot of problems getting our work published because the referees thought that the result must be incorrect. Around this time, I was elected to the US National Academy of Sciences, which allows new members to publish a lightly reviewed inaugural article in the journal PNAS. I decided to give up on a long fight with referees and just publish our findings that way.et al., Phys Rev Lett 2017; 118: 147401] and a number of other proposals related to optical properties. I also proposed that layered transition-metal dichalcogenide (TMD) moiré systems would yield quite different physics compared to graphene multilayers. This part of the moiré field has now really taken off experimentally.Following that paper, I tried to find other instances where interesting moiré superlattice physics would be observable. I proposed the possibility of realizing topological exciton bands  between the electrons plays a dominant role. Once you have strong interactions between the electrons, then many possible many-body ground states  become possible. We are seeing all of these because moiré systems are highly tunable.AM: There are many analogies between strong correlations in graphene multilayers and strong correlations in quantum Hall physics. This connection was clarified by work of Eslam Khalaf, Ashvin Vishwanath and collaborators at Harvard, Mike Zaletel and others. Ultimately it related to topological properties of the electronic bands. At the same time, these systems have an aspect of the quasi-2D Hubbard model [one of the first and simplest lattice models of strongly correlated electrons]. MATBG seems to be a marriage of the quantum Hall effect [which arises in 2D systems] and high-temperature superconductivity—it's an amazing system.NSR: Can you explain the magic-angle effect? What makes certain relative orientations of the graphene sheets ‘special’?It's a whole new paradigm for making artificial tunable crystals, and we’re still just scratching the surface. We’ll see what happens—that's what makes science fun.—Allan MacDonaldPJ-H: The magic-angle effect is a sort of ‘resonant’ condition. One can think of the magic angle as the angle for which the electronic structure is such that it is as ‘easy’ for the electrons to go through one graphene layer as it is for them to ‘tunnel’ to the other graphene layer. In much simpler terms, an analogy for why electrons in MATBG display such varied behavior is this: when electrons have lots of kinetic energy , they barely have time to interact. But in MATBG, the electrons are very slow, and hence they have lots of opportunity to interact with each other as they pass each other by.NSR: The interplay of insulating and superconducting behavior in this system seems to mirror that seen in the copper-oxide high-temperature superconductors. Is similar physics at work? Does this behavior in fact help us to understand the origin of superconductivity in such materials?PJ-H: There are indeed many similarities between the phase diagrams of MATBG and cuprate superconductors, but there are also many differences too. For example, the symmetries of the lattices and the topological properties of the electronic structures are very different. Also, the electrons in the cuprates have degenerate spin [all the same], whereas the spin states for MATBG are somewhat richer. So we do not know yet whether understanding MATBG will help us to understand the origin of superconductivity in cuprates. My intuition is that it will, but it's too early to tell.AM: We do not yet have completely confident answers to these questions, but we are making progress. There are many similarities between high-temperature superconductors and MATBG systems—the proximity to magnetic order and to Fermi-surface reconstructions is most intriguing. In my opinion, there is every reason to expect that we will continue to make progress in understanding MATBG superconductivity by performing new experiments and testing theoretically proposed scenarios, and that this progress will have implications for understanding high-temperature superconductivity. The possibility of tuning the charge-carrier density in situ, and the possibility of adjusting the system properties in other ways  is an important advantage for MATBG.NSR: What is the role of dimensionality here? Are these behaviors dependent on the fact that this is a (quasi-)2D system? And in that regard, does this behavior connect with the work on low-dimensional quantum many-body problems such as the quantum Hall effect (QHE)?PJ-H: Dimensionality is quite important for various reasons. Among them: MATBG is highly tunable electrically because of the 2D geometry; the electronic structure  depends on dimensionality; and interaction effects can be also strongly dependent on dimensionality—for example, screening by electrons is very different in 1D, 2D and 3D. Regarding quantum Hall physics, there is a deep connection in that both the QHE and the electronic bands in MATBG  are topological in nature. That's why the latter can show interesting quantum Hall effects, even at zero magnetic field [unlike the standard QHE].AM: Electronic correlations tend to be stronger in lower-dimensional systems and have more scope to yield surprising many-electron states, including fractional quantum Hall effect (FQHE) systems, MATBG, bilayer and trilayer graphene. The topological picture of the QHE provides a link between MATBG and FQHE physics. One experimental proof of this connection is the common appearance of anomalous quantum Hall states  in MATBG.NSR: How does one study these systems experimentally? Is it now routine to make good-quality single-layer graphene? How do you control the relative orientation of the sheets?PJ-H: It is very standard to obtain very-high-quality single-layer graphene, by mechanical exfoliation of graphite for example. Thousands of groups around the world can do this. What is a lot more tricky is to be able to stack on top of each other two graphene sheets with a very well controlled angle of rotation, and that's even harder for small angles such as the magic angle of 1.1°. There are only ∼15 groups around the world that can currently fabricate MATBG, but more are joining all the time. It's something that can be easily learned if someone shows you. Before the pandemic, we had plenty of groups come to MIT and learn about it, and those groups now have reproduced and extended many of our results.AM: It is amazing what has been accomplished already. If it were possible to develop techniques to control twist angles even more finely, and make the twist angles even more uniform, it would speed up progress in the field.NSR: What are the key questions that still remain to be explored in these systems? What are you personally most eager to study now?PJ-H: There are many key questions remaining to be explored. Perhaps one of the most important is the exact mechanism for superconductivity and the symmetry of the order parameter. Right now, experiments and theory seem to point towards a very unconventional origin for the superconductivity . But we still need to study this in more detail. I’m personally very eager to discover and study new moiré systems, new superconductors and their correlated and topological behavior. I think we have barely scratched the surface of the many hundreds of possible moiré systems we can build, with very different constituents, geometries and complexity.AM: I think that it is important to nail down the origin of superconductivity in MATBG. I am actively working on this question.One important hope is that we will be able to realize fractional anomalous quantum Hall systems  in MATBG or in TMD moirés that show the quantum anomalous Hall effect. Given the flexibility of the moiré superlattices, there is a good chance that favorable conditions could be discovered or engineered. FQH states are one of the possible hosts for topological quantum computing too.NSR: There seem to be many potential degrees of freedom to explore in such systems. For example, there is now interest in going beyond bilayer systems to trilayers—what is predicted and/or observed there? What about using hetero-bilayers, such as with boron nitride or other 2D materials?AM: I am very interested in identifying new layered materials that can host new classes of moiré superlattices—each case will bring its own universe of new physics. Already just with TMD and twisted graphene moirés, we have examples of itinerant [mobile] electron ferromagnetism—but with quite low ordering temperatures. It's interesting to think about how ordering temperatures can be increased and what the ultimate limits are. Because the moiré superlattice systems can be tuned in so many ways, there is some scope for optimism. It's a whole new paradigm for making artificial tunable crystals, and we’re still just scratching the surface. We’ll see what happens—that's what makes science fun.PJ-H: Indeed, the possibilities are pretty much endless. Just earlier this year, two independent groups (Philip Kim's group and my group) discovered superconductivity in magic-angle twisted trilayer graphene (MATTG). The magic angle turns out to be a bit different (∼1.6°), which was theoretically predicted a couple of years ago, so we knew where to look. The superconductivity in MATTG turns out to be even more interesting than in MATBG, because it is stronger and much more tunable. Using hetero-bilayers can indeed add many things, and the discovery of the quantum anomalous Hall effect (QAHE) in MATBG aligned with boron nitride was one of the earliest examples.NSR: More generally, MATBG systems exemplify an explosion of interest in the past two decades in strongly correlated electrons, which has yielded the discovery of a vast zoo of quantum materials, such as topological insulators, Majorana zero modes, Weyl semimetals and so on. What has triggered the explosion of interest? Is there any emerging sense of a unified view of the quantum electronic phases of matter, or are we still very much in the phase of discovery and surprise?PJ-H: Condensed-matter physics saw two ‘revolutions’ in the 1980s, with the discovery of the integer/FQH effects (which brought topology into the field) and with the discovery of high-temperature superconductivity (which brought strong correlations to the forefront). Since then, these two communities  did not interact very closely, because the systems were quite different. Then in the 2000s, three major disruptions occurred: the discovery of graphene and 2D crystalline materials; the theoretical prediction and, soon after, the experimental discovery of topological insulators; and the discovery of a second family of high-temperature superconductors, the iron pnictides. Yet still these communities remained largely separate. MATBG has merged these three communities, because it has characteristics of all of them. This topic of ‘moiré quantum matter’ has stimulated very interesting discussions among all of these people.AM: In my view, we are still very much in a phase of discovery and surprise, but I am very optimistic that these new classes of strongly correlated systems will lead to a broader and deeper view of strong-electron-correlation physics.NSR: Are there any likely practical applications of such systems? In particular, does there seem any likelihood that MATBG might find its way into device technology?PJ-H: This is always very hard to predict. For now, my group and the overall community are motivated by the beautiful fundamental physics that we can explore in these systems. Having said that, MATBG is an electrically tunable superconductor or, in engineering terms, a superconducting field-effect transistor, so one could easily imagine many applications for it, if the community can manage to fabricate MATBG on a large scale. Applications easy to imagine include tunable superconducting quantum bits, tunable quantum photodetectors and classical cryogenic computing.AM: Personally, I am very interested in identifying potential applications—for optical properties perhaps, or for spintronics [information processing using spins]. Interfaces with TMDs may prove useful in adjusting spin–orbit interaction strengths—something that will be key for spintronics.NSR: What is your impression of the research in this area in China?PJ-H: There has been lots of interest in China from the point of view of theoretical physics. In terms of experimental work, China currently has only a few groups with the nanofabrication experience to produce high-quality moiré quantum systems. Those groups (the most renowned of which is that of Yuanbo Zhang (张远波) at Fudan University) are producing very good work. Given the recent rapid evolution of scientific research in China, I expect many more experimental groups will start working on this subject in the coming years.My former student Yuan Cao (曹原) is a very remarkable scientist in many respects. He is very smart, hard-working, creative and effective. He was not only the first author in the two discovery papers I mentioned above, and hence a young leader in the field, but has continued to make outstanding contributions to the field since then. He has received several awards, even at his young age, including the McMillan Award (the most prestigious award for young condensed-matter physicists) and very recently the International Sackler Prize in Physics. I consider myself extremely fortunate to have worked with him. I think I have learned, at the very least, as much from him as he may have learned from me. I believe he will be one of the leading scientists of his generation.AM: Fengcheng Wu (吴冯成), a former student in my group who did important early work on TMD moirés, including optical and electronic properties, and on MATBG superconductivity, is now a professor at Wuhan University and is a leading researcher in the field. Wang Yao (姚望) at Hong Kong University is a leading theoretical researcher on optical properties of TMD moirés. The QAHE was first observed at Tsinghua University in magnetic topological insulators. MATBG has provided a second example, with interesting similarities and differences.Most of the time, the new understanding is just a detail that is already understood by someone—but every now and again it will be something truly new.—Allan MacDonaldNSR: What or who are your key sources of inspiration for this work? And what advice would you give to young researchers entering the field?PJ-H: There are many colleagues whom I find very creative and who have inspired my group's approach to experimental condensed-matter physics. These include Paul McEuen (Cornell), Andre Geim (Manchester) and Amir Yacoby (Harvard). And, of course, my PhD advisor Leo Kouwenhoven at Delft and my postdoc advisor Philip Kim at Harvard had a big influence in my formation. To a young researcher, I’d say: be adventurous and take risks. Try to follow your interests and don’t let others put a ceiling on your ambitions.AM: I’ve been doing this a long time now. I have developed a great appreciation for the ability of experiment to surprise. My approach to doing fundamental theory in materials science is to try to identify exciting new phenomena that are likely to be observable experimentally if someone would just look. My intuition is based very much on known experimental results and by thinking about why different types of theoretical approximations are successful—or unsuccessful—in describing nature. Trying to deepen theoretical understanding of phenomena that have been observed but remain mysterious is nearly as much fun.I would advise young researchers to develop their own independent way of thinking about their scientific area. Whenever you encounter something that you do not understand, dig deeper until you do. Most of the time, the new understanding is just a detail that is already understood by someone—but every now and again it will be something truly new.


The effects of graphene layer and MoS2 morphology on photoelectrochemical (PEC) performance were systematically studied on the basis of electronic structure and transitions, carrier dynamic behavior, and PEC measurements. The heterojunction quality of the graphene/vertical few-layer MoS2 nanosheets was ensured by low-temperature growth at 250−300 °C, resulting in significantly improved charge transfer properties. As a result, the PEC photocurrent density and photoconversion efficiency of the few-layer MoS2 nanosheets significantly increased upon the insertion of a graphene layer. Among the graphene/MoS2 samples, the few-layer MoS2 nanosheet samples exhibited shorter carrier lifetimes and smaller charge transfer resistances than the thin film samples, suggesting that vertically aligned nanosheets provide highly conductive edges as an efficient pathway for photo-generated carriers and have better electronic contact with graphene. In addition, the height of vertical MoS2 nanosheets on graphene should be controlled within the carrier diffusion length (~200 nm) to achieve the optimal PEC performance. These results can be utilized effectively to exploit the full potential of two-dimensional MoS2 for various PEC applications.The morphology of MoS The PEC efficiency of MoS2 is significantly affected by the design and implementation of an appropriate heterostructure that can enhance the separation and subsequent transfer of photogenerated electron–hole (e–h) pairs using a built-in potential generated by the heterojunction [2/TiO2, MoS2/ZnO, and MoS2/CdS [2 nanostructures/graphene has attracted considerable attention as promising PEC cathode and anode material systems [2·V−1·s−1) [2 for efficient charge separation and transfer [2/graphene–CdS composites improves the charge transfer ability and retards the recombination of e–h pairs, thereby enhancing photocatalytic hydrogen evolution reactions [2/graphene hollow spheres for highly efficient photocatalytic hydrogen evolution reaction [2/crumpled graphene enhances PEC hydrogen production [2/reduced graphene oxide heterojunctions suppresses the recombination of e–h pairs and promotes PEC efficiency [2/graphene composites exhibit randomly assembled structures of 2D MoS2 and graphene. The heterostructure of few-layer MoS2 nanosheets aligned vertically on graphene substrates has not been implemented by wet-chemical approaches.The two-dimensional 2D) transition metal dichalcogenides, such as MoSbundance ,2. Two-dD transitjunction ,4,5. AmoMoS2/CdS ,7,8,9, 2 systems ,11. GrapV−1·s−1)  and formtransfer . Chang eeactions . Yu et areaction . Carraroficiency . However2 on electrode substrates is another important factor that can be leveraged to improve PEC efficiency because of its 2D layered structure comprised of a strong in-plane covalent bonding of S–Mo–S and a weak out-of-plane van der Waals interaction between neighboring S–S layers. The vertically standing 2D MoS2 structure has recently been reported to enhance PEC performance considerably, because the highly conductive edges of 2D MoS2 provide an efficient pathway for photo-excited carriers and have good electronic contact with the substrates [2 flakes/TiO2 nanowires improves the photocatalytic hydrogen evolution of MoS2 [2 nanosheets vertically grown on indium-tin oxide (ITO) and TiO2 nanowires [2 affected the adhesion behavior of as-formed gas bubbles. Lu et al. reported that a superaerophobic surface of vertically stacked MoS2 flake electrodes significantly improved PEC hydrogen evolution reactions [The architecture configuration of few-layer MoSbstrates ,16,17. Hanowires . In addieactions .2 nanosheets is also an important factor to maximize its PEC activity, because the bandgap energy is tunable from ~1.2 eV for the indirect gap of the bulk form to ~1.9 eV for the direct gap of the monolayer [2. Velicky et al. revealed that few-layer (5–10) MoS2 flakes provide a good compromise between large surface areas and sufficiently fast charge-carrier transport for energy storage and energy conversion applications [2 on graphene and its systematic electronic, optical, and PEC properties, is minimal. Herein, we report the controllable growth of few-layer MoS2 nanosheets on graphene by using metalorganic chemical vapor deposition (MOCVD) for PEC water-splitting applications. The morphology of MoS2 was successfully manipulated from thin film to vertically aligned few-layer nanosheets on graphene via a controllable and practical manner, that is, by varying growth temperature (2 was grown on graphene at relatively low temperatures (≤ 350 °C) in order to minimize the structural and chemical destruction of the graphene layer. The beneficial effects of the graphene layer were also systematically studied. For PEC water-splitting applications, the optimal structure of MoS2 on graphene, including PEC properties, electronic structures and carrier transfer properties across the graphene/MoS2 heterojunction, was determined by a systematic study.The thickness controllability of 2D MoSonolayer ,21,22. Tications . Howeverperature . Further4 and H2 gases at 950 °C for 5 min. The ICP power and growth pressure were fixed at 200 W and 1 Torr, respectively. The synthesized graphene on Cu was transferred on an ITO glass substrate  through MOCVD reaction with Mo(CO)6 and H2S gas  as Mo and S precursors, respectively. Mo(CO)6 was vaporized at 20 °C and carried into a quartz reaction tube with Ar gas of 25 standard cubic centimeters per minute (SCCM). The flow rate of H2S gas was 75 SCCM. The growth pressure and time were fixed at 1 Torr and 5 min, respectively.The Graphene was grown on Cu foils (Alfa Aesar) using inductively-coupled plasma chemical vapor deposition (ICP CVD) with CHubstrate . The CVD2 were characterized via scanning electron microscopy  and transmission electron microscopy . The crystal structure of MoS2 was characterized by TEM and micro-Raman spectroscopy using an excitation band of 532 nm and a charge-coupled device detector. The optical properties were evaluated by ultraviolet–visible (UV–Vis) spectroscopy  and photoluminescence (PL) spectroscopy (excitation at 532 nm). The photo-excited carrier behavior was investigated by time-resolved PL (TRPL) measurements. The samples were excited using a 467 nm pulsed laser and the transient signal was recorded using a time-correlated single photon counting spectrometer . The energy level of MoS2 was evaluated via UV photoelectron spectroscopy .The morphology and microstructure of MoS2 ITO and ITO/graphene substrates. The working area of the PEC cells was fixed at 0.5 × 0.5 cm2 using nonconductive epoxy to cover undesired areas. PEC characterization was performed using a three-electrode system and an electrochemical analyzer . A Pt plate and KCl-saturated calomel (Hg/Hg2Cl2) were used as counter and reference electrodes, respectively. The electrolyte solution was prepared with 0.3 M KH2PO4 in KOH solution (pH 6.5). The light source used was a 150 W Xe arc lamp that delivered an intensity of 100 mW/cm2 of simulated AM 1.5 G irradiation. The current density–voltage characteristics were recorded using a source meter . Electrochemical impedance spectroscopy (EIS) measurement was performed under constant light illumination (100 mW/cm2) at a bias of 0.6 V while varying the AC frequency from 100 kHz to 100 mHz.PEC cells were fabricated on 1 × 2 cm2 grown on graphene was crucially affected by the growth temperature of the MOCVD process. At 200 °C, an MoS2 film with a thickness of ~50 nm was formed, hereinafter referred to as G/MoS2-200  seemed to be determined by the migration length of impinged Mo adatoms, which bonds two S anions in S-stabilized growth condition. The height of the G/MoS2-300 nanosheet was higher than that of G/MoS2-250 because of the enhanced migration length of Mo adatoms at a higher growth temperature. When the growth temperature was increased further, the ratio of Mo to S adatoms also increased due to the predominant desorption of S adatoms from the growth front surface. Under S-deficient conditions, impinged Mo adatoms tended to bond immediately with the nearest Mo adatoms. As a result, a thin film morphology composed of particles was observed at 350 °C [The morphology of MoSMoS2-200 a. When tMoS2-250 b. The Mohe edges ,4,5. TheMoS2-300 c. HoweveMoS2-350 d. The sit 350 °C ,25,26.2 and graphene were investigated using Raman spectroscopy and TEM. The Raman spectrum of pristine CVD-grown graphene showed a low-intensity ratio of D to G band peaks (>0.15)  of the E12g and A1g peaks [2-250, the RFD value (22.3 cm−1) of E12g (385.0 cm−1) and A1g peaks (407.3 cm−1) corresponds to a few layers of MoS2. The increased RFD value (24.0 cm−1) with a blue-shifted A1g peak (409.3 cm−1) of G/MoS2-300 indicates a slightly increased number of layers. The blue-shifted A1g mode is attributed to the increased restoring force of the interlayer S–S atoms due to the increased number of layers. The thin film samples (G/MoS2-200 and G/MoS2-350) exhibited further increased RFD values with more-blue-shifted A1g modes  a. The lispectrum a. All th spectra b. The E11g peaks ,28. For inset in b. The TEtructure c. The nacture  than ITO/MoS2-250 (0.22% at 0.7 V), as shown in 2-250 was comparable with various recently reported photoanodes, such as TiO2/MoS2 [2 [2 [2/α-Fe2O3 [2 flakes was significantly improved by forming a heterojunction with graphene  yielded significantly higher photocurrent densities compared with the MoS2 thin film samples (G/MoS2-200 and G/MoS2-350). PEC activity was also significantly affected by the size of the few-layer MoS2 nanosheet. Despite its larger MoS2 nanosheet size, G/MoS2-300 yielded a lower photocurrent density than G/MoS2-250. Our previous study on MoS2 nanosheets on TiO2 nanowires demonstrated that for PEC applications, the optimum size of MoS2 nanosheets seemed to depend on the diffusion length of the carriers  [The PEC activities of the MoSal range a, whereaal range . TherefoiO2/MoS2 ,29,30, Z/MoS2 [2 , CoTe/MoS2 [2 [2 , and MoS/α-Fe2O3 . Moreovegraphene c. The phelements . The impelements . Among tctively) .2 nanosheet samples, systematic studies, including EIS, UV–Vis absorption, PL, and TRPL spectroscopy, were conducted. G/MoS2-250 and ITO/MoS2-250 showed nearly the same UV–Vis absorption spectra with two prominent peaks at approximately 607 nm and 663 nm  yielded a red-shifted PL peak at 705 nm with respect to G/MoS2-250. The PL around 705 nm is attributed to the indirect bandgap transitions of the MoS2 bulk form. All graphene/MoS2 samples achieved significantly lower PL efficiencies than ITO/MoS2-250. The considerable PL quenching can be attributed to the reduced recombination of e–h pairs through the heterojunction of graphene/MoS2. The dynamic behavior of photo-generated carriers was further investigated by TRPL spectroscopy  exhibited shorter carrier lifetimes than the thin film samples (G/MoS2-200 and G/MoS2-350), suggesting that vertically aligned nanosheets provided more efficient carrier transport paths, i.e., they have highly conductive edges compared with the bulk form.Both samples exhibited a PL peak at 676 nm b, which troscopy c. The avinset in c. The renterface . In addi2-250 and ITO/MoS2-250 in the dark and under illumination. G/MoS2-250 showed smaller EIS semicircles than ITO/MoS2-250, whose radius mirrors the charge transfer resistance (ctR), indicating that the graphene layer significantly enhanced the charge transfer efficiency. The Nyquist plots can be fitted using a simplified Randles circuit , constant phase element (Q), and diffusion of species in electrolyte solution represented by Warburg impedance (W). G/MoS2-250 yielded values of 3196 Ω and 2069 Ω for ctR in the dark and under illumination, respectively, whereas ITO/MoS2-250 exhibited ctR values of 4597 Ω and 3166 Ω. Moreover, the ctR (dark) to ctR (photo) ratio (1.55) of G/MoS2-250 was greater than that of ITO/MoS2-250 (1.45), suggesting that graphene/MoS2 heterojunction was more beneficial under illumination than in the dark. G/MoS2-250 and G/MoS2-300 showed smaller EIS semicircles than G/MoS2-350, indicating a decrease in ctR and suppression of charge recombination  and the spectrum width, which is measured from the valence band and secondary edges  was 1.50 eV  of MoS2 was approximately 4.38 eV, which is consistent with previously reported values (~4.3 eV) [2 nanosheets is shown in the inset of 2 is close to the Fermi level (~4.6–4.8 eV of work function) of pristine few-layer graphene [2-250 exhibited a small positive water oxidation onset potential  . The estgraphene , resultiransport c. The ap cathode d.2 morphology from thin film to vertically-aligned few-layer nanosheets on graphene in a controllable and practical manner using MOCVD. Desirable vertical few-layer MoS2 nanosheets were synthesized on graphene at relatively low temperatures (250–300 °C). Low-temperature growth was beneficial to the formation of high-quality graphene/MoS2 heterojunctions, which not only significantly enhanced the charge transfer resistance but also exhibited cathodic-shifted water oxidation onset potential (~0.18 V) by lowering a built-in potential barrier for electron transport. As a result, G/MoS2-250 showed approximately three times higher photoconversion efficiency (0.76% at 0.45 V) than ITO/MoS2-250 (0.22% at 0.7 V). The best PEC performance of G/MoS2-250 resulted from the combined effect of (i) a favorable graphene/MoS2 heterojunction, through which photo-generated e–h pairs were efficiently separated and transported; (ii) a desirable architecture of vertically aligned few-layer MoS2 nanosheets, which provided highly conductive edges that serve as efficient carrier pathways and better electronic contact with the graphene substrate; and (iii) a controlled height of few-layer MoS2 nanosheets within the diffusion length of the carriers. These results not only provide the optimal morphology of MoS2 for exploiting the full potential of 2D MoS2 for various PEC applications, but also demonstrate a practical large-scale and controllable 2D MoS2 synthesis approach on graphene.We demonstrated the successful manipulation of MoS


Covalent patterning of graphene opens many application possibilities in the field of photonics, electronics, sensors, and catalysis due to order-dependent optical properties, band structure engineering, and processibility and reactivity improvement. Owing to the low reactivity of the graphene basal plane, harsh reagents  used for covalent functionalization normally result in poor spatial control, which largely compromises the intrinsic properties of graphene. Therefore, precisely spatial control on covalent patterning of graphene is of great importance. Herein, we summarize recent advances for covalent patterning of graphene from the microscale to nanoscale resolution using different techniques such as laser or electrochemical writing, template-directed growth, and tip-induced nanoshaving. Since its first mechanical exfoliation as a freestanding single-layer sheet from graphite by Novoselov and Geim in 2004  nanomaterial with sp in 2004 , graphen in 2004 . The univia both non-covalent and covalent methods. The non-covalent method of graphene functionalization has limited stability. The physisorbed networks, held together through weak non-covalent interactions, hinder its application in graphene-based electronics. In contrast, covalent functionalization is rather robust by forming stable covalent bonds between functional moieties and graphene surfaces. Covalent functionalization of graphene has been proven to be able to open up its band gap  setup after conducting local functionalization with in situ generated radicals from electrochemical reduction of diazonium salt  template with desired patterns, followed by the reductive activation of the regions of unprotected graphene with a Na/K alloy electron beam. Afterward, the sample was exposed to the nitro- or bromo-benzenediazonium salt dissolved in ethanol for covalent functionalization. After removing the excess reagent and the PMMA template, covalent patterning of graphene was successfully achieved with a resolution of few micrometers. By applying thermal treatment of the sample at 400°C, the graphene surface was totally recovered by the complete defunctionalization process, which realized a complete write/store/erase cycle for the management of chemical information. Recently, multifunctional graphene was achieved by stepwise covalent patterning of graphene with multiple diazonium components using a combined technique of electron beam lithography and self-limiting diazonium grafting  via thermal or photoactivation was also an efficient way to produce radicals for graphene covalent functionalization. via heat-initiated cleavage of DBPO with the assistance of a polydimethylsiloxane (PDMS) template. The DBPO decomposed at around 80°C to aryl radicals, which subsequently attached the exposed surface of graphene, forming covalent patterns at the micrometer scale. Four years later, via laser-writing activation, achieving a pattering resolution of 2 μm. Surprisingly, the covalent functionalization process was totally reversible, which allowed the write/read/erase control over the covalent chemical information stored on the graphene surface. In other words, applying a 532 nm laser source on the DBPO precovered graphene enabled writing of chemical information on the substrate. Switching the wavelength of the laser light to 633 nm showed better Raman imaging resolution, providing an excellent opportunity of reading the chemical information of the 2D-patterned graphene. The erasing process was simply performed by thermally removing the covalently attached molecules at high temperatures.Some decent efforts have been devoted to the scalable patterning of graphene substrates um salt . The diaum salt  demonstrgrafting . Figure via self-assembly .Patterning graphene at the nanoscale resolution is of critical importance to meet the requirement of the modern electronics. Some brilliant methods were applied to the covalent functionalization of the graphene surface with the resolution at a few nanometers. assembly . The assvia nanoshaving with the tip of the scanning tunneling microscope (STM). A “nano-man” was created by adjusting the nanoshaving angle and the dimensions. Following this method, in situ pattern, while the multiple directions were observed during the molecular self-assembly process in the ex situ pattern. This finding provides a possibility of controlling molecular crystallization in the desired alignment direction. The corral size of patterns can be controlled by the STM scanning area ranging from 11 to 67 nm.Besides the aforementioned bottom-up approach for covalent nanopatterning, the top-down method is also feasible for creating nanopatterns on the pre-grafted graphitic substrates. At the earliest, in situ growth process of the molecular self-assembly is possible by STM tip-induced nanoshaving. The covalently patterned arrays can potentially introduce a band gap in graphene by maintaining one-dimensional high-speed electron channels. Moreover, further grafting another type of molecule in the empty spaces in the nanopatterned graphene can lead to heterogeneous functionalities for creating hydrophilicity–hydrophobicity or electron donating–electron withdrawing heterostructures for electronic and optoelectronic applications.Covalent functionalization of graphene has been intensively studied in order to widen the application range and improve the chemical reactivity and processibility of graphene. However, precise spatial control on the covalent patterning of graphene has been a challenge. This work provides the overview of covalent patterning of graphene from the microscale to nanoscale including laser or electrochemical writing, template-directed growth, and tip-induced nanoshaving, which shows a great potential for achieving decent resolution even down to few nanometers. This offers an opportunity toward a wide range of applications. For example, the nano-corrals generated on graphitic substrates can be used as microelectrodes for catalytic uses, such as biosensors, or for molecular recognition. Studying the


VCP/p97) cause ALS and exhibit the hallmark nuclear-to-cytoplasmic mislocalization of RNA binding proteins (RBPs). However, the mechanism by which mutations in VCP lead to this mislocalization of RBPs remains incompletely resolved. To address this, we used human-induced pluripotent stem cell-derived motor neurons carrying VCP mutations. We first demonstrate reduced nuclear-to-cytoplasmic ratios of transactive response DNA-binding protein 43 (TDP-43), fused in sarcoma/translocated in liposarcoma (FUS) and splicing factor proline and glutamine rich (SFPQ) in VCP mutant motor neurons. Upon closer analysis, we also find these RBPs are mislocalized to motor neuron neurites themselves. To address the hypothesis that altered function of the D2 ATPase domain of VCP causes RBP mislocalization, we used pharmacological inhibition of this domain in control motor neurons and found this does not recapitulate RBP mislocalization phenotypes. However, D2 domain inhibition in VCP mutant motor neurons was able to robustly reverse mislocalization of both TDP-43 and FUS, in addition to partially relocalizing SFPQ from the neurites. Together these results argue for a gain-of-function of D2 ATPase in VCP mutant human motor neurons driving the mislocalization of TDP-43 and FUS. Our data raise the intriguing possibility of harnessing VCP D2 ATPase inhibitors in the treatment of VCP-related ALS.RNA binding proteins have been shown to play a key role in the pathogenesis of amyotrophic lateral sclerosis (ALS). Mutations in valosin-containing protein (  VCP gene that cause amyotrophic lateral sclerosis result in the mislocalization of RNA binding proteins TDP-43, FUS and SFPQ in human motor neurons. Treatment with a VCP D2-ATPase inhibitor reverses the mislocalization of TDP-43 and FUS, suggesting a gain-of-function of D2-ATPase is driving mislocalization.Harley et al. report that mutations in the  TARDBP, which encodes TDP-43), fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS) and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). Subcellular mislocalization of RBPs is also a pathological hallmark of ALS, with TDP-43 mislocalized from the nucleus to the cytoplasm in 97% of ALS cases.,,Amyotrophic lateral sclerosis (ALS) is an invariably fatal neurological disease in which there is selective and progressive degeneration of motor neurons (MNs). Deregulated RNA metabolism, and in particular RNA binding protein (RBP) subcellular localization and function, play a pivotal role in ALS pathogenesis. RBPs orchestrate the RNA life cycle, regulating transcription, splicing, RNA localization, function and decay. Some ALS-causing gene mutations encode RBPs, including transactive response DNA-binding protein 43 . Pathogenic mutations of VCP are most commonly found in the N terminal domain, which is responsible for cofactor and ubiquitinated substrate binding and are also present in the D1 and D2 domains.Valosin-containing protein (VCP or p97) is an abundant AAA+ ATPase (ATPases associated with diverse cellular activities) with a large variety of intracellular functions that encompass almost all aspects of cellular physiology. VCP functions include protein homoeostasis, mitochondrial quality control and apoptosis.VCP-related ALS.,,,VCP-mutation related ALS to first examine the nucleocytoplasmic distribution of key RBPs compared to control MNs. We reveal that TDP-43, FUS and SFPQ exhibit reduced nuclear-to-cytoplasmic ratios in the VCP mutant MNs, which extends to an aberrant presence within the neurites. We found that treatment of control MNs with a targeted VCP D2 ATPase inhibitor does not recapitulate ALS RBP mislocalization phenotypes, arguing against a loss of its function in this context. Importantly, we find that in VCP mutant MNs the nuclear-to-cytoplasmic mislocalization of both TDP-43 and FUS and the nuclear-to-neurite mislocalization of TDP-43, FUS and SFPQ are reversible by treatment with a pharmacological inhibitor of the VCP D2 ATPase domain. Cumulatively, these findings support a model whereby VCP mutations cause an increase in D2 ATPase activity, which in turn leads to mislocalization of TDP-43, FUS and SFPQ from the nucleus to the cytosol. Our study raises the prospect of harnessing VCP inhibitors that target the D2 ATPase domain in the treatment of VCP-related ALS.We have previously reported robust differentiation of human-induced pluripotent stem cells (iPSCs) into highly enriched and functionally validated spinal cord MNs in which we have identified time-resolved molecular phenotypes of Dermal fibroblasts were cultured in OptiMEM + 10% FCS medium. For iPSC generation, episomal plasmids, pCXLE hOct4 shp53, pCXLE hSK and pCXLE hUL (Addgene) were transfect into dermal fibroblasts. Two control lines used are commercially obtainable (control 2 and control 3) and were purchased from Coriell (cat. Number ND41866*C) and Thermo Fisher Scientific (cat. number A18945). Details of the iPSC lines used in the study can be found in IPSCs were maintained with Essential 8 Medium media (Life Technologies) on Geltrex (Life Technologies), and passaged using EDTA . IPSC cultures were kept at 37°C and 5% carbon dioxide. IPSCs underwent differentiation into spinal cord MNs as described in Hall et al.Motor neuron cultures were treated with 1 μM of ML240  for 2 h.Cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature (RT). For permeabilization and non-specific antibody blocking, 0.3% Triton-X containing 5% bovine serum albumin (BSA) (Sigma) in PBS was added for 60 min. Primary antibodies were made up in 5% BSA and then applied overnight at 4°C. Primary antibodies used were SMI-32 , ChAT , βIII-tubulin , TDP-43 , SFPQ , FUS , hnRNPA1  and hnRNPK . A species-specific Alexa Fluor-conjugated secondary antibody (Life Technologies) at 1:1000 dilution in 5% BSA was added for 90 min at RT in the dark. Cells were washed once in PBS containing DAPI, 4′,6-diamidino-2-phenylindole nuclear stain (1:1000) for 10 min.z-stacks with a z-step of 1 μm. Stacks were processed to obtain a maximum intensity projection. A minimum of 12 fields of view were taken for each well. To calculate the nuclear:cytoplasmic ratio of RBPs in single cells, images were analysed using the Columbus Image Analysis System (Perkin Elmer). A DAPI mask defined the nucleus, and based on nuclear properties a trained machine learning feature selected neurons in an automated fashion. For each individual cell, an average nuclear intensity of the RBP of interest was measured. For the cytoplasmic measurement, a 1.5 µm cytoplasmic region was defined around the nucleus within a cytoplasmic mask and an average intensity was measured. An example of this nuclear and cytoplasmic compartments defined by this analysis can be found in Images were acquired using the Opera Phenix High-Content Screening System (Perkin Elmer). Images were acquired with a ×40 objective as confocal For the nuclear:neurite ratio, we implemented a semi-automated image analysis pipeline combining Ilastik,When examining the nuclear:cytoplasmic or nuclear:neurite ratio, if an increase or decrease in the ratio is detected, it is not apparent which cellular compartment is contributing to the change. To address this for the nuclear:neurite ratio we have utilized the presence of compartment specific markers. The same semi-automated image analysis pipeline was implemented as for the nuclear:neurite ratio as described above, but in addition to the protein of interest, intensity measurements were performed for DAPI and βIII-tubulin and used to calculate the specified ratio.Protein levels of TDP-43, FUS and SFPQ were assessed in whole cells in control and VCP mutant MNs. Cells were subjected to untreated conditions or treatment of 1 μM of ML240 for 2 h prior to protein extraction. The cells were lysed and proteins were extracted with RIPA disruption. Total protein concentration was quantified using BCA protein assay (Sigma). Equal amounts of protein samples were then loaded onto a gel and separated by SDS PAGE and transferred onto a nitrocellulose membrane. Samples were then blocked with PBS, 0.1% Tween, 5% dry milk powder at RT for 1 h followed by primary antibody incubation overnight at 4°C. The following antibodies were diluted in PBS 5% BSA; TDP-43 , SFPQ , FUS , GAPDH . For detection, membranes were incubated with species-specific near infra-red fluorescent antibodies  at RT for 1 h and imaged using an Odyssey Fc Imaging System (Licor).VCP mutant iPSC lines, with details found in t-test was used when comparing between two individual groups with Gaussian distribution. When Gaussian distribution was not achieved a Mann–Whitney test was used. Statistical analysis was conducted by Prism 8. A P-value 0.05 or below was considered to be statistically significant .There are 3 control and 4 The data that support the findings of this study are available on request from the corresponding author.VCP-related ALS, including new hallmarks of ALS such as reduced SFPQ and FUS nuclear-to-cytoplasmic ratio in mutant neural precursors.,,,,VCP mutant iPSC-derived MNs to comprehensively investigate the subcellular localization of 5 ALS-related RBPs. Single-cell analysis of the nuclear-to-cytoplasmic ratio of >70,000 neurons revealed a decrease in TDP-43 and SFPQ in VCP mutant human MNs , SMI-32 and βIII-tubulin (TUJ1) . Importauman MNs , which btant MNs . The pretant MNs . To excltant MNs .VCP disease mutations exert dominant-active or dominant-negative effects. To gain mechanistic insight into the effect of the VCP mutations in human MNs, we utilized ML240, a potent and selective inhibitor of the D2 ATPase domain in the VCP protein.increased the nuclear-to-cytoplasmic ratio of TDP-43  result in a dominant-active effect of the D2 ATPase domain. Application of ML240 in VCP mutant MNs indeed robustly reversed the mislocalization of both TDP-43 and FUS when examining both the nuclear-to-cytoplasmic mislocalization and nuclear-to-neurite mislocalization . ALS-causing VCP mutations recapitulate key hallmarks of sporadic ALS including nuclear-to-cytoplasmic mislocalization of key RBPs including TDP-43, FUS and SFPQ.,,,VCP mutations lead to RBP mislocalization in ALS has remained elusive. In this study, we have used our highly enriched and functionally validated iPSC-derived, patient specific motor neuron model,,ALS is a devastating and incurable neurodegenerative disease. Missense mutations of VCP mutations (R155C and R191Q) cause selective reduction in the nuclear-to-cytoplasmic ratios of TDP-43, FUS and SFPQ in human MNs, where we observe a normal nucleocytoplasmic distribution of both hnRNPK and hnRNPA1. We further show that TDP-43, FUS and SFPQ are also mislocalized into the neurites of MNs  SPFQ is reversible through pharmacological inhibition of the D2 ATPase domain of VCP protein, using the potent and selective inhibitor ML240.,,VCP mutation mechanism has also been shown in relation to other phenotypes.,VCP mutations may result in both dominant active or negative effects, dependent on cofactor binding and subsequent downstream cellular pathways.The main finding of our work, however, is that The underlying molecular mechanisms of how VCP interacts with TDP-43, FUS and SFPQ is unknown and further studies should investigate this. To date, studies have shown a direct interaction between VCP and RBPs, including TDP-43VCP disease mutants exhibit increased D2 ATPase activity have potential important therapeutic implications. Indeed, VCP inhibitors have been found to rescue multiple VCP disease phenotypes in drosophila models and patient fibroblasts.VCP mutations. This extends beyond ALS and IBMPFD into some cases of VCP-related Charcot-Marie-Tooth disease and hereditary spastic paraplegia.,Our findings that Brain Communications online.This work was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK (FC010110), the UK Medical Research Council (FC010110) and the Wellcome Trust (FC010110). R.P. holds an MRC Senior Clinical Fellowship [MR/S006591/1]. A.S. acknowledges the support of the Wellcome Trust [213949/Z/18/Z].The authors report no competing interests.fcab166_Supplementary_DataClick here for additional data file.


Results showed a drastic decrease in richness and diversity of microbial communities after the enrichment with crude oil. Enriched microbial communities were mainly dominated by genera known to degrade hydrocarbons, namely Alcanivorax, Pseudomonas, Acinetobacter, Rhodococcus, Flavobacterium, Oleibacter, Marinobacter, and Thalassospira, without significant differences between geographic areas and locations. Predictive functional profiling of the enriched microbial consortia showed a high potential to degrade the aromatic compounds aminobenzoate, benzoate, chlorocyclohexane, chlorobenzene, ethylbenzene, naphthalene, polycyclic aromatic compounds, styrene, toluene, and xylene. Only a few genera contributed for more than 50% of this genetic potential for aromatic compounds degradation in the enriched communities, namely Alcanivorax, Thalassospira, and Pseudomonas spp. This work is a starting point for the future development of prototype consortia of hydrocarbon-degrading bacteria to mitigate oil spills in the Iberian NW coast.Oil spills are among the most catastrophic events to marine ecosystems and current remediation techniques are not suitable for ecological restoration. Bioremediation approaches can take advantage of the activity of microorganisms with biodegradation capacity thus helping to accelerate the recovery of contaminated environments. The use of native microorganisms can increase the bioremediation efficiency since they have higher potential to survive in the natural environment while preventing unpredictable ecological impacts associated with the introduction of non-native organisms. In order to know the geographical scale to which a native bioremediation consortium can be applied, we need to understand the spatial heterogeneity of the natural microbial communities with potential for hydrocarbon degradation. In the present study, we aim to describe the genetic diversity and the potential of native microbial communities to degrade petroleum hydrocarbons, at an early stage of bioremediation, along the NW Iberian Peninsula coast, an area particularly susceptible to oil spills. Seawater samples collected in 47 sites were exposed to crude oil for 2 weeks, in enrichment experiments. Seawater samples collected  Each year, three million tons of oil are released into the sea due to leaks or accidental oil spills resulting from the exploration, production, refining, transportation and storage of petroleum and its derivatives . Oil spiThe effectiveness of a response to mitigate an oil spill depends on the oil type, location, and the spill size . CurrentCrude-oil is a heterogeneous mixture of more than 17000 complex chemical components with predominance of hydrocarbons . Due to Alcanivorax, Cycloclasticus, Marinobacter, Oleiphilus, Oleispira, Thallassolituus, and generalist microorganisms that can grow on a wider range of carbon sources  (Petroleum degrading microorganisms can be classified into obligate hydrocarbonoclastic bacteria (OHCB), that have the ability to grow on a narrow range of HCs and HCs degradation products, such as the genera  sources . Numerouococcus) , howeverococcus) . In factococcus) . Since dococcus) . Previouococcus) . Therefoococcus) .Oceanospirillales and Pseudomonads appeared first followed by Colwellia, Cycloclasticus, methylotrophic organisms and Flavobacteria, Rhodobacteraceae, and Alteromonadaceae . Samples were then transported to the laboratory in a refrigerated ice box. At the laboratory, samples were filtered through Sterivex–1). The materials for microbial enrichments were sterilized by autoclaving at 121°C for 20 min and plastic materials were sterilized by UV light for 30 min.All glass and plastic materials were chemically decontaminated with 10% (v/v) HCL solution and, afterward, washed with deionized water , after passing through a 0.2 μm sterile filter to remove any microorganisms that could contaminate our samples. The carbon from the petroleum together with the nutrients supplied formed a ratio of C/N/P of 100:10:1.The flasks were incubated for 2 weeks under constant orbital agitation (100 rpm), at room temperature, in aerobic and aphotic conditions to avoid photo oxidation of the petroleum compounds.After 15 days of incubation, 1 mL of medium from each triplicate flask was collected with sterile syringes to determine the abundance of hydrocarbon degraders and afterward all three replicates were combined for DNA isolation.TM filters with a pore size of 0.22 μm, hydrophilic, PVDF, Durapore membrane  with the help of 50 mL sterile syringes, a PowerVacTM Manifold (Qiagen) and a vacuum pump, following the methodologies used in the Ocean Sampling Day event , in a 2-h time frame or until the SterivexTM filters resultant from filtration of natural and enriched communities were used for DNA extraction. DNA was extracted with the DNeasy® PowerWater® Sterivex Kit  and quantified fluorometrically using the Qubit dsDNA HS Assay . The amplification of the V4–V5 region of the genetic marker 16S rRNA gene was performed by using the primer pair 515YF (5′-GTGYCAGCMGCCGCGGTAA-3′) and Y926R-jed (5′-CCGYCAATTYMTTTRAGTTT-3′), according to Earth Microbiome Project protocols . The raw Illumina fastq files for this study were deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB432891.Sequencing of 16S rRNA gene amplicons was performed in Biocant – Biotechnology Park . Two Polymerase Chain Reaction (PCR) rounds were performed to amplify first the DNA with the specific primers and reamplify afterward to add sequencing adapters and dual indexes. The KAPA HiFi HotStart PCR kit was used to perform the PCR reactions, which included 0.3 μM of each primer and 12.5 ng of template DNA in a total volume of 25 μL. Conditions of PCR involved denaturation at 95°C for 3 min followed by 25 cycles of 20 s at 98°C, 30 s at 50°C, 30 s at 72°C with a final extension for 5 min at 72°C. The second round of PCR reactions were performed according to the manufacturer’s suggestions . At GenoFor this work, the Most Probable Number (MPN) procedure was adapted from The QIIME2  bioinformatic platform was used to pre-process, merge, filter by quality and to apply the DADA2 pipeline .https://www.arb-silva.de/download/archive/qiime)  was already demultiplexed, the first step taken in QIIME2 pipeline was to remove the forward and reverse primers of each amplicon by using cutadapt with default parameters . Afterwae/qiime) . The NaïThe prediction of the functional composition of the metagenomes was accomplished by using PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) (version v2.3.0 beta) . The absFor this work, a total of 397 KEGG orthologs (KOs) were identified and analyzed for ten hydrocarbon degradation pathways: Aminobenzoate degradation (00627) , Benzoate degradation (00362) (82 KOs), Chlorocyclohexane and chlorobenzene degradation (00361) (26 KOs), Degradation of aromatic compounds (01220) (98 KOs), Ethylbenzene degradation (00642) (9 KOs), Naphthalene degradation (00626) (15 KOs), Polycyclic aromatic hydrocarbon degradation (00624) (34 KOs), Styrene degradation (00643) (18 KOs), Toluene degradation (00623) (33 KOs), and Xylene degradation (00622) (29 KOs) .--stratified option was added to the picrust2 full pipeline (picrust2_pipeline.py). For each degradation pathway (group of KOs), the absolute contribution of each ASV for each KO (“taxon_function_abun” column in the stratified output table) was added at the genus level and divided by the total contributions of that group of KOs using the group_by and summarize functions of the dplyr R package (v. 0.8.3)  . Unclassn = 16762), using absolute ASV numbers agglomerated at genus level, using the vegan package (v. 2.5-6)   and dive. 2.5-6)  in R (v . 2.5-6) . These i. 2.5-6) , scales . 2.5-6) , dplyr (. 2.5-6) , ggpubr . 2.5-6) , gridExt. 2.5-6) , and cow. 2.5-6) .n = 16762), which allowed to evaluate the structure of the microbial communities in natural and enriched conditions. To evaluate the similarities between all natural and oil-enriched communities, an analysis of similarities (ANOSIM) was performed using PRIMER 6 software   and the software .The taxonomic analyses at both phylum and genus levels were done with R phyloseq package (v. 1.30.0)  and the n = 1624619) and the clam command in vegan package (v. 2.5-6)  , in R, wSequencing analysis yielded a total of 6266779 raw sequences, which after the DADA2 pipeline, decreased to 3867589. The number of raw reads recovered from the natural community DNA samples varied between 42759 and 114733 and the number of raw reads recovered from samples after the petroleum enrichments varied between 31639 and 108914 . After bRarefaction curves  show thap < 2.2e−16) decreased in richness (Number of observed ASVs – p < 2.2e−16) dominated by the most abundant ASVs in comparison to natural microbial communities . In addition, this analysis revealed that these prokaryotic communities are highly homogeneous in terms of sampling area  and location .The biodiversity present in the natural microbial communities significantly , Proteobacteria (≈ 24–66%), Actinobacteria (≈ 0.23–30%), Thaumarchaeota (≈ 0–25%), Verrucomicrobia (≈ 0.06–15%), Cyanobacteria (≈ 0.02–13%), and Euryarchaeota (≈ 0–9%) . In the Of the 1643 distinct taxa identified in the natural and oil-enriched microbial communities, combined, only 55 taxa were found to be equally abundant in both communities, 841 taxa were predominant in the natural communities and only 106 taxa were more abundant in the enriched communities.Alcanivorax, Thalassospira, Pseudomonas, Marinobacter, Alkanindiges, Alteromonas, Cycloclasticus, Halomonas, Joostella, Marinomonas, Rhodococcus, and Thalassolituus. Most of these genera were also detected in the natural communities but in very low number of ASVs. Moreover, a few genera known HC degraders were found mainly in the natural communities , while some others were considered equally abundant in both natural and enriched communities .Specialist analysis allowed to distinguish a group of genera that were significantly more abundant in the oil-enriched communities , revealiAlcanivorax (0.04 to ≈ 70%) and Marinomonas (0.64 to ≈ 5.9%), as well as in other genera like Acinetobacter (0.60 to ≈ 41%), Joostella (0.26 to ≈ 12%), Oleibacter (0.15 to ≈ 28%), Pseudomonas (0.76 to ≈ 45%), Rhodococcus (0.04% increased to ≈ 31%), Salegentibacter (2.81 to ≈ 14%), and Thalassospira (0.01 to ≈ 20%)  .Oleiphilus (0.09 to 0%) and Oleispira (0.25 to 0.08%), and also Ulvibacter (17 to ≈ 7%). Flavobacterium was the only genus that kept its abundance of ≈30% in both communities.Nonetheless, some HC-degrading genera that were present in the natural microbial communities decreased in abundance in the enriched prokaryotic communities, like the OHCB The predicted functional profiling of the microbial communities showed that the natural communities were equipped with machinery to degrade petroleum compounds  and afteRegarding the ten HC degradation pathways analyzed for the 47 sampled sites, there was an increase, on average, in the capability to degrade aminobenzoate (≈ 38%) benzoate (≈ 58%), chlorocyclohexane and chlorobenzene (≈ 37%), aromatic compounds (≈ 59%), ethylbenzene (≈ 197%), naphthalene (≈ 115%), styrene (≈ 10%), and toluene (≈ 64%) from natural microbial communities to enriched microbial communities. However, there was an average decrease in capability to degrade PAH (−27%) and xylene (−2%) compounds.Tenacibaculum for ethylbenzene degradation. On the other hand, in the enriched communities, it was observed a lower number of different taxa (217 genera) contributing to the potential capability to degrade different petroleum aromatic HCs, where a distinct contribution of only five taxa was observed for all 10 degradation metabolisms, namely Alcanivorax, Thalassospira, Pseudomonas, Marinobacter, and Flavobacterium , with the exception for PAH and xylene degradation with a 0% contribution in both pathways.In the natural communities, it was predicted that a high number of different taxa (841 genera) contributed to the potential capability to degrade different petroleum aromatic HCs . Howeveracterium . The cumThe MPN analysis confirmed that, overall, there was an increase in abundance of hydrocarbon-degrading microorganisms during the enrichment period for all sampled sites . ResultsIn the present work, the distribution and selection of native hydrocarbon-degrading microorganisms at different sites along the NW coast of the Iberian Peninsula were investigated.Enrichment experiments allowed the identification and characterization of native microbial consortia with the ability to degrade petroleum compounds, during the early stages of the bioremediation of oil spills.Rarefaction curves showed that the biodiversity present in natural microbial communities decreased due to the oil-enrichment experiments, which is in agreement with the lower values of Shannon diversity index registered in the oil-enriched communities. Since petroleum is composed of recalcitrant and highly toxic compounds, it is expected that crude-oil will induce a selective pressure onto the natural microbial communities and only a small fraction of microorganisms capable of surviving and/or degrading petroleum HCs were able to adapt. This also explains why fewer dominant ASVs account for most of the total number of individuals in the enriched communities, as stated by the Berger-Parker index. Similar results were obtained in a study conducted after the Deepwater Horizon oil spill, where a significant decrease in diversity was observed in oil-plume communities when comparing with non-oil plume communities in the deep-sea . RibicicProkaryotic communities from oil contaminated marine sediments have been previously described has being dominated by Proteobacteria, Bacteroidetes, and Actinobacteria. The dominance of these phyla has been registered in beaches from the northern Spanish coast affected by the Prestige  as well A considerable high number of ASVs were not identified at phylum level in the oil-enriched samples, suggesting that our experimental approach was useful to select new HC degradation competent prokaryotes that are naturally available but difficult to cultivate in laboratory. Future research on these unidentified sequences may reveal novel taxa that may represent a high fraction of the still unknown natural microbial diversity able to degrade HC .Alcanivorax (up to 70%). This observation is consistent with previous studies where seawater containing crude-oil or heavy oil, supplemented with nitrogen and phosphorous fertilizers, were dominated by Alcanivorax  and Flavobacterium (up to 30%) were also found to be highly represented genera in the oil-enriched communities in this study. Flavobacterium also had a relative high abundance in the natural communities (up to 29%), however, some strains in this genus are also described as being capable to mineralize several types of organic matter in aquatic ecosystems , Joostella (up to 12%) and Alkanindiges (up to 10%), previously identified as hydrocarbon-degraders , Oleispira (up to 0.25%), and Oleiphilus (up to 0.09%), were not enhanced by the oil-enrichment experiments performed in the present study. These points to some constrain in the enrichment process developed in the present study. For example, some restraints in enhancing the abundance of Cycloclasticus might have occurred since bacteria from this genus are specialized in degrading more complex HC , since an increase of microorganisms from this genus was only observed for microcosms maintained at 4°C , natural microbial communities had hydrocarbon degrading microorganisms in their composition, so it was expected that by the end of the crude-oil enrichments their abundance would increase, as it was observed. These results are corroborated by In this work, the hydrocarbon-degrading microbial abundance was estimated by the MPN method, for both natural and oil-enriched microbial communities, showing that all sampled sites present capacity to degrade petroleum hydrocarbons. Despite the low abundances obtained  https://www.ebi.ac.uk/ena/browser/view/PRJEB43289.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: European Nucleotide Archive, accession no: CM, AB, CA, and AM: conceptualization. MB, RP, DA, JF, AB, and CA: laboratory and field work. MB, MS, CM, and AM: data curation. MB, CM, and AM: writing – original draft preparation. MB, CM, RP, DA, JF, AB, SR, MC, MS, JL, CA, and AM: writing – review and editing. CM, JL, and AM: supervision. CM, SR, MC, CA, and AM: project administration and funding acquisition. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Different spectral patterns from graphene-riboflavin surface were revealed by SHINERS and GERS techniques. Contrary to GERS method, SHINERS spectra revealed not only ring stretching bands but also vibrational features associated with ribityl group of riboflavin and D-band of graphene. Based on DFT modeling it was suggested that activation of D-band took place due to riboflavin induced tilt and distortion of graphene plane. The ability to explore local perturbations by the SHINERS method was highlighted. We demonstrated that SHINERS spectroscopy has a great potential to probe adsorbed molecules at graphene.Graphene research and technology development requires to reveal adsorption processes and understand how the defects change the physicochemical properties of the graphene-based systems. In this study, shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) and graphene-enhanced Raman spectroscopy (GERS) coupled with density functional theory (DFT) modeling were applied for probing the structure of riboflavin adsorbed on single-layer graphene substrate grown on copper. Intense and detailed vibrational signatures of the adsorbed riboflavin were revealed by SHINERS method. Based on DFT modeling and detected downshift of prominent riboflavin band at 1349 cm Electronic properties and function of graphene depend on the surface structure, origin of the defects ,2, dopinnh symmetry are favorable because of better compatibility with graphene structure [Raman spectroscopy is a non-destructive, sensitive, and powerful technique able to provide detailed structural information about various carbon nanostructures ,13. In atructure . Being dtructure .To observe functional groups of graphene oxide, adsorbed impurities or purposely-adsorbed compounds on graphene, the alternative spectroscopic technique is required. Recently, Tian et al. approached a novel surface-enhanced Raman spectroscopy (SERS) technique and called it “shell-isolated nanoparticle-enhanced Raman spectroscopy” (SHINERS) . The met2 is a water-soluble vitamin that plays an essential role in cellular biochemistry [Riboflavin (Rf) or vitamin Bhemistry . Renewedhemistry . Rf can hemistry . Riboflahemistry . Thus, sThe main aim of our work was to demonstrate the applicability of the SHINERS technique to acquire molecular-level information for adsorbed Rf on the graphene surfaces. The detailed insights into the interaction of Rf and graphene are provided from the analysis of coupled SHINERS, GERS, and DFT modeling approaches.4 · 3H2O, 99%), trisodium citrate dihydrate (HOC(COONa)(CH2COONa)2 · 2H2O, 99%), (3-aminopropyl)trimethoxysilane (H2N(CH2)3Si(OCH3)3, APTMS, 97%), sodium silicate solution . Graphene substrate was received from Graphenea , and riboflavin was obtained from Alfa Aesar . All solutions were prepared with ultrapure water (resistivity of 18.2 MΩ cm) from Direct-Q 3UV  water purification system.All used reagents and solvents  were used without further purifications: gold (III) chloride trihydrate (HAuCl4 solution (0.01 wt%) was brought into a round-bottom flask and boiled in an oil bath (150 °C) with vigorous stirring. The solution reached boiling temperature in 3 min. Then 0.35 mL of sodium citrate (1 wt%) were quickly added into the boiling solution and the mixture was refluxed for 30 min. Next, the solution was left to attain room temperature (about 2 h); so the red colloid of gold nanoparticles (Au NPs) was obtained. For the growth of an ultrathin layer of silicon dioxide over nanoparticles, 0.6 mL APTMS (1 mM) was added to 45 mL of Au NPs. The mixture was vigorously stirred for 15 min; then 4.8 mL of sodium silicate solution (0.54% wt/wt) was added and the mixture was stirred again for 3 min at room temperature. The flask was placed in an 85–90 °C water bath and stirred for 45 min. Then, to stop the reaction process, the flask was placed in the ice bath (3 °C) for 30 min. The cold mixture was transferred into several 1.5 mL test tubes and centrifugated at 5500 rpm for 15 min. After that, the supernatant was removed, the 1.5 mL of water was added, and nanoparticles were dispersed again. Next, tubes were centrifuged for another 15 min and supernatant was removed again to obtain Au@SiO2 nanoparticles as a concentrated colloidal suspension. Therefore, cleaned Au@SiO2 nanoparticles were kept in the dark at low temperature (5 °C) until were used in SHINERS experiments.Initially, 50 mL of an aqueous HAuClHigh-resolution transmission electron microscopy (HR-TEM) image was recorded by Tecnai G2 F20 X-TWIN microscope  equipped with scanning TEM module and high-angle annular dark-field detector for Z-contrast imaging. FEI Helios Nanolab 650 dual beam microscope with an Omniprobe manipulator  was employed to prepare specimens for the measurement.2 solution were dropped onto pure graphene and adsorbed riboflavin onto graphene substrate surfaces and dried in a fume hood for about 30 min.Commercially available graphene substrate grown onto Cu foil was washed with water. Chemisorbed riboflavin layer onto graphene substrate was formed in 1 mM riboflavin water solution for 14 h. Later it was washed with ultrapure water and dried at room temperature in a fume hood for about 30 min. For SHINERS experiments, 5 µL of concentrated Au@SiO−1 or by recording the polystyrene standard (ASTM E 1840) Raman spectrum. Raman spectra were corrected by polynomial function background subtraction. The GERS spectra were recorded by using 532 nm excitation wavelength, while 785 nm wavelength was applied for acquisition the SHINERS spectra. Integration time was 10 s and each spectrum was recorded by accumulation of 10 scans yielding total 100 s collection time. Both SHINERS and GERS spectra were normalized to 1 s intensity. No smoothing procedures were applied to the experimental data.The GERS and SHINERS spectra were recorded by inVia Raman  spectrometer equipped with thermoelectrically cooled to −70 °C CCD camera and a confocal Leica microscope. Two different wavelengths, 785 and 532 nm laser beams were used as the excitation source. The 180 °C scattering geometry was employed in Raman spectroscopy studies. The 50×/0.75 NA objective lens and 1200 lines/mm (for 785 nm) and 1800 lines/mm (for 532 nm) gratings were exploited to record the Raman spectra. Laser power at the sample surface was restricted to 0.9 and 0.6 mW for 785 and 532 nm excitations, respectively. Raman scattering wavenumber axis was calibrated by using the silicon peak at 520.7 cmRaman spectra of riboflavin powder and riboflavin solution were recorded by using HyperFlux PRO Plus  spectrometer at 785 nm excitation. Laser powers were 15 mW for Rf powder and 495 mW for Rf-water solution of approximately 0.3 mM. The overall accumulation time was 37.5 and 1375 s for Rf powder and Rf-water solution spectra, respectively. For excitation and collection of the Raman spectra, the fiber-optic cable was employed. The laser beam was focused to a 100 μm diameter spot. The calibration of the wavenumber axis was confirmed with the polystyrene standard (ASTM E 1840) spectrum.Parameters of the vibrational bands were determined by fitting the experimental spectra with Gaussian-Lorentzian shape components using GRAMS/A1 8.0 software .54H18 and riboflavin in the gas phase were modeled using Orca 5.0.1 software [Ead was calculated according to the relation:EG-Rf, EG, and ERf are energies for graphene-riboflavin complex, graphene, and riboflavin. The basis set superposition error (BSSE) was corrected by a counterpoise method that introduces BSSE to Equation (1) [Molecular interactions of hydrogen-terminated graphene nanosheet Csoftware . We usedtion (1) . Vibratition (1) . Total dtion (1)  were caltion (1) . Visualition (1) .−1 /I(G) can be used for the evaluation of the number of graphene layers [−1 which are characteristic to Cu2O compound [−1 a the domtructure ,45,46; we layers . For ourcompound .−1. Shift of G and 2D bands indicates riboflavin-induced doping of graphene layer. It should be noted that the relative intensity of graphene bands remained similar as before adsorption (I(2D)/I(G) = 1.92). The new bands located at 1351, 1411, and 1536 cm−1 are characteristic for riboflavin ring [2O Raman spectrum  + stretching of rings I, II, III vibrational mode [−1. The band at 1525 cm−1 corresponds to stretching vibration of rings I, II, and III, and the 1457 cm−1 band to CH3 deformation vibration coupled stretching vibrations of rings I and II and ribityl bending mode. The 740 cm−1 and 710 cm−1 modes are ring I in-plane bending and rings I, II, and III in-plane deformation vibrations, respectively. The low-intensity band 1592 cm−1 is associated with graphene G-band vibrational mode. The origin of intense bands at 930, 981, and 1048 cm−1 is not completely clear; these bands might be related with stretching vibration of ribityl chain. More detailed analysis of spectroscopy of adsorbed riboflavin and effects of interaction with graphene substrate is provided below.n powder B. This bnal mode ,48,49,50The energy minimization moves riboflavin into the orientation parallel to the graphene nanosheet, with an average 3.3 Å distance between the polycyclic Flavin moiety and the graphene . The disEad of −22.39 for B3LYP/6-311G and −33.35 kcal/mol for B3LYP/def2-TZVP basis sets. Generally, adsorption energy can be broken down into physically meaningful stabilizing components: electrostatic, dispersion, polarization, and charge transfer energies, and the stabilization components: Pauli repulsion and preparation energies [Here, we studied interactions in the gas phase. Calculations predict sufficiently negative adsorption energy energies . The disenergies ,53. Hirsenergies ,54,55. Fenergies . First, EHOMO) and electron affinity (related to ELUMO). The energy difference between HOMO and LUMO is regarded as the fundamental energy gap Efund, which quantitatively determines chemical potential (μ), molecular hardness/softness, and electrophilicity [Efund signifies the higher molecular reactivity and lower kinetic stability. The predicted Efund for the molecular complexes is 2.39 eV at 6-311G and 2.31 eV at def2-TZVP basis sets calculations. These values are smaller compared to those of Rf optimized in the gas phase.To investigate the electronic complex structure, density of states (DOS) plots were constructed. DOS shows the distribution of energy levels that electrons can occupy at a given molecule. DOS could be plotted as total DOS (TDOS) for the full complex and as a partial DOS (PDOS) for individual components of the complex. hilicity . The smaEopt of the G-Rf complex , followed by transition at 639, 433, and 428 nm. These transitions start at different orbitals in the 267−270 range that are mainly located at graphene nanosheet and are targeted to the 271st molecular orbital located at Flavin group. Compared to the sole Rf, the introduction of graphene perturbs its electronic structure that shits absorption to the red and increases the transition probability.TD-DFT at B3LYP/6-311G was used to calculate the optical gap energies  complex . Usuallyhe Efund . TD-DFT −1 is assigned to the νs(C2-N3-C4) + stretching motion of rings I, II, and III ) used for modeling. A clear 4 cm−1 redshift, which is also supported by DFT, is observed for the Rf transfer from aqueous solution to graphene surface. Such a frequency downshift of the vibrational modes of atoms involved in delocalized π orbitals has been previously linked to π-stacking [−1 compared to the one in the solution spectrum. Comparing experimental spectra obtained from Rf in different environments, the bands in the experimental G-Rf spectrum at 1158, 808, and 740 cm−1 are assigned to Rf. However, DFT modeling does not predict strong bands in the 900–1000 cm−1 spectral region.stacking ,59, ther−1 band of Rf was found to increase by a factor of 26 in SHINERS spectrum comparing with GERS. In the fingerprint spectral region SHINERS spectrum exhibited three well-defined bands , which were tentatively assigned to vibrational modes having a high contribution from vibration of atoms in ribityl chain  in the graphene structure. Our work highlighted the ability of SHINERS spectroscopy to probe the local structural perturbations in graphene.In this paper, we provided detailed vibrational spectroscopy study of graphene-riboflavin surface. Several experimental vibrational spectroscopy methods, including ordinary Raman (riboflavin solution and powder spectra), resonance Raman (graphene spectra), graphene-enhanced Raman, and shell-isolated nanoparticle-enhanced Raman spectroscopies were employed to probe the structure and bonding of both single-layer graphene grown on copper and riboflavin adsorbate. We found that riboflavin adsorption induces the blue-shift of the 2D and G bands by 11 and 3 cm


Fluorinated graphene contains F atoms with high levels of chemical activity, and the application of fluorinated graphene in energetic materials may greatly contribute to the progress of combustion reactions. However, there is a lack of research on the thermal properties of fluorinated graphene and its application on nitrate esters. In this paper, theoretical calculations and experiments were used to study the thermal properties of fluorinated graphene and its application on nitrate esters. The anaerobicity and poor thermal stability of fluorinated graphene were proved by ab initio molecular dynamics (AIMD) calculations and TG-DSC experiments. The ester weakening effect of fluorinated graphene on nitroglycerin was determined via wavefunction analysis, with the greater the fluorination degree, the stronger the ester weakening effect. The existence of fluorinated graphene can significantly increase the heat dissipation of the composites, which was concluded by TG-DSC experiments and TG-DSC-MS-FTIR. The research in this article provides an important reference for the application of fluorinated graphene in energetic materials. Fluorinated graphene (FG) has been known to be a typical derivative of graphene since it was first prepared in 2010 , adjusta3/B coated with FG. They not only proved that the existence of FG can promote the combustion of boron powder, but also revealed the mechanism for promoting combustion, which involves FG eliminating the inert B2O3 shell at a low temperature.Aluminum (Al) and Boron (B) are often added to solid propellant ,7 as higHigh-energy explosives can significantly increase the energy level of solid propellants ,16,17,18In this article, in order to understand the characteristics of FG and its influence on the thermal decomposition of nitrate esters, theoretical calculations and experiments were used to study both the thermal stability and thermal oxidation of FG and the effects of FG on the thermal decomposition of absorbent powder (the main ingredient is nitrate esters).In this paper, CP2K 8.2  was mainIn CP2K 8.2, PBE-D3 functionals ,25,26 coIn order to explore thermal stability, PBE-D3/DZVP-MOLOPT-SR-GTH was used to obtain the 4000 fs AIMD trajectories (step-size is 1 fs) of graphene, whole-fluorinated graphene and half-fluorinated graphene under the CSVR  thermostat and micro-canonical ensemble (NVE). In order to explore the thermal oxidation stability, GFN1-xTB  was usedIn ORCA 5.0.1, the RIJCOSX technique  and the Few-layer graphene: its diameter was 0.5–5 μm with a purity of around 99.8wt%. The thickness was 0.8–1.2 nm.Fluorinated graphene: its diameter was 0.4–5 μm with a purity of 98% The molar ratio of F/C is 29.68–36.63%.Absorbent powder: this was prepared by mixing uniform and via cooling with centrifugation. 55.5% nitrocellulose, 30.9% nitroglycerin, 12.6% glycerol triacetate and 2% 1.3-Dimethyl-1,3-diphenylurea were its main components.The composites of absorbent powder and graphene or fluorinated graphene were obtained by mechanical stirring. After being stirred and standing at room temperature for 24 h, the graphene (fluorinated graphene) and powder are mixed in the mass ratio of 2:3.2 or O2.A simultaneous thermal analyzer NETZSCH STA 449 F3  with a platinum furnace was used for TG-DSC analysis. The heating rate was 5 °C/min and the atmosphere was N2O3 furnace was used for TG-DSC-MS-FTIR analysis. The heating rate was 5 °C/min and the atmosphere was N2.The coupling device NETZSCH STA 449 F3-QMS 403-FTIR  with a AlThermo Fisher Scientific Nexsa  was used for the testing of X-ray photoelectron spectroscopy (XPS), and the charge correction was carried out with C1s = 284.80 eV as energy standard.2 and CF3 groups is around 291–293 eV in the XPS C1s spectrum [2 at 291.7 eV, CF3 at 293.8 eV, respectively. This matches the results of the O1s XPS spectrum and F1s XPS spectrum in 2 groups and CF3 groups in the fluorine graphene sample are 70.5%, 26.2% and 3.3%, respectively.spectrum ,43. TherAfter fluorinating, π-π packing was destroyed and the layer spacing increased in graphene. Usually, the change in structure will cause a change in thermal stability and thermal oxidation stability. In terms of the application of fluorinated graphene in energetic materials, it is very important to understand the thermal stability and thermal oxidation of FG, but there are few studies on the thermal properties of FG. In this section, the thermal stability and thermal oxidation of graphene and FG are studied via theoretical calculations and experiments.2 at 25–600 °C were studied, and the results are shown in In order to verify our speculation regarding thermal stability, which arose as result of theoretical calculations, the TG-DSC curves of graphene and fluorinated graphene under NIn order to explore the thermal oxidation of fluorinated graphene, we obtained the AIMD trajectories of oxygen molecules near the graphene, whole-fluorinated graphene and half-fluorinated graphene at 2000 K with a CSVR thermostat. The 0 fs, 300 fs and 900 fs models were selected, whose front and top viewed at 0 fs, 300 fs and 900 fs are shown in 2 at 25–750 °C were studied, and the results are shown in In order to further understand the thermal oxidation properties of fluorinated graphene, the TG-DSC curves of graphene and fluorinated graphene under OThe results relating to the binding energy of these structures  at wB97M-V/def2-TZVP in combination with counterpoise correction can be seen in According to First of all, according to In In this section, the strength of the nitrate groups in NG which belong to different structures  are discussed. The electron density difference between graphene  and NG are used to explain the difference in strength.2 in NG which belong to different structures. The positions of O-NO2 are shown in Commonly, the integration of the electron localization function (ELF)  in overl2, but it will weaken the bond strength of left O-NO2 and right O-NO2 in NG, which is defined as the ester weakening effect. Secondly, comparing the magnitude of this effect, graphene shows the strongest ester weakening effect, and half-fluorinated graphene shows the weakest ester weakening effect. Finally, comparing the ester weakening effect of half-fluorinated graphene and whole-fluorinated graphene, it can be explained that the greater the fluorination degree, the stronger the ester weakening effect. In order to further explain the mechanism of the ester weakening effect, electron density difference maps were analyzed. In 2 and right O-NO2 in NG is where the electron density decreases and the middle area of NG exhibits polarized characteristics. However, it is worth noting that 2 and right O-NO2, and the wider the electron exchange, the greater the decrease in bond strength.In In In this section, the thermal decomposition of the composites  is researched for the purpose of understanding the effect of fluorinated graphene on the thermal decomposition of nitrates. TG-DSC and TG-DSC-FTIR-MS are used to achieve this.xOy , CO and CO2. Finally, −1 [−1 when the temperature is 650 °C, and this corresponds to the vibration of C-F bonds produced during the decomposition of the composite (fluorinated graphene + absorbent powder). According to the MS and FTIR images in 2 and fluorine-containing substances [ally, −1 ,54. In Fbstances .In this paper, both the thermal stability and thermal oxidation of fluorinated graphene and the effect of fluorinated graphene on the thermal decomposition of nitrate esters were studied via theoretical calculations and experiments, and the following four conclusions were obtained.(1)The thermal stability of fluorinated graphene is poor compared with graphene in daily experiments.(2)Fluorinated graphene is anaerobic, and the degree of anaerobicity increases as the degree of fluorination increases.(3)Fluorinated graphene influences the nitrate group in nitrate esters, and the deeper the fluorination degree, the greater this influence is.(4)Fluorinated graphene greatly increases the heat released during the decomposition of the composites when it is added to the absorbent powder.In the future, new preparation methods for FG-EM composites and theoretical calculations will greatly promote the application of fluorinated graphene in the field of energetic materials.