We reportfull control over charge separation states and thus photogating fielddirection by electrically tuning the defect occupancy. Our resultsshow that electron occupancy of the two in-gap states, presumablyoriginating from sulfur vacancies, can account for the observed richinterfacial charge transfer dynamics and electrically tunable photogatingfields, providing microscopic insights for optimizing optoelectronicdevices.Bond-free integrationof two-dimensional (2D) materials yieldsvan der Waals (vdW) heterostructures with exotic optical and electronicproperties. Manipulating the splitting and recombination of photogeneratedelectron–hole pairs across the vdW interface is essential foroptoelectronic applications. Previous studies have unveiled the criticalrole of defects in trapping photogenerated charge carriers to modulatethe photoconductive gain for photodetection. However, the nature androle of defects in tuning interfacial charge carrier dynamics haveremained elusive. Here, we investigate the nonequilibrium charge dynamicsat the graphene–WS Such artificial material manipulation provides fertileground for exploring fundamental physics and enabling novel optoelectronics.Among the vast gamut of vdW heterostructures, graphene–transitionmetal dichalcogenide (TMDC) heterostructures have received particularattention because they inherit strong light–matter interactionsand high charge carrier mobility from their constituent building blocks.17 It has been reported that photoinduced charge transfer (CT) in graphene–TMDCheterostructures occurs within ∼100 fs, and the activated CTtrajectories critically depend on the pump photon wavelength. Whiledirect hole tunneling from TMDC to graphene dominates the CT mechanismfollowing resonant or above-bandgap photoexcitation,17 photothermionic emission (PTE) governs the CT process upon sub-bandgapphotoexcitation.19 In the PTE scenario, the absorbedlow-energy photons efficiently heat the electron bath in graphene,resulting in a thermalized hot carrier distribution. The thermalizedhot carriers with sufficiently high energy can be emitted over theSchottky barrier at the graphene–TMDC interface.18As the backbone of these applications,understanding and tuningthe spatial distribution of photogenerated electron–hole pairsat the vdW interface is paramount for photocurrent operation. Forthat, recent spectroscopic studies have investigated photoinducedinterfacial processes in graphene–TMDC heterostructures usingultrafast pump–probe techniques.20 In our previous work,10 we revealedthat the charge separation time is closely related to defect statesin TMDCs: electrons populating the excited states of WS2 are rapidly and efficiently trapped by empty defect states within∼1 ps and subsequently stored there for more than ∼1ns. This leads to long-lived charge separation and efficient photogatingin graphene, in line with the ultrahigh photoresponsivity observedin high-performance graphene–TMDC-based photodetectors.1 Rational defect engineering and manipulationrepresents one of the key strategies to further optimize the performanceof optoelectronic devices. Nonetheless, realizing the full potentialof defect engineering for optoelectronics is nontrivial, as it requiresconcerted efforts to understand the origin of defects, develop effectivemethods to control their nature/density, and elucidate their impacton microscopic interfacial charge carrier dynamics. So far, limitedefforts have been devoted to tackling these issues.Following optical excitationsand CT, transferred electrons andholes reside in different materials: transient charge separation statesemerge. The resulting charge separation time is critical in determiningthe photoconductive gain and operation bandwidth for many graphene-basedoptoelectronic devices such as photodetectors.2 heterostructuresby operando optical-pump terahertz (THz)-probe (OPTP) spectroscopy.Electrochemical gating effectively controls the chemical potentialin graphene and, thus, the interfacial energetics for CT. More importantly,it allows modulation of the filling of electronic defect states atthe interface, which strongly affects the photogating mechanism. Wereport the first experimental implementations of electrical controlof photogating field direction  and efficiency in vdW heterostructures: for p-doped heterostructureswith empty in-gap defect states, photoinduced electron injection ortrapping into defects in WS2 governs the photogating mechanism,leading to an “electron photogating” effect; for n-dopedheterostructures with fully filled in-gap defect states, photogeneratedholes are injected into defects, resulting in a “hole photogating”scheme. Such reversal of the photogating mechanism is of relevanceand importance for optoelectronic applications based on vdW heterostructures.By simulating the hot carrier distribution in the heterostructurefollowing photoexcitation, we further provide compelling experimentalevidence that the observed rich interfacial CT dynamics and electricallytunable photogating fields at graphene–WS2 vdW interfacescan be rationalized by electron occupancy of two distinct in-gap states,presumably originating from sulfur vacancies. Our results not onlyreveal fundamental interfacial photophysics across the vdW interfacebut also provide insights into the design of advanced (opto)electronicapplications through defect engineering.Here, wetrack and manipulate nonequilibrium charge carrier dynamicsin electrochemically gated graphene–WS2 heterostructure(see details in the Supporting Information). The monolayer nature of the bare WS2 monolayer is manifested by two pronounced optical resonances at2.04 and 2.43 eV , correspondingto the well-established A- and B-exciton transitions in WS2.21 In addition, the Raman spectrum ofthe bare WS2 monolayer shows resonances at ∼355and ∼419 cm–1 , corresponding to its in-plane (E2g1) and out-of-plane (A1g) vibration modes, in line with previous reports.23 When brought in contact with graphene, WS2 exhibits red-shiftedexciton resonances  due tothe dielectric screening effect of the graphene layer.24 Meanwhile, the line width of the A-exciton resonanceof WS2 in the heterostructure broadens , which we attribute to the static interlayer CTprocess.25 These optical signatures indicategood electronic coupling at the vdW interface. For the heterostructure,along with the well-preserved vibration modes of WS2, weobtain the expected G- and 2D-bands originating from graphene . The single symmetric 2D-band confirmsthe monolayer nature of graphene, and the G-band position at ∼1586cm–1 suggests an initial EF of ∼0.15 eV in the graphene layer .As schematically shownin 2 vdW interfacewhile tuning the filling of defect states,we perform OPTP measurements on electrochemically gated graphene–WS2 vdW heterostructures (see details in the Supporting Information). As depicted in E/E) scales linearly with the photoconductivity26 (Δσ) and is recorded as a functionof pump–probe delay time. The heterostructure used in thisstudy is made of polycrystalline graphene and WS2 layersgrown by chemical vapor deposition (CVD) with typical domain sizesof a few micrometers. As the diameter of the probe beam used for OPTPmeasurements is ∼1 mm, the measured photoconductivity dynamicsrepresent the average response of heterostructures with mixed twistangles.To investigate the interfacialcharge dynamics at the graphene–WSVg – VD). The resistance reaches a maximum value of ∼1 kΩ inthe vicinity of the Dirac point and decreases in both the p-dopedand n-doped regimes. 2 vdW heterostructure at different gate voltages. Notably,the photoconductivity dynamics in the first 10 ps  in peak while tuning EF has been wellreported in graphene29 and can be simply understood as follows: in intrinsicor low-doped graphene, photoexcitation leads to a substantial increasein the charge carrier density, thereby increasing the conductivity. In contrast, in highlydoped graphene, photoexcitation elevates the electron temperature,leading to increased momentum scattering of charge carriers and, thus,a transient reduction in conductivity . Altogether, the evolution of resistance  and beyond 100 ps  under three different pump fluences, as shown in We now focus on photoconductivity dynamics on longer timescalesbeyond 20 ps. Because the intrinsic hot carrier response of bare grapheneis negligible within 10 ps,20ps upon electrochemical gating, from positive for the p-doped (referringto graphene) regime to negative for the n-doped regime. We understandthe observed gating-modulated photoconductivity as CT-induced transientelectron loss  in graphene,which downshifts EF and thus increases(decreases) the conductivity of p-doped (n-doped) graphene at allgate voltages employed. We also observe that Δσ>100ps is positive regardless of the gate voltage, indicating a transitionfrom CT-induced electron loss to gain while tuning graphene from thep- to n-doped regime. This result further indicates a reversal ofthe interfacial photogating field, from pointing away from graphenein the p-doped regime to pointing toward graphene in the n-doped regime.Our previous study10 reported that electronstrapped in empty defects in WS2 are responsible for photogatingin p-doped heterostructures. In the following, we show that holestrapped in the electrochemically filled in-gap defects in WS2 are responsible for photogating in n-doped heterostructures. Furthermore,by comparing Δσ20ps  CT occurs at the graphene–WS2 vdW interface only via PTE following sub-bandgap excitation in graphene,based on previous reports;34 (ii) the conduction band of WS2 and empty in-gap defect states serve as electron acceptors,while the valence bands of WS2 and electrochemically filledin-gap defect states are hole acceptors, whenever the interfacialenergetics is favorable; and (iii) following CT, the back-transfermakes the system relax back to initial conditions and dictates thecharge separation lifetime. We assume that the lifetime of chargecarriers trapped in the defect states is substantially longer thanthat of charge carriers populated in the conduction and valence bandsof WS2 because of their localized nature.10 Within this framework, we simulate thermalized hot carrierdistributions in graphene at different initial EF following photoexcitation and thermalization by taking intoaccount EF-dependent heat capacity, energyconservation, and particle number conservation (Supplementary Section 2). A fixed portion of the thermalizedhot electrons (holes) energetically distributed above (below) thecorresponding acceptor states are assumed to be able to transfer acrossthe vdW interface.To describe theelectrically tunable CT-induced photoconductivityand its evolution over time, we propose a defect-mediated CT and photogatingmechanism. In this mechanism, electrochemical gating modulates thedefect occupancy in WS35 and ab initio theoretical calculations36 revealed that sulfur vacancies (VS) in WS2 forms two energetically narrow unoccupied defectstates inside the bandgap because of an exceptionally strong spin–orbitcoupling in WS2. Furthermore, the presence of defect statesin WS2 has been supported by ultrafast spectroscopic studies,where defects lead to rapid trapping in WS246 and long-lived interfacial charge separationat the graphene–WS2 interface.10 The typical densities of these defects lie in the rangeof 1011–1013 cm–2.36 Given the operating range of the electrochemical gating method,we electrically tune only the filling of the in-gap defect states,while that of the filled defect state in the valence band of WS2 is beyond our reach. In our simulation, we consider threescenarios: (i) none, (ii) one, and (iii) two in-gap defect states.The defect density is realistically varied between 1011 and 1013 cm–2, and its (or their) energeticsare fully relaxed within the bandgap of WS2 for simulation.Based on the model, a critical and open questionconcerns the density,filling state, and quantity of defect states involved in CT  and photogating processes . Recent scanning tunneling microscope (STM) studiesSupplementary Sections S3 and S4, we find that models involving no or only one in-gap defectstate are unable to reproduce the main features shown in 2 are the only CT channels to harvest thermalized hotelectrons and hot holes, respectively, from graphene. We calculatethe majority conducting carrier density change  induced by CT in graphene using the difference between thenumber of thermalized hot electrons distributed above the conductionband minimum of WS2 and the number of thermalized hot holesdistributed below the valence band maximum of WS2 (Supplementary Section 3). As shown in Figure S3, the pure PTE model, without consideringany defect states, fails to reproduce the main features of the experimentalresults, in particular for Δσ>100ps: (i)inthe n-doped regime, the absolute value of the CT-induced (negative)photoconductivity continuously increases with the upward shift of EF because the increasing EF makes hot electron transfer energetically more favorablethan hot hole transfer; (ii) following the sub-100 ps back-CT process,the photoconductivity in the hybrid system is expected to relax backto the equilibrium state without exhibiting photogating effects , which is in contradiction with theexperimental results. Similarly, the PTE model involving only onein-gap defect state also fails to reproduce our experimental observations.This is because, following back-CT at ∼100 ps, ΔNmajor immediately flips sign when EF crosses the Dirac point and the defect energy . On the otherhand, the two-defect model illustrated in Nmajor is zero in the undoped regime and becomespositive in the doped regimes, as shown in As discussed in EF liesbetween these two in-gap defect states, electron trapping to the emptyin-gap defect state and hole trapping to the occupied in-gap defectstate can take place simultaneously, resulting in approximately nullnet gating charges residing in WS2 and negligible photogatingeffects  following the above-mentioned schemes,we can reproduce all the main features shown in 2 is energeticallymore favorable than that of thermalized hot holes to the valence bandof WS2. On longer time scales (>100 ps), in line withourassumption and previous reports, the nature and density of defectsdictate the photogating mechanisms. As shown in 2, the photoinjected or trappedelectrons in the empty in-gap defect states in the p-doped heterostructurelead to an “electron photogating” scheme in graphene,while trapped holes in the initially filled in-gap defect states causea “hole photogating” mechanism. Furthermore, we noticethat the effect of pump fluence on Δσ>100ps depends largely on EF. In the dopedregimes, Δσ>100ps increases sublinearlywithpump fluence: it gets gradually saturated with fluence , limited by the defect density (fixedfor a given sample) used for the photogating. In the undoped regime,Δσ>100ps shows no obvious pump fluence dependenceand is close to zero. This is a result of the trade-off between electronloss and gain in graphene, as EF liesbetween the two defect states. Our proposed model simplifies somedetailed processes that may contribute to CT. For instance, we haveassumed that the quantum yield of CT is EF-independent and linearly scales with ΔNmajor, which neglects the rate competition between CT and hotcarrier relaxation in graphene (which is EF-dependent). The slightly asymmetric gating-dependent photoconductivityshown in 38On this basis, we further discuss the PTE modelinvolving two in-gapdefect states following the schemes shown in 2 leads to electron gatingto graphene10), our study provides a novelscheme to electrically control the photogating field direction bytuning defect occupancy without changing the CT direction.The results shown in S) can explain the electrically tunable photoconductivitydynamics shown in ab initio theory,VS in WS2 has a typical density of 1011–1013 cm–2 and forms two low-lyingin-gap defect states,36 which perfectly match the inferredin-gap defect density of ∼1012 cm–2 and state quantity of 2. Furthermore, on the basis of simulations,we deduce that the energy positions of the two defect states shouldbe relatively symmetric with respect to the Dirac point of graphene.The inferred energetics are in good agreement with recent STM studies35 of graphene–WS2 vdW heterostructures.Wenow discuss the nature of the defects that govern interfacialphotophysics in our study. While other possibilities cannot be completelyruled out, we show compelling evidence that considering only the energeticsand density of in-gap defect states generated by sulfur vacancies(V40 Recent advances in defect engineering enable fine-tuning of defectdensity and species in TMDCs,44 which is relevant for optimizing photogating andthus photodetection applications of graphene–TMDC heterostructures.For instance, VS can be either generated in a controlledfashion by electron beam irradiation45 orchemically repaired by rational use of superacids and/or sulfur-containingagents.47 The ability to precisely control the initialdefect type/density and implement electrical control of EF and defect filling in graphene–TMDC heterostructuresprovides a new degree of freedom for manipulating interfacial dynamicsat the vdW interface and developing high-performance optoelectronicdevices. On the other hand, rational passivation of VS andfabrication of undoped heterostructures can suppress defect-inducedlong-lived photogating at the interface, which is relevant for designinggraphene–TMDC-based modulators with fast modulation speed andwide bandwidth.Sulfur vacancies are known to be prevalent in both exfoliated andCVD TMDCs due to their low formation energy.in situ the interfacialcharge dynamics in graphene–WS2 vdW heterostructuresunder electrochemical gating. The EF modulationreversibly controls the defect occupancy in WS2 and thehot carrier distribution in graphene, allowing precise tuning of thedirection and efficiency of the photogating field at the vdW interface.This work provides a fundamental understanding and control of thecomplex interfacial charge dynamics in vdW heterostructures, openingup new possibilities for developing optoelectronic devices via defectand doping engineering.In summary, we investigate


The growth parameters for epitaxial growth of grapheneon siliconcarbide (SiC) have been the focus of research over the past few years.However, besides the standard growth parameters, the influence ofthe substrate pretreatment and properties of the underlying SiC waferare critical parameters for optimizing the quality of monolayer grapheneon SiC. In this systematic study, we show how the surface propertiesand the pretreatment determine the quality of monolayer graphene usingpolymer-assisted sublimation growth (PASG) on SiC. Using the spin-ondeposition technique of PASG, several polymer concentrations havebeen investigated to understand the influence of the polymer contenton the final monolayer coverage using wafers of different miscut anglesand different polytypes. Confocal laser scanning microscopy (CLSM),atomic force microscopy (AFM), Raman spectroscopy, and scanning electronmicroscopy (SEM) were used to characterize these films. The resultsshow that, even for SiC substrates with high miscut angles, high-qualitygraphene is obtained when an appropriate polymer concentration isapplied. This is in excellent agreement with the model understandingthat an insufficient carbon supply from SiC step edge decompositioncan be compensated by additionally providing carbon from a polymersource. The described methods make the PASG spin-on deposition techniquemore convenient for commercial use. Such relaxed measurementconditions have made graphene an outstanding candidate for AC andDC quantum metrology applications.5 Additionally, the fabrication of two-dimensional(2D) heterostructures and the intercalation of graphene layers withother materials have opened routes for engineering new electronicmaterial systems.7 Nevertheless, for all the requirementsmentioned before, consistent methods for producing large-area homogeneousgraphene layers of high quality are necessary. Epitaxial growth onSiC substrates fulfills all these requirements and, moreover, avoidsthe transfer to another substrate and is compatible with existingfabrication technologies.11 The key prerequisite to obtaining high-quality epitaxialmonolayer graphene films is to prevent step bunching of the SiC substrateduring growth, since high terrace step edges favor bilayer growthalong the edges and deteriorate the electronic properties of the graphene.This is challenging since on one hand high process temperatures arenecessary for homogeneous growth of the graphene layer which on theother hand promotes SiC step bunching. Therefore, the control of theprocess parameters 12 is a severe issue. Moreover, other factors suchas wafer properties 14 as well as pretreatment techniques 21 prior to high-temperature growth are crucial for the growth result.Single-layer epitaxialgraphene is well-known for its extraordinarystructural and electronic properties. Graphene on SiC is a potentialcandidate in a variety of applications such as radio frequency (RF)transistors, integrated circuits (ICs), field-effect transistors (FETs),and sensors.23 A polymer pretreatment is applied on the SiC substrate that suppliesextra carbon during the initial growth stages of the buffer layer23 by thermal decomposition. Theaccelerated buffer layer growth stabilizes the SiC terraces, and sub-nanometersubstrate steps are conserved throughout the whole growth process.Using the PASG technique, ultrasmooth monolayer graphene films canbe fabricated, showing excellent resistance isotropy,23 and metrology-grade quality is demonstrated by their successfulapplication as quantum Hall resistance standards.24 This raises the following questions: to what extent doesthe intrinsic carbon supply from the thermal decomposition of SiCstill play a role in graphene growth and how can the external polymeramount be precisely controlled for homogeneous and reproducible graphenegrowth?Recently, the so-called polymer-assisted sublimation growth (PASG)technique was developed in our lab, which is able to effectively avoidSiC step bunching by a simple improvement of the existing sublimationgrowth technique.25 Moreover,by using 4H- and 6H-SiC substrates the impact of the polytype on thegraphene growth is highlighted.In this study, we investigate in detail the interplayof the twocarbon sources for the PASG technique to obtain precise growth controlfor the fabrication of high-quality graphene layers. On the one hand,the polymer amount was varied, which directly controls the externalcarbon supply. On the other hand, by using SiC substrates with differentmiscut angles, the number of terrace steps was varied. Since the thermaldecomposition of the SiC preferably takes place at the terrace stepedges, the amount of SiC-related carbon is directly related to thesubstrate’s miscut angle.We show that only by controllingthe amount of polymer, the graphenegrowth can be easily optimized and adapted to the miscut angle ofthe substrate. A detailed investigation of the spin-on technique ispresented, which turns out to be a reliable and reproducible polymerdeposition technique compared to the liquid phase deposition (LPD)technique. These findings will facilitate implementation into existingproduction lines and pave the way for improved graphene device applications.22.1In this study, three different SiC (0001) substrates of hexagonalpolytype were used with small miscut angles of 0.1 and 0.03°against the [1–100] direction (perpendicular to the primaryflat), because only this guarantees optimal starting conditions withstep heights of about one SiC monolayer (∼0.25 nm). The miscutangle was verified by atomic force microscopy (AFM) inspections. Fora reliable comparison of the results, a miscut of about 0° waschosen against the [11–20] direction (perpendicular to minorflat), which avoids a roughening of the terrace edges during growthsee 1. Allwa22 was applied in two different ways: liquid phase deposition (LPD)23 and spin-on deposition.26 In the caseof LPD, the SiC substrates were ultrasonicated in a highly concentratedsolution of AZ5214E photoresist in IPA (25% solution) for 15 min,followed by rinsing the substrate to remove the excess polymer usingisopropanol from a spray bottle. Solution concentration, sonicationtime, and the final rinsing time are the three main parameters thatcan control the size and amount of carbon adsorbates in the LPD techniquethat finally influence the quality of monolayer graphene.10 The solution concentration (25%) and the sonicationtime (15 min) were kept constant in this study. Two different rinsingtimes were used: a shorter (6–8 s) time and a longer (10–12s) time. Details on optimum rinsing times are provided in the The samples were cleaned in acetone and isopropanol(IPA) priorto the polymer treatment. The PASG techniqueThe second way toapply the PASG technique is by spin-on depositionof a weak solution of AZ5214E photoresist and isopropanol on SiC substratesprior to growth. The volume ratios of polymer solutions ({AZ(μL)/IPA(ml)})used were 5.1, 3.4, 2.2, 1.5, and 0.75 and were named C5–C1,respectively see 221 Initially the reactor was evacuated to 1 × 10–6 mbar. Ar gas was introduced at 400 °C with agas flow of 20 sccm and a heating rate of ∼13 K/s. For graphenegrowth, the samples were annealed at 1750 °C for 6 min at 1 barAr pressure. All of the samples presented in this study were subjectedto the same growth parameters of temperature, time, and gas flow forthe fabrication of high-quality graphene.The spin-on process involves spin coating of the highlydilutedsolutions with a high acceleration ramp to 6000 rpm within 1 s anda final speed of 6000 rpm that is held for 30 s. Immediately afterthis polymer treatment step, the samples were introduced into theinductively heated reactor for graphene growth.2.227 In this work, we used anOlympus LEXT OLS5100 system equipped with the following objectives:×2.5, ×5, ×10, ×20, ×50, and ×100. Eachobjective has the option for an additional digital zoom of up to ×8.Hence, it was possible to image large areas such as 2560 μm× 2560 μm to small areas of 16 μm × 16 μm.The 3D laser scanning microscope is equipped with a 405 nm wavelengthviolet semiconductor laser that scans in the X–Y direction and a photomultiplier tube that generates imagesup to 4096 × 4096 pixels. The laser intensity image combinesa series of images in the Z direction to create a2D intensity image.CLSM is a fast and efficient way to characterize the quality ofthe samples.2.3AFMwas performed using a Witec Alpha 300 RA and Park NX10 instrumentsin noncontact mode. AFM achieves simultaneous mapping of the topography,feedback, and phase information, which is a measure of the energydissipation between the probe and sample, thus providing details onthe material and height information. The Point Probe Plus Silicon-SPM-Sensortips (PPP-NCLR-20 type) manufactured by Nanosensors with a resonancefrequency of 146–236 kHz were used for the measurements.2.4The Raman measurementswere performed using a LabRAM Aramis (Horiba) confocal Raman spectrometerwith an excitation wavelength of 532 nm and diffraction grating of600 grooves per mm. The full width at half-maximum (fwhm) values ofthe graphene-specific 2D peak were extracted from the individual Ramanspectra using a Lorentzian fitting algorithm. The distribution ofthe fwhm values of the 2D peak of graphene is plotted in a Raman map(area scans) by integrating 7000 individual Raman spectra. Micro-Ramanmappings of 20 μm × 20 μm were recorded for all samples.The lateral resolution was about 0.5 μm.2.5The SEM measurements were performed using a Supra 40 field emissionscanning electron microscope (FESEM) from Carl Zeiss GmbH. This SEMis equipped with Zeiss SmartSEM software (version 5.0708) and twotypes of detectors an in-lens and a secondary electron (SE) detectorfor capturing images. To resolve the contrasts between buffer layer,monolayer and bilayer graphene, very low acceleration voltages (around1 keV) were used. A working distance of 3–3.5 mm and an in-lensdetector was used for image capturing.3First, the graphenegrowth results for the polymer deposition byLPD technique are presented. Since the IPA rinsing after the polymerapplication is the most critical step, the rinsing time was variedand investigated for two 6H-SiC wafers with low (−0.03°)and high (−0.1°) miscuts. The surface morphologies ofthe final graphene layers were visualized by confocal laser scanningmicroscopy images (CLSM) see 1.28There are three major contrasts that can be observedin The difference between longer and shorter rinsing usingisopropanol shows the impact of the polymer amount on the qualityusing two different miscut substrates. For the shorter  rinsingtime (6–8 s), shown in Figures S1–S4 in the SupportingInformation for a better understanding of the critical steps in theLPD technique.This comparison demonstrates that the polymer amount thatdetermineshow much carbon is supplied to obtain an optimal growth result mustbe adapted to the substrate’s miscut angle. However, the maincontrol of the amount of polymer adsorbates is decided by the rinsingstep, which is manually operated and thus user dependent and challengingto reproduce. Since the sample handling during rinsing is criticalto the growth result, the LPD technique is limited when it comes tothe growth optimization on SiC substrates with different propertiesand when it comes to the operation of different users. Apart fromthe CLSM images, SEM, AFM, and Raman data of these samples are providedin In the following, we show the results of PASGof epitaxial graphenewith spin-on deposition of the polymer on the SiC substrate, whichallows for improved controllability and reproducibility. First, theimpact of different polymer amounts on the graphene morphology isexemplarily examined by using a 6H-SiC wafer with a −0.1°miscut see 2 and 3. 29 The contrast identifies graphene on terraces with different layerterminations within the 6H-SiC unit cell. It is also an indicationof very low step heights of one or two SiC layers (0.25 and 0.5 nm),and it proves that step bunching was effectively suppressed. The brightline contrast reveals terrace step edges between SiC terraces withequivalent terminations with a height of a half 6H-SiC unit cell,∼0.75 nm.29 Note that in the SEMimages the buffer layer appears white (white spots) and bilayer grapheneis hard to distinguish from the monolayer because of a small workfunction difference.27 Although the investigationof the high polymer concentrations C5 and C4 for PASG has shown thatvery smooth monolayer graphene can be obtained, the formation of abuffer layer and polymer aggregates indicates that these are not theoptimum concentrations for this wafer.In the following, thegrowth results for the remaining three polymerconcentrations C3–C1 for the same wafer are analyzed see 3.The CLSM images for the moderate polymer concentrationsC3 andC2 shown in 30 With the additional carbon that is externally applied through thepolymer in our study, we can conclude that an optimal concentrationof carbon supports a uniform decomposition of the SiC surface, well-definedterrace steps, and an improved parallelity of the terraces. Therefore,moving away from the optimum concentration C3 makes the terraces curvierin nature.A comparison of the surface morphology from theSEM images forthe polymer concentration series . From the Gaussian frequencydistribution of the FWHM values a mean value of 34 ± 2 cm–1 was extracted, which proves the high quality of thegraphene monolayers.10 The incomplete graphenelayer of the sample with the lowest polymer concentration, C1, showsareas with a narrow fwhm of ∼30 cm–1  are observed. They are attributed to small graphene bilayerdomains, in agreement with the CLSM and the SEM images, and they haveno detrimental effect on the resistance standards.33 For the best samples (for example C3 and C2) mobilitiesare in the range ∼5000–7000 cm2/(V s) andthe typical charge carrier densities are close to ∼5 ×1011 cm–2 after lithography and no additionaldoping of the processed graphene devices. These are essential criteriafor the fabrication of electrical devices such as electrical resistancestandards for our calibration needs in metrology. The values suggestthat high-quality homogeneous graphene monolayer films have been madethat are suitable for these magnetotransport precision measurementapplications.34As we use our graphene for metrological purposes and for fabricatingdevices to perform precision quantum Hall measurements for resistanceand impedance standards,The examination of themonolayer coverage for the overall polymerconcentration series from high to low values, C5 to C1, gives a consistentpicture from slightly overgrown to undergrown. It is in very goodagreement with the model that the polymer acts as an additional carbonsource for graphene growth and that the graphene quality can be controlledby fine-tuning the polymer concentration. After a detailed analysisof the graphene growth on substrates with a miscut angle of 0.1°toward primary, it is interesting to investigate the influence ofmiscut variations also for the polymer spin-on technique.Figure S6 in of the Supporting Information. Thistrend is in very good agreement with the model that higher miscutsubstrates with higher step density can supply more carbon by thermaldecomposition of SiC at the terrace edges and therefore require lesspolymer externally. For miscut values lower than 0.1°, the resultsare described below in detail.Toverify our hypothesis of a correlation between miscut angleand polymer concentration derived from the previous results for 0.1°miscut angles shown above, we also extended our spin-on depositiontechnique to 6H-SiC substrates with miscuts higher and lower than0.1°. For the higher miscut angle of −0.3° towardprimary flat, concentration C2 was sufficient to get fully coveredhigh-quality graphene monolayers, whereas for the 0.1° miscut,the polymer concentration C3 was optimum. This shows already thata higher miscut angle requires a lower absolute polymer concentrationto produce equally good graphene monolayers. The results for the high-miscut−0.3° 6H-SiC with C2 concentration are provided in In the following, we concentrateon the growth results on 6H-SiCsubstrates with a very low miscut angle of −0.03°. 16With an increase in polymer concentration to C2and C3 see 4c,d, the15). However, even for the highest polymer concentration,areas with a buffer layer (dark gray) remain uncovered with graphene.This is in contrast to the complete graphene coverage on substrateswith higher miscut angle of 0.1°  is faster than that of a comparable value butnegative miscut 6H polytype (−0.03°). The influence ofthe miscut direction is not analyzed here. The overall trend of theeffect of polymer concentration on miscut angle remains unalteredfor both polytypes.This tendency is the same as that observed forthe graphene growthon 6H-SiC substrates. However, there is a noticeable difference betweenthe growth results on the two polytypes for the same polymer concentrations.For the same concentration C3 applied to the 6H-SiC substrate witha comparable miscut angle, an incomplete graphene layer has formedsee 4c. This4In this work, we demonstratedthe concentration-dependent effectof polymer-treated SiC substrates on the graphene growth quality usingspin-on deposition for the PASG technique. The prerequisite for homogeneousand complete graphene layers involves a complex interplay of carbonsupply from the substrate and the external source. The observationsdemonstrate that low polymer concentrations may lead to incompletegraphene monolayer coverage and that giant step bunching may occur,whereas when the concentration is optimal, the layer becomes moreand more complete. On the other hand, an excess of externally appliedcarbon can lead to polymer aggregates and other growth defects. Suchfine control on the layer quality is the main advantage of this spin-ondeposition technique. For each SiC polytype, there are differencesin the optimum amount of externally applied carbon to effectivelysuppress step bunching during the graphene growth process. The concentrationof polymer that is necessary for an ideal monolayer coverage dependssignificantly on the miscut angle of the SiC wafer. The surface densityof step edges defines the number of weak points in the crystal andin turn the miscut significantly affects the amount of carbon thatis released during the decomposition of the SiC wafer surface. Thefindings also suggest that the required polymer concentration alsodepends on the polytype. 4H-SiC requires less carbon from the polymerin comparison to its 6H equivalent for a complete monolayer graphenecoverage. This study of the dependence on graphene quality with respectto the substrate properties and external carbon supply by using PASGis an important baseline for optimizing the control of the graphenemorphology for electronics, surface science, and intercalation applications.


The theoretical study for the first time reveals that the introduction of chlorine accelerates the decomposition of methanol and thus promotes the VG growth in PECVD. Finally, as an excellent filler framework in polymer matrix, VG arrays are used to construct a free‐standing composite TIM, which yields a high vertical thermal conductivity of 34.2 W m−1 K−1 at the graphene loading of 8.6 wt% and shows excellent cooling effect in interfacial thermal dissipation of light emitting diode.With the continuous progress in electronic devices, thermal interface materials (TIMs) are urgently needed for the fabrication of integrated circuits with high reliability and performance. Graphene as a wonderful additive is often added into polymer to build composite TIMs. However, owing to the lack of a specific design of the graphene skeleton, thermal conductivity of graphene‐based composite TIMs is not significantly improved. Here a chloroform‐assisted method for rapid growth of vertical graphene (VG) arrays in electric field‐assisted plasma enhanced chemical vapor deposition (PECVD) system is reported. Under the optimum intensity and direction of electric field and by introducing the highly electronegative chlorine into the reactor, the record growth rate of 11.5 µm h −1 was achieved and vertical graphene arrays with a height of 100 µm was successfully synthesized. Vertical graphene together with polydimethylsiloxane were used to construct a free‐standing thermal interface material, which yielded a high vertical thermal conductivity of 34.2 W m−1 K−1 and excellent cooling effect in light emitting diode.With a chloroform‐assisted method, a record growth rate of 11.5 µm h However, graphene is always used as a thermal additive mixed with polymers to construct a composite thermal interface material (TIM). Unfortunately, graphene framework will not significantly improve the performance of TIMs without special structural design. Actually, to transfer intrinsic thermal property of nano graphene sheets into macroscopic graphene‐based TIMs, especially in vertical direction, the assembly strategy and structural design method of graphene framework play an important role. In principle, vertical graphene (VG) may significantly improve heat transfer capability. However, in top‐down method including rolling‐cutting method, directional‐freezing method, oriented hydrothermal reduction method and shrinking‐compressing method, VG is synthesized with graphene oxide or reduced graphene oxide dispersion assisted by polymer and solution, in where defects in graphene sheets and interfaces between graphene and polymer are harmful to the thermal properties of TIMs. Instead, plasma enhanced chemical vapor deposition (PECVD), as a typical bottom‐up method, can directly grow VG structure on substrates retaining the intrinsic thermal properties in macro‐scale vertical materials. Therefore, PECVD can be regarded as an effective method to prepare thin TIMs embedded with vertically aligned graphene framework at a film thickness in the range of several hundred micrometers.With the rapid development of electronic devices toward high power density and miniaturization, thermal interface materials (TIMs), which function between a heating device and a heat‐sink, is getting more and more important along with the technology evolution.2, 3 MW and RF are often chosen as plasma sources to improve the growth rate of VG because of their high electron density and electron energy. However, VG grown by this method still faces the dilemma that the morphology of the grown graphene is difficult to control. DC PECVD and capacitively coupled PECVD are advantageous to the directional growth of VG due to their symmetrical electrode set‐up. Whereas, these methods are still stuck with limited growth rate. Apparently, achieving rapid and directional growth of VG simultaneously using PECVD is still a long‐standing bottleneck.As a well‐established industrially compatible thin‐film deposition technique, PECVD including radio frequency (RF), microwave (MW), and direct current (DC) plasma have been used to grow VG.21 MW −1. Furthermore, chloroform was used to accelerate growth rate of VG. When appropriate proportion of chloroform was introduced into methanol carbon source, the growth rate of VG could be increased to 11.5 µm h−1, resulting in a near 100‐µm VG array. Density functional theory (DFT) calculation results further proved that active species decomposed from chloroform consume the excessive gas‐phase H radical and remove excessive terminating H atom of a growing graphene edge for rapid growth of VG arrays. Finally, using the grown VG arrays, we prepared a VG composites‐based thermal interface material (TIM) by adding silicone. This TIM yield a high vertical thermal conductivity of 34.2 W m−1 K−1 at the graphene loading of 8.6 wt%, giving an ultrahigh thermal conductivity enhancement (TCE) per 1 wt% graphene over 2200%, superior to almost graphene composite‐based TIM. In addition, its better cooling effect than that of commercial thermal pads makes it has great potential in interfacial thermal dissipation of electronic products.Herein, we developed a halogen‐regulated method for rapid growth of VG in an adjusted electric‐field‐assisted PECVD technique. Theoretical simulation was adopted to investigate the effect of electric field direction on growth rate. When the in‐built electric field is set to vertical upward, the grow rate of VG can be increased into 5 µm h2Figure  As shown in Figure As is shown in Figure 27 As 2.1Figure −1 at the voltage of 60V−+ and increases to 4.5 um h−1 when the electric field direction is inversive (60V+−), suggesting that the direction of electric field affects the growth rate of VG. Besides, the growth rate gradually accelerated with the increased voltage intensity from 100 to 250 V+−  is invariant even if the direction was reversed. While for the electric field induced by RF coil (ERF), its intensity and direction vary continuously with time Sine  has also been used to simulate the electric field distribution in the complicated plasma system. The vector diagram Figure , left sh, Figure  suggesti2.2 Based on this, considering the strong etching effect of fluorine plasma on silica, chlorine is regarded as the best choice for decomposition of methanol. Figure −1 when the ratio of methanol to chloroform is 20:1. After that, the growth rate gradually slows down.  was conducted directly to monitor the two different growth systems. wth rate.39 In G Figure .2.33OH to CHCl3 is 20:1. In the PECVD reactor, the plasma produces various active radicals such as CHx*(x = 0–3), H* and *OH in the gas phase. Moreover, *CCl3 and Cl* radicals are expected to exist considering the dehydrogenation of reactor CHCl3, which is also in good agreement with the OES signal of Cl*  and TEM elemental analysis show that the graphene is chlorine‐free  in Figure 3 in fast CVD growth of VG: first, in a plasma‐enhanced environment, the presence of CHCl3 will introduce some chlorine species, to consume the excessive gas‐phase H* radical, ensuring the concentration of *OH radical, which could assist the fast growth of VG; Second, active Cl* radical can also remove excessive terminating H atom of a growing graphene edge with no barrier. The two key roles ensure the fast growth of VG.For the VG growth, all the reactions occur on the top edges of the graphene layers and thus the gas‐phase reaction is vital, which is similar with that of the vapor‐solid growth mechanism of graphene growth on the insulating substrates.40 As 2.4Figure −1 K−1) with a same bond line thickness (BLT) of 60 µm and a lateral size of 5 × 7 mm were placed between the heat sink and ceramic heater (5 × 7 × 1.2 mm), respectively, with a constant pressure of 60 psi. The circulating cooling water was continuously pumped into the heat sink for the full extraction of heat generated by the testing system, and the real‐time temperature variation was recorded by a thermocouple. As shown in Figure −1 K−1 at the graphene loading of 8.6 wt%, achieving an ultrahigh specific thermal conductivity enhancement (TCE) over 2200% with per 1 wt% graphene content in polymer matrices, which is better than most graphene composite‐based TIM. Figure −2 °C−1 (without TIM), respectively, demonstrating that the cooling efficiency of VGA‐TIM is ≈1.89 times higher than that of state‐of‐the‐art thermal pad.In our work, the grown vertically aligned, covalently bonded vertical graphene arrays (VGA) were used as the filler skeleton for enhancing the through‐plane thermal conductivity of soft silicone matrix (polydimethylsiloxane (PDMS)) to form a high‐performance elastomer TIM (VGA‐TIM), whose real cooling efficiency was investigated by a home‐built measurement system developed for simulating the actual heat dissipation process of electronic devices, as shown in ased TIM.48 Figκeff) and thermal contact resistances (Rc) of two samples, with an applied power of 10 W. The κeff of different cases can be obtained according to the steady‐state heater temperatures shown in Figure −1 K−1, which is nearly 3.7 times higher than that of 5000S35 (0.81 W m−1 K−1). Therefore, we can calculate the Rc of the two samples by the following equation:κbulk is the through‐plane thermal conductivities of VGA‐TIM/5000S35. As a result, the Rc value of VGA‐TIM is determined to 18.2 K mm2 W−1, showing a significant decrease of 70.7% compared with that of 5000S35 thermal pad (62.1 K mm2 W−1) under the same testing conditions  was employed to in‐depth investigate the effective thermal conductivities  to evaluate its heat transfer capacity in real operating conditions. In a typical experiment process, the LED lamp was fixed on the aluminum heat sink by four fastening screws, and the VGA‐TIM as well as 5000S35 with the same 3−1. Later, 100‐µm VG arrays was achieved within 10 h. This study provides new insights into plasma and gas‐phase reaction engineering to obtain aligned graphene sheets and paves the way for the rapid growth of VG with high quality. More importantly, using the designed vertical graphene structure, we fabricate a VG based composite for TIM application, which exhibited excellent thermal properties and outstanding cooling effect, showing huge potential in interfacial heat dissipation of electronic devices.In conclusion, we demonstrated a halogen‐regulated method for rapid growth of VG in an adjusted electric‐field‐assisted PECVD system. When appropriate proportion of chloroform was introduced into methanol carbon source, the maximum growth rate of VG could be increased to 11.5 µm h4A conventional inductively coupled PECVD with a frequency of 13.56 MHz was adopted as the equipment for growth of vertical graphene (VG). Then a built‐in electric field was introduced into the system for alignment of graphene sheets. The electric field was provided by two electrodes connected to a direct current power supply. Voltage can be adjusted from 1 to 250 V. During the growth process, silicon wafers were placed in the heating zone of a tube‐type PECVD. After the growth temperature was up to 650 °C, the mixture carbon sources of methanol and chloroform were introduced into the system. Plasma was then generated with a radio frequency source power of 250 W. Following an electric field was set as a specific value, oriented VG arrays can be prepared.−1 for the energies and forces of the system, respectively. To fully include the effects of radicals and the graphene edge, spin‐polarization in the calculations was used. To give the reaction energy barrier, the climbing image nudged elastic band (CI‐NEB) approach was applied.The geometry relaxation and energy barrier calculations were performed under the framework of density functional theory (DFT) as implemented in Vienna ab‐initio Simulation Package (VASP). For the treatment of exchange‐correlation potentials, the Perdrew–Burke–Ernzerhof (PBE) functional within the generalized gradient approximation (GGA) was adopted. A zigzag graphene nanoribbon with the unit cell of 14.75 × 30.00 Å was constructed. A kinetic energy cutoff of 400 eV was used, and give a threshold convergence of 10–4 eV and 0.01 eV Åα X‐ray source). The thermal diffusivities (α) of the sample were measured using LFA 467 MicroFlash system . The specific heat capacity of the sample evaluated by using a DSC . The infrared (IR) photos were captured by using an infrared camera .The morphology and detailed structure of VG was investigated by SEM ; Raman spectroscopy , XPS  were used for the statistical analysis of the data presented in this work. Calculation method was detailed descripted in The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


We show twisted 2D materials contain a torsional displacement field comprised of three transverse periodic lattice distortions (PLD). The torsional PLD amplitude provides a single order parameter that concisely describes the structural complexity of twisted bilayer moirés. Moreover, the structure and amplitude of a torsional periodic lattice distortion is quantifiable using rudimentary electron diffraction methods sensitive to reciprocal space. In twisted bilayer graphene, the torsional PLD begins to form at angles below 3.89° and the amplitude reaches 8 pm around the magic angle of 1. 1°. At extremely low twist angles (e.g. below 0.25°) the amplitude increases and additional PLD harmonics arise to expand Bernal stacked domains separated by well defined solitonic boundaries. The torsional distortion field in twisted bilayer graphene is analytically described and has an upper bound of 22.6 pm. Similar torsional distortions are observed in twisted WS In twisted 2D materials, spontaneous lattice reconstructions mean that twist angle alone provides an incomplete description. Here, using electron diffraction, the authors show that the displacement field in twisted bilayer graphene can be described as a superposition of three periodic lattice distortion (PLD) waves with wavevectors oriented at 120° from each other, forming a “torsional" PLD. PLDs are typically intrinsic to a crystal4, Fermi-surface driven5, accompanied by a CDW, and have periodicity spanning a few unit cells (~1–2 nm). However, recently extrinsic van der Waals (vdW) driven superlattices with tunable periodicity (up to a few 100 nm) were discovered in twisted bilayer graphene (TBG)6. TBG has been spotlighted for extraordinary correlated electron behaviors for a twist at the so-called “magic” angle (~1.1°)7. Yoo et al. showed that magic angle TBG is not a simple superposition of two constituent layers6, but rather a 2D crystal that periodically restructures at the mesoscale. Subsequent reports showed moiré superlattices of other vdW systems with similar periodic restructuring11. Furthermore, this restructuring has a dramatic effect on the band structure, magnetism, and superconducting properties12. Therefore, understanding twisted 2D materials requires a full description of the atomistic structure down to picoscale displacements. However, a systematic depiction of restructured moiré superlattices is nearly absent and limited to descriptions of local stacking geometry.Periodic lattice distortions (PLD) are at the heart of correlated electronic behavior such as superconductivityθ) of 1.1° and 1.2°, respectively. We report an upper bound for the PLD amplitude of twisted bilayer graphene to be 22.6 pm based on interlayer interaction energy. In addition, we show that the torsional PLD amplitudes can be accurately predicted across all twist angles using an analytic model of the vdW stacking and elastic energies. Lastly, we show that this torsional PLD exists across a variety of other layered 2D materials.Here, we show the atomic structure of 2D moiré superlattices at and near the magic angle are concisely and accurately described by a torsional PLD comprised of three transverse displacement waves. In this way, the complexity of low-twist moiré crystal restructuring is reduced to a single PLD order parameter with an amplitude and wave vector. Each layer in the bilayer system has an equal and opposite torsional PLD amplitude. From quantitative diffraction of low twist angle and magic angle graphene, the atomic displacements of the larger superlattice can be measured. In twisted bilayer graphene, we report a torsional PLD amplitude of 7.8 ± 0.6 pm and 6.1 ± 0.4 pm for twist angle , half the atoms in one layer are atop atoms in the layer below . Despite the complex super-structure of moiré stacking, the diffraction pattern of TBG is a simple superposition of two rotated single-layer Bragg peaks16—validated by quantum mechanical scattering simulation in Fig. 17.Moiré patterns emerge for two rotated lattices. In TBG, two single layers of graphene are stacked with a small interlayer twist Fig. c. The mo13 and later 4D-STEM19 revealed that this superlattice corresponds to a triangular array of AB/BA domains. However, domain boundaries soften near or above the magic angle (θ ≈ 1°) and a simple array of perfect AB/BA domains fails to correctly capture the full atomistic structure of the twisted system21.In low twist angle bilayer materials, a striking restructuring of the moiré lattice emerges. Dark-field (DF-) TEMr0 are deformed and original atomic positions, q is the wave vector, and A is the displacement vector). Both longitudinal (A∥q) and transverse (A⊥q) distortion waves naturally emerge in various charge-ordered crystals, including 2D materials .Here, we show that a PLD model provides a precise and concise description of lattice restructuring in TBG. PLDs are sinusoidal displacements of atomic positions  to accommodate the symmetry of the moiré pattern . Importantly, this torsional wave occurs in both layers, however, the direction of the field in each layer is reversed such that distortions are opposite. Transverse distortions, Δ1) is typically sufficient to describe the system, however, more generally, PLDs with higher-order harmonics (n > 1) are permissible and the total displacements become the sum of multiple harmonics (as discussed later).A torsional PLD succinctly and accurately describes the relaxed structure of TBG Fig. b. The toΔ1) from a torsional PLD in one layer of twisted bilayer graphene. The arrows show the direction and magnitude with which atoms displace from their expected lattice sites. The torsional field is a nanoscale trigonal lattice of rotational distortions spaced 1/∣q∣ apart. The distortion field exhibits behaviors desired for relaxation of TBG—twisting AA regions in one direction and anti-twisting AB/BA regions in the other. The vdW interaction between layers strives to locally twist (anti-twist) AA (AB/BA) regions to minimize (maximize) interlayer registration and reduce the total interaction energy. The relaxed structure ∣2 where Jα is a Bessel function of the first kind, k is the position of the superlattice peak in reciprocal space, and α is an integer25. For typical values of k and A, the Bessel function monotonically increases with ∣k∣ and decreases inversely with the integer α. The appearance of strong superlattice peaks at high-order Bragg spots  is a signature of periodic lattice distortions (PLD)  reveals the transversity of PLDs in twisted bilayer materials. In reciprocal space, the transverse PLDs manifest as a distribution of superlattice peaks that are stronger along the azimuthal direction . We report a torsional PLD amplitude (A1) of 7.8 ± 0.6 pm for θ = 1. 1° and 6.1 ± 0.4 pm for 1.2° near the magic angle in TBG. The PLD amplitude was quantified by matching experimental and simulated diffraction intensities . The periodic relaxation of TBG results from competition between interlayer van der Waals stacking energy benefit (VvdW) and elastic cost of distortion (VEl)26. Elastic energy cost to accommodate a torsional PLD with amplitude A1, assuming Hookean elasticity, is G is the shear modulus of graphene (9.01 eV/Å2 20) . VvdW has two salient features: first, energy per moiré supercell is proportional to the area of the cell (∝θ−2) and second, VvdW minimum is at A1 = Amax = 20.35 pm . A non-linear least squares fit gives a semi-empirical model of v0 = 0.0732 eV Å−2 and Amax = 20.35 pm. Notably, Amax corresponds to energetically allowed maximum A1. The total energy of TBG with a torsional PLD is Vtot with respect to A1 gives a Lorentzian function:A1 extracted from experimental data and simulations , TBG relaxes with more complexity, thus roughly defining the extreme low-twist regime. Comparing diffraction patterns at higher θ  of Fourier harmonics to accommodate sharper boundaries:We report a lower twist angle θ ≲ 0.°, TBG reAn dictates the texture of a torsional PLD. Figure N, see Supplementary Note N). Figure N field pattern that matches previously reported experimental results of local twist fields18. Although each harmonic PLD wave contributes elastic energy independently −); analogous to a smooth ‘sawtooth’-like wave in a one-dimensional wave  are calculated by minimizing the total interlayer and intralayer energies (See Methods). Each higher-order term becomes non-negligible incrementally at smaller angles. A2, A3, and A4 will exceed 1 pm at θ ≲ 0.9°, 0.45°, and 0.3°, respectively , however, this demarcation is imprecise. An decays exponentially with coefficient κ(θ) linearly proportional to the twist angle (An = A1eκ(θ)⋅(n−1)−). Thus, decreasing θ retards decay of An and higher harmonics becomes more significant , remains well described by the empirical Lorentzian, Eq. . Figure Multislice simulation of SAED Fig. g–o showsθ nears zero, many higher harmonics (N) are needed and the Fourier basis is more cumbersome. Instead, a hard-domain model, where the superlattice is treated as a quilt of AA, AB, and BA domains with dislocation boundaries, may also become suitable. In the limit of zero angle twist, boundaries become the stacking fault boundaries reported by Brown et al. for untwisted bilayer graphene17.As 28. Figure 2 (4L-WS2), (c) twisted double bilayer CrI3 (2L + 2L CrI3), and (d) twisted WSe2/MoSe2 heterostructure.Torsional PLDs in twisted 2D materials are a universal phenomenon and not limited to TBG2) and trihalides (MH3). Figure 2 with equal twist angles between layers. Surprisingly, a strong torsional PLD is observed, despite having a large twist angle (θ ≈ 4°)10. For a multi-layered system, relaxation may not be equivalent between layers. For 4L-WS2, for example, the PLD amplitudes are strongest for the innermost layers. Here equal and opposite PLDs of the inner two layers matches simulated diffraction patterns . This is also consistent with diffraction simulations , the reciprocal structure of the top layer (Vtop(k)) is described by:Vbot(k)) has the same form with Vtop(k) but with equal and opposite PLD amplitude (Vtop(k) + Vbot(k)∣2. The first term represents Bragg peaks. The second term is first-order superlattice peak. Here k ⋅ A is small (less than one) and the higher-order terms In the presence of a single torsional PLD with three wave vectors a = 2.46 Å, c = 3.34 Å) and assumed both graphene layers are perfectly flat. The energy at each atomic site was calculated by summing overall interactions with atoms in the opposing layer. VvdW was calculated by integrating registry energies over a moiré unit cell. The calculation was done over many twist angles (0.463°–6.009°) with up to 61,348 atoms per layer.vdW interlayer registry energies were calculated using Kolmogorov–Crespi potential with known lattice parameters for graphitic systems (VEl) were analytically derived from elasticity tensor assuming plane stress deformation and using previously reported shear modulus value (G = 9.01 eV/Å2 20). See Supplementary Note Elastic energies (An) were calculated by minimizing the total energy using the simplex algorithm implemented in MATLAB (fminsearch).Higher harmonic PLD amplitudes  substrates. Monolayer graphene was identified by optical contrast and Raman spectroscopy. After preparing graphene and h-BN flakes, the thin single crystallite h-BN layer was first picked up at 70 °C using poly(Bisphenol A carbonate) coated on a polydimethylsiloxane stamp. Then, the h-BN layer was engaged to half of the graphene flake. By lifting the stamp off the substrates, we pick up only the part of the graphene which was covered by the top h-BN layer, leaving the remaining part of the graphene on the substrate. After the detachment, the substrate was rotated at a controlled angle. Engaging the graphene/h-BN stack on the adhesive polymer to the other half piece of graphene on the substrate makes the artificial bilayer graphene with a controlled twist angle. The whole stack was then transferred onto a thin SiN membrane TEM grid.Graphene and h-BN are mechanically exfoliated onto Si/SiO2 single crystals were synthesized by metal-organic chemical vapor deposition (MOCVD) on 300 nm oxide silicon wafers34. A single triangular domain from the growth was selected, then tear-and-stacked four times with a twist of 4 degrees and delaminated onto a Si/SiO2 substrate by a vacuum assembly robot10. To prepare the twisted four-layer WS2 for TEM imaging, the sample was spun coated with PMMA and floated in 1 M KOH until the substrate detached from the PMMA, then wet transferred onto a 1 μm holey carbon/copper TEM grid. Lastly, the TEM grid was solvent cleaned in acetone to remove all polymer (from stamp residue and PMMA).Large monolayer WS3, the bilayer CrI3 flakes were firstly exfoliated and identified inside a nitrogen gas-filled glovebox and assembled with a desirable twist angle by the ’tear-and-stack` method. tDB CrI3 samples were double encapsulated with thin hexagonal boron nitride (h-BN) flakes (~5 nm) to prevent the degradation from the oxygen and moisture from the ambient condition. The prepared tDB CrI3 samples were finally transferred onto the TEM grids, cleaned with chloroform solvent, and dried in nitrogen gas.For the fabrication of twist double bilayer (tDB) CrIMonolayers of TMDs were prepared via mechanical exfoliation from bulk crystals (from HQ Graphene) onto a PDMS stamp (PF Film X0 from Gel-Pak). The PDMS stamp was pre-treated and treated by oxygen plasma to minimize the PDMS residue left on the monolayers. Heterostructures were fabricated using the PDMS-assisted dry transfer technique operating under a home-built setup with micromanipulators, and a rotational stage, and the monolayers were aligned under an optical microscope. Heterostructures were annealed in a vacuum at 130 °C for 1 h.Peer Review FileSupplementary Information for Torsional Periodic Lattice Distortions and Diffraction of Twisted 2D Materials


The recovery of metagenome-assembled genomes is biased towards the most abundant species in a given community. To improve the identification of species, even if only dominant species are recovered, we investigated the integration of flow cytometry cell sorting with bioinformatics tools to recover metagenome-assembled genomes. We used a cell culture of a wastewater microbial community as our model system. Cells were separated based on fluorescence signals via flow cytometry cell sorting into sub-communities: dominant gates, low abundant gates, and outer gates into subsets of the original community. Metagenome sequencing was performed for all groups. The unsorted community was used as control. We recovered a total of 24 metagenome-assembled genomes (MAGs) representing 11 species-level genome operational taxonomic units (gOTUs). In addition, 57 ribosomal operational taxonomic units (rOTUs) affiliated with 29 taxa at species level were reconstructed from metagenomic libraries. Our approach suggests a two-fold increase in the resolution when comparing sorted and unsorted communities. Our results also indicate that species abundance is one determinant of genome recovery from metagenomes as we can recover taxa in the sorted libraries that are not present in the unsorted community. In conclusion, a combination of cell sorting and metagenomics allows the recovery of MAGs undetected without cell sorting. Metagenomics is a standard technique used to study microbial community composition and functional potential ,2,3,4,5.The limitations of current metagenomics approaches, such as access to rare species, introduce bias to the taxonomic composition of complex microbial communities in genome-centric metagenome studies. Eliminating this bias is particularly interesting in wastewater treatment plants because rare species, such as nitrifiers, may be underestimated or ignored. The efficiency of the system depends on the presence of certain species in the activated sludge (AS) , which iA potential strategy to circumvent the challenges of recovering low-abundant species in metagenomes is to reduce the complexity of a target microbial community. Greib and collaborators  revealedThis study aimed to improve MAG recovery by combining flow cytometry, cell sorting, and metagenomics from activated sludge microbiomes. Briefly, we sequenced metagenomes of the unsorted and sorted communities from a batch-grown activated sludge microbiome collected in a wastewater treatment plant. After MAG recovery, we dereplicated the different MAGs into genome operational taxonomic units (gOTUs) and compared their phylogeny and abundances to 16S rRNA genes (rOTUs) reconstructed from the different libraries. We used our data to check if the recovered MAGs belonged to the dominant microbial community in our cell culture. In addition to improving genome recovery from sorted and unsorted communities, we used our dataset to extrapolate the factors controlling the recovery of MAGs, including genome coverage and relative abundances. Furthermore, we defined a detection limit for MAG recovery and a strategy to define cut-off limits for MAG recovery in metagenomes.600nm, d = 0.5 cm), each, and cultivated in the same way.We used the cells of a microbial community originating from an activated sludge basin of a wastewater treatment plant  as our model microbial community described in Liu et al. . The ent−1 peptone (from meat), 0.2 g L−1 meat extract, 0.219 g L−1 yeast extract, 0.1 g L−1 glucose, 0.49 g L−1 Na-propionate (filtered), 0.0059 g L−1 CaCl2·2H2O, 0.0294 g L−1 KCl, 0.06 g L−1 NaCl, 0.04 g L−1 K2HPO4, 0.2156 g L−1 KH2PO4 and 0.0196 g L−1 MgSO4·7H2O, purchased from: Merck KGaA , SERVA Electrophoresis GmbH  and Carl Roth GmbH . The medium was a mixture of 98% synthetic wastewater and 2% peptone. The medium constituted 0.198 g Lg at 4 °C) for 10 min. The supernatant was discarded. We washed the cells with phosphate-buffered saline once  and stabilized them by adding 2 mL paraformaldehyde solution  to the cell pellet and incubated for 30 min at room temperature (RT). Next, the samples were centrifuged , re-suspended in 4 mL of EtOH (70%), and then stored at −20 °C. The wastewater microbial community samples were fixed and stained for cytometric analyses, as described by Liu et al. . Brieflyg, 10 min, 4 °C) with PBS. The cell solutions were adjusted to an OD of 0.035 (dʎ700nm = 0.5 cm) using PBS. A quantity of 2 mL of an adjusted sample solution was centrifuged . The pellet was re-suspended with 1 mL of solution A  and incubated at RT for 10 min in an ultrasonication bath  and 10 min without any further treatment. After another centrifugation step , the cells were stained with 2 mL solution B [0.24 μM DAPI  in phosphate buffer (289 mM Na2HPO4 and 128 mM NaH2PO4 in distilled water)] overnight at RT in the dark.We washed the stored samples twice by centrifugation  to visualize the community structure as described by Liu et al. . Samples6 cells per tube. We tested the number of cells needed and reached the value of 4.95 × 107 cells (>50 ng). Therefore, it was necessary to pool 22 tubes per sub-community. Next, we concentrated these tubes (22 per sub-community) by centrifuging twice the sorted cells. We first centrifuged the cells  and kept only 80 µL in each tube. The cells were vortexed for 10 s and pipetted up and down several times before pooling them into one tube. Next, we centrifuged  the cells and discarded the supernatant completely. We stored the pellet directly at −20 °C.Cell sorting was conducted as described by Koch et al. . BrieflyGates were affiliated based on their cell number in relation to the average cell number per gate (7658.1 cells). Dominant (DG) sub-communities consist of gates whose cell counts are above the average cell number per gate, while low abundant (LA) sub-communities consist of gates whose cell numbers are below the average cell number per gate. Additionally, Outer (OG) sub-communities are those in the master cell gate template, but outside of DG and LA sub-communities (OG = master cell gate − (DG + LA)), g and 4 °C. Finally, the supernatant (50 µL) was carefully collected into a sterile DNA-free tube without any Chelex beads. The DNA concentration was measured with Qubit® 3.0  after DNA extraction. The pellet was stored at −20 °C before library preparation and sequencing. The extracted genomic DNA was submitted to StarSEQ® GmbH  for Illumina library preparation and sequencing. We extracted DNA from the UC and the three sub-communities  using the Chelex method as described by Koch et al. . In shor® GmbH. The sequenced libraries were imported in FASTQC format. The reads were quality controlled by removing low-quality reads and adapters using Trim Galore v0.4.3 implemented in metaWRAP pipeline [The DNA extracts from the four metagenomes  were used for library preparation and sequencing. The libraries were prepared using the Nextera XT DNA preparation kit from Illumina according to the manufacturer’s instructions. Next, metagenomes were paired-end sequenced using Illumina  at a minimum of 20 million reads per library. Finally, high-throughput sequencing was done by StarSEQpipeline . The tripipeline . Prokaryotic MAGs were generated using the metaWRAP v0.7 pipeline  with metIn Equation (1), completeness is the estimation of genome quality based on the presence or absence of single-copy marker genes, and contamination is the evaluation of genome quality as revealed by multiple copies of marker genes .MAGs were classified as medium quality if their quality score was greater than 50 and with completeness between 50% and 80% and less than 10% contamination. MAGs with a quality score greater than 50, completeness above 80%, and contamination below 5% were considered high-quality.https://itol.embl.de/, accessed on 8 September 2022).We used GTDB-tk v0.3.2 ,28 to asSphingobacterium (at the genus level) recovered in the DG and Comamonas B-9 (classified at the species level according to EZBioCloud.net) recovered in the UC were not included in further analyses. To assess a better representation of our genomes across sorted sub-communities and unsorted communities, we dereplicated the recovered MAGs into gOTUs using MuDoGeR . BrieflyThe quality-checked reads from the four libraries were mapped to the gOTUs even if they were not recovered in a given library. Read mapping for all libraries was done using Bowtie2 , and mapN is the number of reads per genome and G is the genome size (in base pairs).In Equation (2), L is the average size of reads per library, We reconstructed nearly full-length phylogenetic marker genes (16S rRNA) from metagenomic libraries using the Mapping-Assisted Targeted Assembly for Metagenomics (MATAM) pipeline v1.6.0 . The paiFurthermore, MATAM mapped the reads of each marker gene to the metagenomic libraries and calculates the relative abundances of each 16S rRNA reconstructed sequence. After running MATAM, we removed all 16S rRNA sequences smaller than 900 bp using SeqKit  since thThe relative abundances of rOTUs were determined in three steps. First, the relative abundances of 16S rRNA sequences obtained from MATAM were calculated by dividing the counts of each 16S rRNA sequence by the total number of sequences in each library . Second,A wastewater microbial community was cultivated in sequential batch culture prior to cytometric analyses. In a cytometric histogram, a total of 200,000 cells per sample were measured using cell information on scattering light and fluorescence signals attached to the DNA contents of cells . A totalWe recovered 24 MAGs of high and medium quality from all communities (sorted and unsorted). Three MAGs were retrieved from DG , seven MAGs from LA , and six from OG . A total of eight MAGs were recovered from UC  . The quaElizabethkingia ursingii and Escherichia flexneri, classified up to species level, and Sphingobacterium sp., classified to genus level  distances . Comparius level . The OG us level .Escherichia flexneri, C.omamonas terrigena, A. bouvetii, A. baumannii, Sphingobacterium sp., Empedobacter falsenii and Variovorax sp.)   . A totalunities) . The numunities) .Elizabethkingia ursingii) from DG was of high quality with estimated completeness of 82.8%, contamination of 0.06%, and quality score of 82.50%  affiliated with four species were exclusively recovered in the sorted sub-communities . Of thesf 82.50% . Two MAGmination . One med gerneri .C. B-9) was found below the detection limit in all groups  and OG (26.47%) but not in UC and LA. The most dominant species in UC and LA were A. baumannii (24.92%) and Variovorax sp. (30.90%). On the other hand, coverage of Comamonas terrigena was almost 2-fold lower in OG (5.67%) compared to LA (10.90%) and UC (10.63%). The gOTUs with the smallest coverage in the UC are A. gerneri and Sphingobacterium sp. (3.03%). Further, nine gOTUs were found below the detection limit in one or more sub-communities: six gOTUs in the DG, two gOTUs in the LA, and one gOTU in the OG. However, these taxa were found with coverage above the threshold in one or more sub-communities (munities  (see Secmunities . The genWe reconstructed 16S rRNA genes (rOTUs) from the unsorted and sorted sub-communities to compare their microbial community diversity to that of MAGs in all groups. A total of 57 rOTUs affiliated with 29 taxa at species level were generated from the entire dataset: 9 rOTUs from UC, 16 rOTUs from DG, 15 rOTUs from LA, and 17 rOTUs from OG. Escherichia flexneri, A. bouvetii, A. baumannii, A. pittii, Acinetobacter bereziniae, Citrobacter pasteurii, Elizabethkingia miricola, Sphingobacterium multivorum, and Stenotrophomonas pavanii. However, the relative abundances of these taxa varied in the different sub-communities  was retrieved only in the LA ,Delftia acidovorans , and Enterobacter hormaechei (relative abundances of 0.16% and 1.80% in LA and OG) (n the LA . Brevund and OG) .Achromobacter anxifer, A. gerneri, Comamonas terrigena, Chryseobacterium artocarpi, Chryseobacterium geocarposphaerae, Klebsiella huaxiensis, Moraxella osloensis, and Pseudomonas qingdaonensis) were recovered in multiple sorted and unsorted communities  (Escherichia flexneri and Comamonas terrigena were found in the exact same sub-communities) in both gOTUs and rOTUs. Furthermore, the relative abundances of Escherichia flexneri and Comamonas terrigena were all greater than the average detection limit of relative abundance in all sub-communities for both gOTUs and rOTUs. A. bouvetii and A. baumannii rOTUs were present in all sub-communities, while they were only recovered in UC as gOTUs.Eight of taxa found as gOTUs were also found as rOTUs (gerneri) . HoweverEmpedobacter falsenii was recovered in all sub-communities as gOTU except DG. In contrast, Empedobacter falsenii rOTUs were only found in UC . Elizabethkingia miricola and A. pittii gOTUs were only obtained in LA sub-communities. Although Elizabethkingia miricola gOTU was only recovered in LA communities, their relative abundances in OG and UC communities were greater than the average detection limit. In the case of A. pittii, the relative abundances in all sub-communities were all greater than the detection limit despite only being recovered in LA. A. gerneri was only found as gOTU in OG, while the rOTUs were found in all sub-communities except OG. Although A. gerneri gOTUs were only recovered in OG, their relative abundance in LA (2.81%) was greater than the detection limit. All A. gerneri rOTUs obtained in DG, LA and UC had a relative abundance greater than the average detection limit . Sphingobacterium sp., Elizabethkingia ursingii and Variovorax sp.—classified gOTUs were not recovered in the rOTUs. Sphingobacterium sp. was not recovered in DG . In contrast, the relative abundance of Elizabethkingia ursingii gOTUs in all other sub-communities was greater than the average detection limit. Variovorax sp. classified gOTUs were recovered in all sub-communities except DG, with the relative abundances all above the average detection limit except for DG. nd in UC . Relatived in DG , but exhA. bereziniae, Chryseobacterium pasteurii, Sphingobacterium multivorum and Stenotrophomonas pavanii being recovered in all subcommunities  in rOTUs, while A. bereziniae relative abundance was below the average detection limit in DG and LA sub-communities. Our data indicated that relative abundance, although relevant, may not be the most important feature for the recovery of MAGs.A total of 21 taxa were only found in rOTUs, with munities . The relOur study combined high-throughput flow cytometry cell sorting and metagenomics to recover genomes in a cell culture of a wastewater microbial community. For this purpose, we performed Influx high-throughput cell sorting on the microbial community after separating cells virtually into sub-communities and sorting them via FACS before sequencing. By sorting cells of sub-communities, we could recover more gOTUs with higher quality than in whole communities. This phenomenon is related to the limited ability of metagenomics to recover low-abundant species ,44. By dIn the present study, we recovered 16 MAGs from three sorted sub-communities (3 libraries), after staining the cells with DAPI and eight MAGs from the unsorted community (1 library). The study by Alteio et al.  also couGammaproteobacteria (six unsorted and nine sorted MAGs) and Bacteroidia (two unsorted and seven sorted MAGs), was classified at family, genus, and species levels. We found some species in these bacterial clades unique to sorted sub-communities. Interestingly, the number of taxa observed in the unsorted community and sorted sub-communities showed that some MAGs overlapped between all groups. For instance, Escherichia flexnerii was constantly present in all pooled sub-communities and the unsorted community, and three taxa  found in the unsorted community were also recovered from the low abundant and outer sub-communities.The taxonomy of the genomes of different bacterial clades present in the wastewater treatment plant, including Escherichia flexneri) was present in all communities and showed a high genome-coverage in all of the communities where it was recovered, indicating that Escherichia flexneri is one of the most abundant species in the community. Furthermore, we could recover several species  across multiple communities; however, their coverages were, in some cases, below the detection limit. Interestingly, other species only found in a single community exhibited higher coverages in the libraries where they were not recovered . We observed a similar trend for Elizabethkingia ursingii, which showed higher coverages in the communities where it was not recovered . In other cases, species showed a higher coverage in libraries where they were not recovered as a MAG . Some species showed coverages below the detection limit in the libraries where they were recovered . This trend suggests that coverage alone is not the primary driver in genome recovery. A study by Meziti and collaborators [Empedobacter falsenii and Elizabethkingia ursingii were recovered as high-quality MAGs, with coverage of 5x in LA and 8x in DG.One species . In contrast, A. baumannii rOTUs were recovered in all sub-communities. By comparing our rOTUs to that of Liu and collaborators [Acidimicrobiia, Actinobacteria, Anaerolineae, Blastocatellia, Caldilineae, Chlorobia, Chloroflexia, Clostridia, Cytophagia, Deltaproteobacteria, Gracilibacteria, Halobacteria, Nitrospira, Thermotogae, ML635J-21, Verrucomicrobiae, and Saccharibacteria.A study by Liu and collaborators , using tpipeline  to deterEscherichia flexneri, A. baumannii, A. bouvetii, A. pittii and Elizabethkingia miricola) were recovered in all sub-communities. Furthermore, these species have relative abundances above the rOTUs average detection limit in all groups (The average detection limit of relative abundance of recovered rOTUs was 0.15% (±0.20) in the unsorted and sorted sub-communities, almost 20 times lower than the detection of our prokaryotic metagenome-assembled genomes . Five rOTUs . Although we recovered species with genome coverage below the threshold (10x genome coverage), it would be important to test if those species were real with the addition of genome standards  prior to sequencing. Furthermore, we suggest that combining single-cell sorting with separating sub-communities using flow cytometry may increase the recovery of underrepresented species in unsorted microbial communities.


Functionalized 2D materials have unique properties, but are currently not used for the assembly of van der Waals heterostructures. Here, we present the controlled transfer of artificially synthesized, polar and highly transparent oxo-functionalized graphene, which can decouple graphene layers. A method is described to transfer and integrate transparent and polar 2D materials into heterostructures. Such heterostructures can exhibit properties not seen in individual building blocks such as exotic superconductivity, modified band structures, and superlattice effects.8–12The fast-growing family of two-dimensional materials includes diverse members ranging in properties from insulators .19 In particular, we here use our recently introduced oxo-functionalized graphene (oxo-G), which is a graphene oxide derivative with a very low defect concentration.14,20,21 Its density of in-plane lattice defects as well as the surface chemistry are well controlled.20,22–24 Oxo-G possesses hydroxyl, epoxy and organosulfate groups with hydronium counter ions as surface functional groups, resulting in a highly polar and protic surface with a negative zeta-potential.17,25 After reduction of oxo-G to a certain quality of graphene (red-oxo-G) only vacancy and hole defects remain. The presence of defects leads to the characteristic photoluminescence (PL) spectrum that can be used as a fingerprint of the material in a heterostructure.26–28 In this field, a graphene/ozone-treated graphene heterostructure was already prepared as a transparent conductive electrode for an organic light emitting diode.29 However, no controlled transfer of chemically modified graphene was demonstrated .First, we tested to make oxo-G/oxo-G and red-oxo-G/oxo-G structures by using the Langmuir–Blodgett technique. However, the approach is random and thus limited in terms of controlling the appropriate size, shape and quality of flakes used for the assembly. Accordingly, the assembly of specific stacks of oxo-G derivatives using this method is highly unlikely. The details of this approach are summarized in the ESI Fig. S1.2 wafers. The size and quality of flakes are subsequently probed by optical microscopy, atomic-force microscopy (AFM) or Raman spectroscopy. Flakes of interest are selected, as shown in co-n-butylmethacrylate) (short term: Elvacite 2550), as reported in previous work.36 A schematic representation of the transfer slide is shown in 37 The release is achieved by heating the substrate to 120 °C melting the Elvacite polymer  was conducted to prove that other 2D materials, here in particular graphene can be transferred onto the flakes of oxo-G. In The average spectra in 45 However, we propose that a random orientation of the graphene layers prevents perfect stacking as observed for bilayer graphene. Also impurities on the CVD graphene layer, as seen in et al. determined the critical distance for decoupling graphene layers to be 1.66 nm maximum depending on their orientation.45 It is clearly seen in the height profile of the AFM image  leads to decoupling. The structure was then heated to 100 °C so that impurities could diffuse out more easily and the structure could relax. The 2D peak measured  is the se Fig. S6 that oxoWe introduced the controlled transfer of highly transparent oxo-G and demonstrated the specific assembly of oxo-G/oxo-G, oxo-G/red-oxo-G, oxo-G/G and also G/oxo-G/G, which identifies oxo-G as an interfering layer. We solved inherent problems of transferring oxo-functionalized graphene derivatives, which are barely visible and may crack or fold. We propose that a layer of water between the surface and oxo-G derivatives allows successful transfer. Moreover, by preparing a marker labelled glass substrate for the transfer stamp the precise orientation of transferred flakes becomes possible. The method is compatible also for transferring graphene and very likely other 2D materials, such as h-BN, TMDs or functionalized 2D materials in general. In future research the developed technique can be used to incorporate functionalized 2D materials into heterostructures to fabricate optoelectronic devices. In addition, the influence of defects in graphene on the properties of heterostructures can be studied. In general, with the presented method, novel 2D heterostructures with unexplored properties become accessible.There are no conflicts to declare.NA-002-C9NA00594C-s001


Ocular sarcoidosis is an inflammatory disease that manifests as uveitis, and is often difficult to distinguish from other forms of uveitis based on nonspecific findings alone. Comprehensive proteomic analyses of vitreous humor using LC-MS/MS were performed in each patient with ocular sarcoidosis, vitreoretinal lymphoma (VRL), and controls with epiretinal membrane or macular hole. Differential expression proteins (DEPs) were identified by comparing with VRL and controls, and functional pathway analysis was performed. The candidate biomarker proteins for ocular sarcoidosis were validated using enzyme-linked immunosorbent assay. A total of 1590 proteins were identified in all samples. Of these, 290 and 174 DEPs were detected in vitreous of ocular sarcoidosis compared with controls and VRL, respectively. Enrichment pathway analysis revealed that pathways related to the immune system were most upregulated. Validation of two candidate biomarkers for ocular sarcoidosis, neutrophil gelatinase-associated lipocalin (NGAL) and junctional adhesion molecules B (JAMB), confirmed upregulated NGAL and JAMB protein expressions in ocular sarcoidosis compared to controls and VRL. The results of this study revealed that altered vitreous protein expression levels may discriminate ocular sarcoidosis from other uveitis diseases. Vitreous NGAL and JAMB are potential biomarkers and may serve as an auxiliary tool for the diagnosis of ocular sarcoidosis. Uveitis is a sight-threatening intraocular inflammation and the leading cause of irreversible blindness in the developed world . IntraocCutibacterium acnes (C. acnes) is suspected to be a cause of ocular sarcoidosis [Proteomics is a promising bioinformatics tool aiming at advancing the diagnosis and elucidating the pathogenesis of diseases. Proteomic analysis in the ophthalmology field has been performed on various ocular tissues including aqueous humor, retina, and vitreous humor ,9,10,11.coidosis , detaileEighty-nine patients were included in this study, comprising 28 with ocular sarcoidosis, 25 with VRL, seven with Bechet’s disease (BD), and 30 controls with epiretinal membrane or macular hole diagnosed at Tokyo Medical University Hospital in 2017–2019. Patients with ocular sarcoidosis or VRL who had not been treated with corticosteroids within the past one month before vitrectomy were enrolled. Excluded from the study were patients with preoperative trauma, vitreous hemorrhage, pre-existing macular pathologies including age-related macular degeneration, and immunodeficiency. Ocular sarcoidosis, VRL, and BD were diagnosed according to current diagnostic criteria based on clinical characteristics, radiographic examination, blood tests, vitreous cytopathological examination, and molecular genetic (such as gene rearrangement) analyses, as described previously ,22.First, we performed a comprehensive proteomic analysis of 10 cases each of ocular sarcoidosis, VRL, and controls. Then we validated the results with 27 ocular sarcoidosis cases, 22 VRL cases, 20 control cases, and 7 BD cases . The strategy of this study is summarized in g for 3 min, and the supernatants were collected and stored at −80 °C until use.Vitreous humor samples (approximately 0.5 to 1.0 mL) were collected from the mid-vitreous region at the start of a standard 3-port 25-gauge vitrectomy. The samples were removed with a vitreous cutter before intraocular infusion. The vitreous samples collected were delivered immediately to the Cytology and Molecular Laboratory  for tests for the diagnosis of VRL and ocular sarcoidosis. The samples were centrifuged at 3000× Sample processing for proteome analysis was performed according to the previous report . Brieflyhttp://jvenn.toulouse.inra.fr/app/index.html, accessed on 18 June 2022) and the figures were generated using BioRender . Volcano plots were analyzed and generated using GraphPad Prism 9. Principal component analyses were performed using MetaboAnalyst 5.0 . Hierarchical clustering analyses were performed and visualized using Morpheus . The functional interaction pathways were evaluated by enrichment pathway analysis using Reactome . Protein–protein interaction networks were visualized using Metascape .To detect the changes in protein expression in each disease, the proteins identified by LC-MS/MS were analyzed by various methods including Venn diagram, volcano plot, principal component analysis, and hierarchical clustering analysis. Venn diagrams were analyzed using jVenn  was performed on vitreous humor samples using Human Lipocalin-2 ELISA Kit (Abcam) and Human JAM-B ELISA Kit , according to the manufacturers’ instructions.p value less than 0.05 was considered significant. Data are expressed as mean ± standard deviation. Comparison of the expressed proteins between diseases was performed using Student’s t-test and adjusted using the false discovery rate (FDR). Differential expression protein (DEP) was defined as a protein that satisfied the criterion of FDR-adjusted p value less than 0.05. The protein concentrations of each candidate biomarker measured by ELISA among diseases were compared using Kruskal–Wallis test followed by post hoc Mann–Whitney test. Bar plot and receiver operating characteristic (ROC) curve were generated using GraphPad Prism 9 .All statistical analyses were performed using SPSS  and R (4.0.0.) . A p value less than 0.05 were extracted as proteins with differential expression. Eventually, 290 and 174 proteins were detected as DEPs in ocular sarcoidosis compared with controls and VRL, respectively  between ocular sarcoidosis and controls or VRL are listed in p values less than 0.01 indicating significant differences when compared with both controls and VRL. Next, to quantify the vitreous expression levels of these two candidate proteins, ELISA assay was performed in a patient cohort much expanded from the cohort used in high-throughput proteomic analysis. The validation population comprised 27 patients with ocular sarcoidosis, 20 with VRL, 22 controls, and seven with BD. Since a larger volume of vitreous is required for measuring JAMB due to the small amounts of protein in some samples, the number of patients recruited for JAMB was lower compared to NGAL. The NGAL protein concentrations were 18,402 ± 14,612, 4610 ± 6459, 2266 ± 5104 and 2229 ± 1572 pg/mL in ocular sarcoidosis (n = 27), VRL (n = 20), controls (n = 22), and Behçet’s disease (n = 7), respectively (n = 20), VRL (n = 10), controls (n = 10), and Behçet’s disease (n = 6), respectively, showing significant upregulation in ocular sarcoidosis compared to VRL and controls (p < 0.0001) (p = 0.0001), respectively  C and 0.8ectively D. These Rapid and accurate diagnosis is essential for ocular sarcoidosis because the treatment is fundamentally different from other types of uveitis including Behçet’s disease and VRL . HoweverMany studies have used comprehensive analysis to identify serum biomarkers for the differentiation of uveitis ,27,28,29Our study detected 290 significant DEPs in ocular sarcoidosis compared with controls, most of which were upregulated. Notably, pathway enrichment analysis of the upregulated pathways in ocular sarcoidosis showed that most of the top 10 pathways involved the immune system. In particular, “Neutrophil degranulation”, the most significantly upregulated pathway in ocular sarcoidosis, is associated with the innate immune system. Sarcoidosis is a complex disease involving both the innate and adaptive immune systems . There aIn this study, the expression levels of NGAL and JAMB in vitreous humor were confirmed using ELISA. NGAL, also known as lipocalin-2, is an adipokine associated with various processes including metabolic homeostasis, apoptosis, infection, immune response, and inflammation ,42,43,44In addition, JAMB, which is a member of the junctional adhesion molecule of the immunoglobulin superfamily, is mainly expressed in vascular endothelial cells and plays a role in supporting leukocyte recruitment to the extravascular space . Liang ePathways related to the downregulated miRNAs in biopsied specimens of patients with orbital MALT lymphoma compared to IgG4-ROD were mostly related to the MAPK signaling pathway. This signaling is a well-investigated pathway involved in inflammation due to a pathway regulated by the translocation gene of MALT lymphoma.The analysis of pathways enriched in the target genes in the significantly expressed miRNAs confirmed the essential role of these transcripts and their relative targeting miRNAs in the pathogenesis of IgG4-ROD and orbital MALT lymphoma.n = 5 each) in the control group, 80% of patients with ocular sarcoidosis were females. A reason for the high percentage of female patients with ocular sarcoidosis is that the disease tends to show female preponderance. In addition, vitrectomy is not frequently performed in ocular sarcoidosis due to the invasive nature of the procedure. For these reasons, adjustment for sex difference in the proteomic specimens was difficult. However, the vitreous concentrations of NGAL and JAMB measured by ELISA were not significantly different between males and females . Hence, whether sex difference exists in these biomarkers remains unknown. We plan to further validate the present findings with a larger number of cases in future study.While this study has revealed several interesting findings, the single-institute setting and retrospective design are the main limitations of this study. In addition, although the subjects with ocular sarcoidosis or VRL were not treated with corticosteroids within one month before vitrectomy, as per the selection criteria, the vast majority of the subjects had undergone steroid treatments including eyedrops, sub-Tenon’s injection and oral medications for vitreous opacity prior to the one-month pre-vitrectomy period. These treatments could have affected the vitreous samples. A study on treatment naïve eyes would be ideal. However, analysis of vitreous humor samples from patients who had a recent history of corticosteroid treatment as in this study is commonly encountered in actual clinical practice, because eyes with acute inflammation such as ocular sarcoidosis usually have received prior steroid treatments before undergoing vitrectomy. Further study that excludes patients with inactive ocular sarcoidosis and analyzes the changes in proteomics over the course of treatment for ocular sarcoidosis may promote understanding of the pathogenesis and identification of diagnostic biomarkers. Furthermore, there was sex bias in the patient groups in proteomic analysis. While there were equal numbers of males and females (We report for the first time a comprehensive proteomic analysis of vitreous humor in patients with ocular sarcoidosis compared with VRL and other vitreoretinal diseases. Identification of the DEPs contributes to our understanding of the pathogenesis of ocular sarcoidosis relative to other vitreoretinal inflammatory diseases. In particular, NGAL and JAMB expressions were significantly upregulated in ocular sarcoidosis compared to VRL and controls. This result suggests that these two proteins may be potential biomarkers for ocular sarcoidosis.