Both theoretical and experimental researches on gas response of graphene-based sensors to these typical gases are summarized. Finally, the future research trend about graphene synthesis technique and relevant perspective are also given.Graphene-based materials have aroused enormous focus on a wide range of engineering fields because of their unique structure. One of the most promising applications is gas adsorption and sensing. In electrical engineering, graphene-based sensors are also employed as detecting devices to estimate the operation status of gas insulated switchgear (GIS). This paper reviews the main synthesis methods of graphene, gas adsorption, and sensing mechanism of its based sensors, as well as their applications in detecting SF The groundbreaking fabrication of graphene [2/g, graphene displays a wonderful performance for surface chemistry [The past several decades have witnessed increasing attention by researchers to carbon-based materials, especially those formed by spgraphene  has arougraphene ,4. Givenhemistry ,6,7. Thehemistry ,10,11,12hemistry ,16,17,186) gas, a most commonly utilized kind of insulation gas in high voltage equipment of power systems, especially gas-insulated switchgear (GIS) due to its excellent chemically inert and wonder dielectric strength [2, H2S, SO2F2, CF4 and SOF2 [6 in order to estimate the operation status of GIS, guarding against more serious insulated faults.It has been well-known that sulfur hexafluoride . Results indicated that, although some twisted parts at the graphene do exist, the thick places account for the most proportions.Stankovich  prepared2, NH3, H2O, and CO and proved that these gaseous chemicals can be detected individually, since graphene-based sensors have such required sensitivity.For carbon materials, gas adsorption is primarily depended on physisorption on their surfaces, and sometimes the electrostatic and dispersion reactions that can be tuned by chemical substitution can happen as well. Two factors, namely the surface characteristic of the adsorbent and the properties of the target gas molecules, can influence the interaction strength and further determine the adsorbing rate . Specifi2/graphene oxide sheet was employed to study its sensing property to H2, and they found that high sensitivity (92%) and fast response (no more than 20 s) could be obtained at 180 °C when the concentration of hydrogen reaches 500 ppm. In another research [2 nanorods arrays on graphene sheets enhanced sensitivity to H2S, which is mainly attributed to the outstanding electronic characteristics of graphene and the synergism of the large surface area of SnO2 nanorods arrays. Based on the first principles theory, Congcong et al. [2 and NO.In addition, the flexibility of modification and functionalization in graphene surfaces has opened up many possibilities for the development of tailored functional materials, so that they possess varied physicochemical properties to be employed as gas adsorbent, gas sensors, or some other electrochemical devices. Liscio et al. , with grresearch , it was g et al.  investig2 significantly improved compared with the unmodified one, as Pd has a good affinity toward H2 detection. In the research by Fowler et al. [2 and NH3. Results showed that NO2 receives electrons from graphene, while NH3 donates electrons to graphene, resulting in different sensing mechanisms of these two gases. That is, holes induced conduction and electrons induced conduction for NO2 and NH3, respectively.In terms of the application of graphene-based materials for gas sensors, its fundamental principle is based on the conductivity change after the adsorption of gas molecules . In detar et al. , the graTherefore, the nature physical property of a high specific surface area allows graphene to be prepared as gas sensors for detecting individual gaseous chemicals. More importantly, the chemical modification by functional groups, metal or nonmetal atoms, as well as nanoparticle decoration can enhance the sensitivity and selectivity of graphene-based sensors to one kind of gas in the environment with mixed gases, and this improves its availability for gas sensing in related industrial applications.6 decomposed gases. Much research has been conducted both theoretically and experimentally to investigate the adsorption mechanism and response characteristics. This section is divided into two parts to separately summarize theoretical calculations and experimental analyses.In view of excellent physicochemical properties of graphene-based materials discussed above, their application for preparing sensing devices have never failed to be emphasized, without the exception of fabricating related sensors employed in electrical engineering to detect SF6 decomposed gases. Based on first principles theory, relevant adsorbing parameters including total adsorption energies (Ead), charge transfer (Qt) value, interaction distances (D), energy gap (Eg), and total or local density of state [2F2, SOF2, H2S, CF4, and SO2, and the graphene model should be set up and then optimized to obtain their most stable geometric structures. Then, targeted gas molecules enable us to approach the surface of graphene so as to proceed with the adsorption process. After optimizing whole system, the related adsorbing configurations and parameters mentioned above can be gained. One thing should be mentioned that the adsorbing energy of each system could not be obtained directly. Instead, it is calculated by the following equation:Egraphene/molecule, Egraphene, and Emolecule, respectively, represent the systemic energy after reaction, and the total energies of the graphene and the gas molecule before the reaction. A negative Ead indicates spontaneity of the corresponding reaction. Similarly, Eg of each reaction is calculated asELUMO and EHOMO represent the Lowest Unoccupied Molecular Orbital and the Highest Occupied Molecular Orbital, respectively. 6 decomposed gases, which are all geometrically optimized.As a complete modeling and simulation software, a materials studio was designed to help predict and understand the relationship of a material’s atomic and molecular structure with its properties and behavior ,46. Hereof state ,47 can bof state ,49. Firs6 decompositions. In the theoretical research [2S on intrinsic graphene is proven to be quite weak as a consequence of the small binding energy, the large bonding length, and the small net charge transfer; while for Pt-doped graphene, it has the ability to adsorb up to seven H2S molecules on its surface according to larger binding energy and a shorter Pt H2S bonding distance. Such results provide powerful proofs for the conclusion that metal-doped graphene has better gas response to gaseous chemicals analyzed before. In fact, Pt that naturally has a strong catalytic activity undoubtedly helps its doped graphene with good gas sorption [In terms of theoretical calculations, plenty of research has been conducted for the purpose of discovering a novel kind of graphene-supported material that has a good gas response to SFresearch , adsorptsorption  if it ac2S sensing, and they found that, compared with the film sensors prepared by intrinsic graphene, those decorated with copper (Cu) manifested better sensing behavior. Moreover, the adsorbing ability of Cu-graphene to H2S depends on the concentration of decorated Cu. Too much or too little Cu concentration would deteriorate the gas response of graphene-based sensors to H2S. Another interesting phenomenon is that the Cu decoration perfectly changes the type of the semiconductor, i.e., the intrinsic graphene-based sensors present a p-type semiconductor, while the dopant of Cu present an n-type one. Generally speaking, the doping of metal that has redundant electrons enables electrons to pad the holes and form a duplet. Once all the holes are full of electrons and certain free electrons still exist, it shows an n-type conductor; conversely, if the holes fail to be filled, it remains a p-type conductor. It is no wonder that, in this study, the Cu atom in a proper concentration provide enough electrons, leading to the transformation of the conductor type. However, too little Cu only reduces the concentration of carriers, lowering the conductivity of graphene-based sensors, while too much Cu improves the number of electrons, enhancing dramatically the conductivity of the prepared sensors. Such changes are, on the contrary, detrimental to the gas response as the conductivity changes inconspicuously after the gas adsorbing reactions. That is, they present an insensitivity to their environment. However, it is not contradictive to our previous analysis that the modification of metal can enhance sensing performance. If the dopant concentration is proper, the modified complex presents a further improved response to the target gas.Berahman et al.  designed2 onto light metal-doped (Li and Al) graphene oxide (GO) was investigated by Chi Chen et al. [2 adsorption performance, with a binding energy of up to 1.08 eV, as the light metals that stably anchor on the GO via hydroxyl and epoxy groups provide many active adsorption sites for gas molecules. Rad et al. investigated the SO2 adsorption on pristine graphene, N-doped graphene [Ead comes to the adsorption when N-graphene acts as adsorbent (−19.6/mol), followed by B-graphene (−11.2 kJ/mol) and finally pristine graphene (−9.1 kJ/mol), which suggests that N-doped graphene has the best adsorbing ability to SO2. B-graphene shows as an n-type semiconductor because of a highly negative charge of C-atoms neighboring the boron dopant. When the SO2 molecule is adsorbed on the B-graphene surface, the energy gap reduces by 0.60 eV from 2.90 eV for insolated B-graphene, implying a sharp increase for conductivity of the B-graphene-based sensor. Even so, the adsorption of SO2 on B-graphene or intrinsic is physisorption, on N-graphene is chemisorption, as their different adsorbing cases in these two systems. The inter-molecule bond of SO2 witnesses little change after adsorption in SO2/B-graphene system due to weak sorption; while deforms destructively in SO2/N-graphene system because of their strong binding force.Based on first principles theory, adsorption of SOn et al. , and thegraphene , and B-dgraphene   and d2 (1.467 Å) present no significant change after adsorption compared with that before reaction , the M5 site for H2S adsorbed on intrinsic graphene is the most negative (−0.617 eV), indicating that the adsorption in this site occurs most easily; upon Au-graphene, the reaction for H2S is the most likely to take place when the S atom is oriented to the Au-graphene . In terms of the other gases, the lowest Ead among several adsorbing sites are −0.467 eV for SOF2/graphene with the B site, −0.961 eV for SOF2/Au-graphene with 2F- and S-oriented site, −0.472 eV for SO2F2/graphene with 2O- and F-oriented site, −1.790 eV for SO2F2/Au-graphene with 2F- and O-oriented site, −0.302 eV for SO2/graphene with the T site, and −0.587 eV for SO2/Au-graphene with O-oriented site, respectively. These results reveal that a much stronger adsorption reaction happens in the Au-graphene system than that of the intrinsic one. Moreover, the adsorptions between the Au-doped graphene and gas molecules are proven to be chemisorption since their Ead are far negative than the critical value (−0.8 eV) of the chemical adsorption reported elsewhere [6 decomposition gases, coming up with a new method to research the adsorption process and a new type of possibly workable sensing material.It can be seen that, although all of the 2F2, SOF2, and SO2 were set up, to study the adsorption behaviors and gas response of Ag modified graphene to these gases. To attain a better understanding of these adsorbing reactions and adsorbing capacity of Ag-doped complex to an individual gas, both single gas molecules and double ones were investigated in this research, with optimized geometrical structures shown in In other representative research  based on6 decompositions. Among three systems, SO2F2 shows the most active effect to Ag-doped complex with the lowest adsorption energy (−1.448 eV) and the shortest adsorption distance (2.013 Å), which means that Ag-doped graphene sensors have the best sensitivity to SO2F2 compared with the other two types of gases. Furthermore, all negative Qt values indicate that the electrons transfer from the Ag-graphene to the gas molecules; the largest number of charges that transfer from the adsorbent to the SO2F2 molecule further verifies their strong reaction, proving that SO2F2 can be easily adsorbed on the Ag-doped complex.In terms of one gas molecule adsorption, it can be seen that new bonds are formed in every system between certain atoms of gas molecules and Au atom on the surface of graphene, suggesting the strong binding forces of Ag-graphene to SF2F2 and SOF2 molecules adsorption, the processes appear similar to single gas adsorption, namely that only one in two molecules interacts with Ag-graphene and adsorbed on its surface, and the other one have little interaction with it. While for double SO2 molecule systems, the adsorption seems to be much stronger as the Ead become more negative and the Qt doubled. Such conditions imply that Ag-graphene has an adsorbing capacity to SO2 that is superior to SO2F2 and SOF2. The adsorption to double SO2 molecules can be regarded as chemisorption, and that to SO2F2 or SOF2, physisorption. Even so, the Ead and D in the SO2F2 system still indicate their relatively strong reaction with Ag-graphene.Taking two gas molecules’ adsorption into consideration and comparing their adsorbing parameters with those of single gas molecule adsorption systems, it can be concluded that, for doubled SOEg) about different systems can be obtained to clearly understand the influence of the adsorbing gas molecules on the conductivity of Ag-graphene prepared sensors. Through analyses, it can be obtained that Eg of the SO2F2/Ag-graphene and Ag-graphene/SO2 systems increases sharply when the number of adsorbed gas molecules increases from 1 to 2, revealing a decreased conductivity of the graphene sensors; as for the Ag-graphene/SOF2 system, the number of adsorbed gas molecules may have little impact on the conductivity of graphene sensors. Overall, it was eventually concluded that the sensitivity of Ag-graphene to diverse gases agrees with the following order: SO2F2 > SO2 > SOF2, since the energy gap has little change with respect to the SOF2/Ag-graphene system.Based on further studies on frontier molecular orbital theory, the HOMO, LUMO, and energy gap . The adsorptions were performed at different temperatures in order to study its influence on the adsorption process of SO2 on GO. Near ambient pressure, the adsorption abilities of GO to SO2 are 156 mg/g (15 °C), 153 mg/g (25 °C), and 135 mg/g (35 °C), respectively. Given the decreased adsorbing ability with the increasing temperature, the predominate adsorption of SO2 on GO gives rise to physisorption, which depends on a relatively strong van der Waals interaction due to the large dipole moment of SO2. Upon N2 plasma treatment on GO, a certain amount of N functional groups for ~3.5 atom % indeed is gained; however, it fails to noticeably facilitate the SO2 adsorption on treated GO.Babu et al.  prepared6 decomposition components, H2S, via an electrochemical workstation. The sensors were prepared by repeatedly dropping the graphene suspension onto the film with concomitant drying treatment, for the sake of obtaining a dense, uniform, and smooth graphene film on the substrate. Afterward, the sensor was exposed to environments in a sealed steal tank with different concentrates of H2S to determine its change in resistance.Yu lei et al.  introduc2S against the resistance measured without the presence of sulfuretted hydrogen, expressed as [R represents the real-time resistance of the sensors immersed in H2S, and R0 represents resistance measured in the initial vacuum environment. In this study, three concentrations, namely 300 ppm, 50 ppm, and 100 ppm, of H2S were detected to obtain the sensitivity–time curves for further analysis, expressed in The response of sensors is characterized as the percentage of the resistance change due to the exposure to a gas containing Hessed as (3)S=R−R2S environment; (ii) their sensitivities to H2S reach as high as −7.93%, −10.63%, and −15.78%, respectively, despite their poor performances in terms of long response time to H2S (about 10 min); (iii) the higher the concentration of H2S is, the higher the sensitivity of graphene-based sensors is. Through detection of H2S by graphene-based sensors, it can be proved that graphene-based sensors have a relatively high sensitivity to H2S, and the concentration of such decomposition can be approximately estimated.From 2S and SOF2 adsorption performances. Two concentrations of each gas  are taken into consideration, with the sensitivity–time curves obtained under ambient condition. For H2S adsorption, an increased resistance of 28.15% and 18.73% are obtained for 100 ppm and 50 ppm, respectively; while upon SOF2 adsorption, 100 ppm and 50 ppm result in 23.83% and 15.36% decrease in resistance, respectively. These results manifest that Au-graphene can realize the selectively detection for SOF2 and H2S. Moreover, the probable concentration of individual gas can be estimated, given the dependence between resistance change and gas concentration. In comparison with the above sensitivity of graphene to H2S, Au-modified graphene obviously presents superior sensing behavior, leading to a better response.In , Au-dope2, as shown in 2, the existed target gas is expelled until its inexistence and subsequently begins the nest round of detection. From 2S and SOF2. Overall, it can be concluded that Au-graphene is a promising sensing material for detecting H2S and SOF2 according to its remarkable sensitivity, selectivity, and reusability. It is hoped that Au-doped graphene sensors can selectively detect these two gases in GIS.To investigate the repeatability of such material, the recover lines that used to explore its recovery property are obtained by injecting N6 decomposition components in order to estimate the insulation state of the power system has also been reviewed.In this paper, the main approaches for the synthesis of graphene, its composites, and its based materials have been summarized; simultaneously, the application of such materials in electrical engineering for the detection of SFThe technique of exfoliation, showing good promise to industrially produce graphene, TCVD, which can reproduce graphene on a centimeter scale substrate, and PECVD, which exhibits a fine feasibility to synthesize graphene on any substrate together, constitute the three main synthesis means for graphene preparation.6 decomposed products for use in power systems, which, to a certain degree, broadens their applications, contributing to the further exploitation of their properties.Based on previously theoretical and experimental researches, it can be confirmed that graphene-based materials might be a novel category of sensors because of their exceptional properties such as large specific surface area, high sensitivity, and selectivity to gases. Their outstanding sensing performances enable SF6 decomposition component detection, more high-accuracy experiments that lower the minimum response concentration as much as possible to meet industrial demands are still required. Not only do repeatable experiments need to be performed, but experiments about gas response to SO2F2 and SOF2 also need to be implemented, improving the probability of comprehensively understanding this issue.However, essential work focused on the effective preparation of graphene and relevant experiments aimed at repeatedly verifying the feasibility of graphene-based sensors should be conducted in order to realize its application on the diagnosis of GIS. First, in terms of synthesizing graphene, the inadequate ability for controlling the layer of graphene and realizing large-scale production ought to be solved. Besides, though the theoretical results prove their suitability for SF6 decomposition detection can be built up, detection precision and accuracy can be greatly developed. That is, the individual gases of SF6 decomposed products could be qualitatively and quantitatively detected and, with a short response time, a high sensitivity, environmental friendliness, and chemical stability can to a large extent substitute current electrical devices.For all of these efforts, the ultimate aim is to find a series of graphene-based sensors in which each sensor is quite sensitivity to a specific gas. It is expected that, if the sensor array prepared by graphene-based materials applied for SF


Graphene functionalization with organics is expected to be an important step for the development of graphene-based materials with tailored electronic properties. However, its high chemical inertness makes difficult a controlled and selective covalent functionalization, and most of the works performed up to the date report electrostatic molecular adsorption or unruly functionalization. We show hereafter a mechanism for promoting highly specific covalent bonding of any amino-terminated molecule and a description of the operating processes. We show, by different experimental techniques and theoretical methods, that the excess of charge at carbon dangling-bonds formed on single-atomic vacancies at the graphene surface induces enhanced reactivity towards a selective oxidation of the amino group and subsequent integration of the nitrogen within the graphene network. Remarkably, functionalized surfaces retain the electronic properties of pristine graphene. This study opens the door for development of graphene-based interfaces, as nano-bio-hybrid composites, fabrication of dielectrics, plasmonics or spintronics. Organic functionalization is key to the development of graphene-based functional composites, yet selective covalent functionalization is hindered by graphene chemical inertness. Here, the authors demonstrate a versatile route to graphene covalent bonding with amino-terminated organic molecules. Graphene, one of the most relevant materials of the decade, has to strengthen several drawbacks before it may step from fundamental physics to technology. Particularly, the absence of an electronic band-gap and its extreme chemical inertness significantly compromise their use as an active element in electronic devices. Development of highly precise and selective chemical functionalization strategies is understood as a breakthrough to circumvent this issue. A ruled covalent functionalization will provide added value to pristine graphene to develop applications in domains as important as band-gap engineering, creation of magnetism in graphene for applications in spintronics, fabrication of dielectrics, formation of nano-bio hybrid composites for improved biosensors or tailoring optical-plasmonic properties. To this aim, wet chemistry protocols  have been settled12345678910111281415-SiC(0001) by used chemical vapour deposition at 1,600 °C under an argon (Ar) laminar flow in a hot-wall Aixtron VP508 reactor182 V−1 s−1. The present work has been performed using both, SLG and QFSMG. Both surfaces exhibit similar results, and therefore, we will refer to them indistinctly.For our highly selective covalent organic functionalization we used high quality grown epitaxial graphene on 4HTo validate our mechanism, we have chosen p-aminophenol (p-AP) as a model molecule as it incorporates an aromatic ring and two different ending groups. The only requirement for our methodology is to use a molecule terminated in an amino group, as the mechanism we will describe hereafter is selective to this particular group. The terminal group is the one that has to be chosen paying attention to the possible applications. In our case the phenol-linked molecule can be considered as a chemical spacer or linker for anchoring more complex molecular nanoarchitectures in the search of designing functional structures2361020In this work we show an unprecedented chemical mechanism, described at an atomic level, by which the excess of charge located in unsaturated dangling bonds of C atoms around single-atomic vacancies (SAV) induces a selective oxidation reaction of any molecule containing an amino group, in which the N gets incorporated into de graphene lattice followed by a redistribution of the charge of the molecule in the neighbourhood of the vacancy. This mechanism can be used as a rational functionalization strategy to covalently couple amino-terminated organic molecules, as p-AP, to the surface of graphene epitaxially grown on SiC without disrupting its outstanding electrical properties over mm distances. Our method is based on making use of the localized electrons appearing around atomic vacancies. The physics of induced point defects on graphene have emerged in the last years as a source of new controlled properties, giving rise to magnetism27+ ion irradiation. These vacancies have been previously observed and isolated by transmission electron microscope (TEM) and STM, and their formation protocol is well established29303132333435We have performed First-principles density functional theory (DFT)-based calculations on a fully relaxed model of 4H-SiC(0001) SLG/(6✓3 × 6✓3)R30° with a unit cell with 13 × 13 SLG on topRegarding the SAV structural morphology, it has been recently shown that two distinct configurations of a SAV can be found in graphene32We observe that the apparent height of the SAV in STM images is about 1±0.2 Å and its width about 1±0.2 nm. These values are unaltered for the case of the QFSMG surface and are in good agreement with previously published valuesOnce SAVs are created they appear stable and spread all over the graphene surface. The next step is dosing about four Langmuirs of p-AP. STM images recorded after this process show that the surface topography has importantly changed. All the localized threefold radiating 1 Å high bumps become larger and higher, losing their starring appearance. The average apparent height is now 1.3±0.6 Å . Upon fus core-level peak derived from N in a graphitic network has been reported to appear at 400.5 eV  spectra recorded on the surface after dosing are indicative of the existence of N and carbonaceous species on the surface, suggesting that the bumps depicted in STM images corresponds to p-AP molecules anchored to the SAVs with an unique adsorption site for N atoms . The bin eV ref. , howeverRemarkably, if p-AP is dosed on pristine graphene (no SAVs) no absorption takes place, as depicted by STM and XPS measurements . Thus, wWe have checked the thermal stability of the anchored molecules, and no significant changes were found neither in the line-shape and intensity of the XPS spectra nor in the STM images appearance up to temperatures above 250 °C, indicating the covalent nature of the bonding, and allowing us to disregard any unspecific molecular adsorption . Moreove9Interestingly, a recent work explores the catalytic activities promoted by the unsaturated dangling bonds that may remain on a surface after SAV formation2 groups. We have computed the height of the barrier, ΔE, at the transition state (TS) in the double (−NH2) dehydrogenation process of the p-AF molecule. This TS has been investigated within the climbing-image nudge elastic band approach (see full details in the 2) dehydrogenation (around 1.5 eV), besides the fact that the remaining dehydrogenated oxygen would not be able to perfectly integrate within the SAV into the graphene lattice, only getting attached to one (of three) of the C atoms of the SAV. All calculated energy barriers are deeply discussed in the With all this chemical information in mind we have carried out a large battery of DFT-based calculations to confirm the above-mentioned scenario  molecule transfers 0.56e− towards the graphene lattice, while the N atom (considered as belonging to the molecule) accumulates an extra electronic charge of around 0.82e− with respect to its neutral state, coming from a charge rearrangement in the molecule, leaving the rest of the molecule with a net loss of almost 1.4e−. This is related to the fact that the N atom integrates into the graphene lattice, stabilizing its charge state by adsorbing and accumulating charge on the N atom from the rest of the molecule, and acting as a drain-pipe towards the C atoms at the neighbourhood.Interestingly, the N atom in the proposed structure has the charge unbalanced, since it is surrounded by four C atoms . To evalThe left panel of N region—within the solid line–, the resembling of pristine graphene DOS is quite poor, and some induced features arises just above and below associated to a reflection of the electronic properties of the positively charged molecule. In particular, the one arising between −1 and 0 eV—labelled as (*) in right panel of N on their closest C atoms. On the other hand, the highly hybridized molecular HOMO and LUMO positions are indicated as blue and red shadowed region, respectively, and they have visibly spread out by effect of the chemical interaction. The tunnelling region, where experimental images were recorded (−0.7/−0.3 eV) is also indicated in the right panel of To evaluate the spatial extension of the electronic charge transferred from the molecule towards the graphene lattice, right panel of Nevertheless, as we move away from the N atom, the C-atoms set (crown)—between the dot and solid lines in 12 cm−2 but it can be controlled by changing the duration of the ion irradiationThe density of immobilized groups is an important point when focus on applications12 molecules per cm2. For doses leading to higher coverage many events overlap and the characteristic electronic properties of the system collapse.This mechanism can be theoretical described with a simple phenomenological calculation based on random numbers. We have computed the density for different fluencies removing from the counting all overlapping events. The overlapping criterion has been taken in such a way that isolated events are considered if a minimum lateral distance larger than 1 nm separates them. This simple computational model reproduces quite perfectly our experimental data, and let us to conclude, both from calculation and experiments, that the maximum number of independent molecules that can be covalently linked is 8 × 1013 molecules per cm2. The statistical nature of our methodology only allows us to have a density four times lower.It is important to note that assuming a purely geometrical model of hard spheres with the experimental radius of 1nm, the maximum number of immobilized molecules is about 3.2 × 10Moreover, from R) versus inverse temperature measurements at zero magnetic field recorded for a QSG sample. Of paramount importance is the fact that the functionalized system does not exhibit a measurable gap at low temperatures (below 34 K); the thermally activated regimes at intermediate (between 34 and 140 K) and high temperature (above 140 K) regimes exhibit low-activation energies  curves evidence the presence of Landau Levels in p-AP functionalized samples. Shubnikov–de Haas oscillations are evident in Upon cooling below 34 K, Moreover, from a fundamental point of view, the electronic band structure of the sample does not seem to be altered (at most slightly modified) after p-AP functionalization. This is shown in 38Atomic force microscopy (AFM) measurements further prove that this functionalization process did not either alters the morphology of the graphene samples. Topographic and surface potential images of the functionalized samples look very much alike to pristine graphene on SiC, with no evidences of large-scale damage .2-terminating molecules, with the doubly dehydrogenated N atom incorporated within the graphene lattice (as in the present case): 4-aminothiophenol, 4-aminobenzaldehyde, 4-(oxiran-2-yl)aniline, among many others, where the same previous behaviour has been also observed. For all these cases the molecules transfer a net electronic charge of around 0.5–0.6e− to the graphene, while N atom accumulates an extra electronic charge of around 0.9–1e−. These calculations support the experimental observed generality of the process.To validate our mechanism, we have chosen p-AP as a model molecule as it incorporates an aromatic ring and two different ending groups. The only requirement for this methodology is to use an amino-terminated molecule, as the mechanism is highly selective and efficient to this particular group. The other ending group has to be chosen towards potential applications. Several variations of this molecule are nowadays commercially available or possible to synthetize by several laboratory routes. We have experimentally confirmed that our protocol perfectly works also with aminothiophenol and p-Phenylenediamine molecules , and the2 per molecule  during 45 s at normal incidence angle. The acceleration energy of the electrons inside the ion gun was 140 eV, the current sample was 1 μA and the gas pressure during this process was kept at 1 × 10−7 mbar. Aminophenol was dosed from a crucible hold at room temperature inside the UHV chamber.Growth, STM, XPS and ARPES experiments were performed XPS spectra were recorded using a Phoibos 150 electron analyser equipped with a 2D-DLD detector and an Al-Kα monochromatic X-Ray source. ARPES spectra have been acquired at RT at BaDElPh beamline located at ELETTRA Synchroton in Trieste using a SPECS Phoibos 150 with a 2D-CCD detector system2 square samples using the Van der Pauw configuration in 9 T and 14 T Quantum Design PPMS, using Wimbush press-on contacts on Cu-sheet pads placed at the sample corners. The magnetic field was applied perpendicular to the graphene sheet. Resistance measurements were taken in three configurations: two of them optimized for resistivity measurements along nominally orthogonal directions in the sample while the remaining configuration maximized the Hall resistance. Further information is given in the Experimental Section of the Resistance as a function of magnetic field and temperature (1.8 K–360 K) was measured on 1 × 1 cmFirst-principles computational simulations have been carried out within the basis of different DFT-based implementations. In particular, we have combined the localized-basis-set and plane-wave DFT-schemes as implemented in the FIREBALL50The data that support the findings of this study are available from the corresponding author on request.How to cite this article: Bueno, R. A. et al. Highly selective covalent organic functionalization of epitaxial graphene. Nat. Commun.8, 15306 doi: 10.1038/ncomms15306 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Notes, Supplementary Methods and Supplementary References


The co-doping of graphene with nitrogen and sulfur was investigated aiming at understanding their interactions with the presence of oxygen in graphene. The co-doped graphene (NS-G) was synthesized via a one-pot hydrothermal route using graphene oxide as starting material and L-cysteine, an amino acid containing both N and S, as the doping agent. The obtained NS-G with a three-dimensional hierarchical structure containing both macropores and mesopores exhibited excellent mechanical stabilities under both wet and dry conditions. As compared to N or S singly doped graphene, the co-doped sample contains significantly higher concentrations of N and S species especially pyrollic N groups. The co-doped sample considerably outperformed the singly doped samples when used as free-standing electrode in supercapacitors due to enhanced pseudocapacitance. The simultaneous incorporation of S and N species with the presence of oxygen significantly modified the surface chemistry of carbon leading to considerably higher doping levels, although directly bonding between N and S is neither likely nor detected. Hence, the synergetic effect between N and S occurred through carbon atoms in neighboring hexagonal rings in a graphene sheet. The incorporation of heteroatoms in carbon materials can significantly modify their surface and bulk properties and considerably broaden their applications in various fields. Among others, nitrogen doping of graphene has been intensively investigated in the last few years. Sulfur and boron are among other commonly used heteroatoms for the doping1−1, which are far below the theoretical electrochemical double-layer capacitance of 520 F g−19Recently, graphene has been widely explored as electrode materials in supercapacitors due to its large specific surface area and high electronic conductivity3456Much progress has been made in the synthesis of graphene with different morphologies so far. For example, two-dimensional graphene nanosheets, one-dimensional graphene nanoribbons, and zero-dimensional graphene quantum dots have been obtained91112182045141516171819Other than the morphology control, another effective way in improving the capacitive performance of graphene is the introduction of heteroatoms222425It has been reported that nitrogen doping can improve the specific capacitance and cyclability of graphene in supercapacitors142728Although co-doped carbon materials potentially possess promising properties for various applications, there are only a few reports about graphene that are simultaneously doped with sulfur and nitrogen31323334353632Recently, we used GO as precursor and nine different amino acids as environment-friendly nitrogen sources for the synthesis of nitrogen-doped graphene via hydrothermal method, where the influence of acidity of amino acid on the capacitive performance was investigated. The charged amino acid, and the ensuing electrostatic interactions between amino acid and GO affect the morphology and specific surface area of nitrogen doped graphene (N-G), and ultimately affect its electrochemical performance. The specific capacitance for the obtained N-G samples are following the order of acidic amino acids > neutral amino acids > basic amino acids−1, an energy density of 29.4 Wh kg−1 and a power density of 10.0 kW kg−1, which significantly outperformed the nitrogen doped graphene (N-G) and sulfur doped graphene (S-G). The location, interaction and synergetic effect of nitrogen and sulfur on carbon surface were discussed.The present study aims at the understanding of the interaction between nitrogen and sulfur as dopants in graphene. Graphene oxide and L-cysteine were used as starting material in the hydrothermal synthesis. The obtained NS-G exhibits three-dimensional hierarchical structure containing macropores. The NS-G hydrogel with high mechanical stabilities was used as an additive/binder-free electrode and showed an excellent electrochemical performance with a high specific capacitance of 566 F g−1, indicating a predominantly elastic response. The G″ of NS-G hydrogel is slightly sensitive to the frequency and does not cross over G′, implying a high degree of covalent or non-covalent cross-linking in the hydrogel13The co-doped NS-G, singly doped N-G and S-G hydrogels were successfully synthesized via the one-pot hydrothermal method. When preparing using the same reactor, the dimension of the hydrogel with different doping elements appears to be rather similar . We alsoThe morphology of the NS-G, S-G and N-G aerogel samples was investigated by SEM. As displayed in The results from XRD measurements are shown in −1 corresponding to the D and G band, respectively , S-G (1.03) and N-G (1.00) are essentially equal, which means the defect levels of these samples are similar. In the doped samples, the weak and broad peaks at around 2800 cm−1 can be assigned to the combination of 2D-band and D+G-band. The peaks at around 2680 cm−1 are related to the second-order two phonon mode 2D-band and the weak contribution at around 2920 cm−1 is a combination of the D-and G-band, which are related to the disorder-induced feature of carbon material−1 is also observed. The presence of the broad peak in RGO, NS-G, N-G and S-G confirms the presence of defects.ectively . The relectively  and d, i0 = 0.4-1). The co-doped samples prepared from different cysteine to GO ratios show similar pore structure but with less pronounced hysteresis in isotherms (2 g−1) is slightly lower than those of S-G and N-G. It is also lower than the nitrogen-doped graphene prepared using acidic amino acid in our previously study (367.1 m2 g−1)2 g−129332 g−1 for NS-G005 and 156.9 m2 g−1 for NS-G1).The BET surface area and pore structure of the freeze-dried samples were investigated by nitrogen physisorption, and the obtained isotherms are shown in sotherms . As a wh3 g−1. This is considered as a positive feature in this work, since mesoporous structure with large pore size facilitates electrolyte diffusion and therefore can potentially enhance the capacitive performance when used as electrode.The pore size distributions are listed in The doped samples were further investigated by high resolution XPS. As shown in 4138n–C (n = 1 or 2) and conjugated –C = S– bonds respectively3031n– species, which usually form at the edges of grapheneThe surface atomic concentrations of C, O, S and N were derived from the corresponding peak areas of the XP spectra and the results are summarized in Above-mentioned results demonstrate the unique features of the co-doped NS-G, including the high specific surface areas and pore volume, 3D porous structure, and high nitrogen and sulfur contents. These features strongly suggest that NS-G could have a better electrochemical performance than the singly doped samples.−1. The voltammograms of the three samples show approximately rectangular shape, which are typical for double layer capacitors. Obviously, the NS-G has the highest capacitance in the three samples. −1. The curves show an approximately triangular form for the three samples, indicating the electric double-layer performance. The time of accomplishing a charge/discharge cycle is clearly longer for NS-G than that for N-G and S-G. The mass specific capacitances (Csc) calculated by using the equation Csc = (IΔt)/(mΔV) were 566, 282 and 371 F g−1 for NS-G, S-G and N-G, respectively. The Csc value of NS-G is higher than that of single doped graphene, RGO  is higher than the theoretical electrochemical double-layer capacitance of 520 F g−1The doped graphene hydrogels were used as free-standing electrodes, and tested using cyclic voltammetry (CV) and galvanostatic charge-discharge (GCD) techniques with a three-electrode system. It has been reported that pyridinic N in graphene layers results in the attraction of ions, and pyrrolic N has an enhancing effects on the capacitance due to its pseudocapacitive contributions46−1 is shown in −1 for 2000 cycles (−1) was obtained after 2000 cycles, which demonstrates the excellent cycling performance of the NS-G electrode.The calculated specific capacitance (Csc) at different current densities from 0.5 to 10 A g0 cycles . The capElectrochemical impedance spectroscopy (EIS) provides information on the internal resistance of the electrode material and the resistance between the electrode and electrolyte. The real part of resistance (Z′) measured is the ohmic resistance derived from the electrolyte and the contact between the electrode and the current collector. As shown in 30−1 was determined to be 385 and 263 F g−1 for NS-G005 and NS-G1, respectively  was obtained after 2000 cycles.Finally, the power density and energy density of NS-G was investigated by a symmetrical two-electrode system in 6 M KOH. The CV and GCD curves of NS-G at different scan rates and current densities are shown in 0 cycles  in a sym−1 at 0.5 A g−1. The enhanced pseudocapacitance in the co-doped sample can be assigned to higher contents of pyrrolic N groups and S species.In summary, nitrogen and sulfur co-doped hydrogels and aerogels were successfully synthesized through a hydrothermal method by using amino acid and GO as precursor. The obtained NS-G with a three-dimensional hierarchical structure containing both macropores and mesopores exhibited excellent mechanical stabilities. The simultaneous incorporation of S and N species with the presence of oxygen significantly modified the surface chemistry of carbon leading to considerably higher doping levels, although directly bonding between N and S is neither likely nor detected. Hence, the synergetic effect between N and S occurred through carbon atoms in neighboring hexagonal rings in a graphene sheet. When used as free-standing electrode in supercapacitors, the co-doped sample considerably outperformed the singly doped samples due to an enhanced pseudocapacitance and a lower charge transfer resistance. The specific capacitance of NS-G reached 566 F g−1, 30 ml) containing 0.05 mmol L-cysteine (C3H7NO2S the mass ratio of L-cysteine to GO is 0.1:1) was first treated by sonication for 10 min. The resulting stable suspension, sealed in a Telfon-lined autoclave, was hydrothermally treated at 160°C for 4 h. After that, the autoclave was naturally cooled to room temperature and the as-prepared NS-G hydrogel was removed from the autoclave and immersed in deionized water for seven days, in which deionized water was renewed once a day to remove any unreacted reagents. Finally, the NS-G hydrogel was freeze-dried overnight to obtain the aerogel. In addition to the L-cysteine to GO ratio of 0.1:1, a lower mass ratio of 0.05:1 and a higher ratio of 1:1 was investigated and the obtained samples are designated as NS-G005 and NS-G1, respectively. For comparison, N and S singly doped hydrogels (N-G and S-G) were prepared using the same procedure. L-alanine  and 3-mercaptopropionic acid  with 30 ml GO suspension (2 mg/ml) were employed for N-G and S-G, respectively. The hydrothermal synthesis was also performed without doping agent and the obtained sample is designated as RGO. The water content was determined by weighing the samples before and after drying.Graphene oxide was prepared from graphite powder using a modified Hummers methodα, (λ = 1.5418 Å) radiation. Raman spectra were recorded with a Bruker spectrometer with 532 nm laser, and X-ray photoelectron spectroscopy (XPS) measurements were carried out using a ESCALab220i-XL electron spectrometer (VG Scientific) using a 300W Al Kα radiation. Nitrogen physisorption was carried out at 77 K with an ASAP 2020 Physisorption Analyzer. The mechanical properties of the prepared hydrogels were investigated using a dynamic mechanical analyzer (DMA Q800).All of the samples were characterized by scanning electron microscopy (SEM) using a Hitachi S-4800 system and transmission electron microscopy . X-ray diffraction (XRD) measurements were performed with a D8 Bruker diffractormeter with a Cu K2 and the area of the prepared electrode is 1 cm2. In the three-electrode system, the graphene hydrogel samples were used as the working electrode, platinum foil as the counter electrode and standard Hg electrode as reference one, and 6.0 M KOH aqueous solution as electrolyte. CV and GCD test in the potential range of −1.2 to −0.2V was performed at different scan rate and current densities. For the electrochemical test in the two-electrode system, two slices of hydrogel with the same mass were pressed onto a nickel foam sheet (3 × 1 cm) at 10 MPa for 30 s and used as electrodes. The electrode contained approximately 2.0 mg/cm2 of active material. The two electrodes were separated by a filter paper, which was fully soaked with electrolyte. The potential range for CV and GCD tests was 0–1.0 V. Electrochemical impedance spectroscopy (EIS) measurements were carried out by applying an alternating current with the voltage of 5 mV over a frequency range from 0.01 Hz to 100 kHz. The gravimetric capacitance, CCV (F g−1), was calculated from CVs using the following equation: CCV = Σ|I|/2νmΔV, where Σ|I| is the area of the current (A) profile, m is the mass of active material in the electrode (g), ν is the scan rate (V/s) and ΔV is the potential window (V). The specific discharging capacitances were determined using the equation Csc = (IΔt)/(mΔV) from three-electrode measurement, and in the two-electrode system the energy density and power density were calculated using the equation E = 1/2 Csc ΔV2 and P = E/Δt, respectivelyCyclic voltammetry and galvanostatic charge/discharge of doped graphene electrodes were performed with a CHI660D electrochemical workstation with both two-electrode and three-electrode systems. The working electrodes were fabricated by the prepared hydrogels in the wet state without using any binder or conducting additives. A slice of the wet hydrogel was cut and blotted with a filter paper to remove excess water; then it was pressed onto a nickel foam sheet (3 × 1 cm) at 10 MPa for 30 s. The mass loading of active material on each current collector was about 2 mg/cmSupplementary Information for Interaction between Nitrogen and Sulfur in Co-Doped Graphene and Synergetic Effect in Supercapacitor


Moreover, performance on word order and social-pragmatic scores independently predicted both groups' later performance on wh-question comprehension. Our findings indicate that both grammar and social-pragmatics are implicated in the comprehension of wh-questions. The “grammatical-origins” argument is supported because the ASD group did not reveal earlier and stable comprehension of wh-questions; furthermore, their performance on SVO word order predicted their later success in linguistic processing of wh-questions. The “pragmatic-origins” argument is also supported because children's earlier socialization and communication scores strongly predicted their successful performance on wh-question comprehension.Compared to typically developing children, children with autism (ASD) show delayed production of wh-questions. It is currently controversial the degree to which such deficits derive from social-pragmatic requirements and/or because these are complex grammatical structures. The current study employed the intermodal preferential looking (IPL) paradigm, which reduces social-pragmatic demands. The IPL paradigm can help distinguish these proposals, as successful comprehension promotes the “pragmatics-origins” argument whereas comprehension difficulties would implicate a “grammatical-origins” argument. Additionally, we tested both the linguistic and social explanations by assessing the contributions of children's early grammatical knowledge  and their social-pragmatic scores on the Vineland to their later wh-question comprehension. Fourteen children with ASD and 17 TD children, matched on language level, were visited in their homes at 4-month intervals. Comprehension of wh-questions and SVO word order were tested via IPL: the wh-question video showed a costumed horse and bird serving as agents or patients of familiar transitive actions. During the test trials, they were displayed side by side with directing audios . Children's eye movements were coded offline; the DV was their percent looking to the named item during test. To show comprehension, children should look longer at the named item during a where-question than during a subject-wh or object-wh question. Results indicated that TD children comprehended both subject and object wh-questions at 32 months of age. Comprehension of object-wh questions emerged  According to the DSM-V , usually occurring in the beginning of the sentence (in English). Syntactically, these wh-words stand for information that is missing in the sentence. Wh-questions probe for missing arguments  or adjuncts . Furthermore, argument wh-questions can ask for the grammatical subject of a sentence  Who __ likes Mary?) or the grammatical object of the sentence  Who does Mary like __?). Notice that both subject and object wh-questions involve wh-movement from the original argument location; however, the movement for subject wh-questions does not change the canonical word order of English sentences  above), whereas the movement for object wh-questions changes the word order of the sentence to OSV , indicating that children's proportion of looking to the named object was different for the “what” and “where” questions. There was no main effect of visit , nor a significant group × trial interaction . A significant group effect emerged , with greater overall looking to the named object by the TD group than by the ASD group. Given these significant trial and group effects, the next set of analyses investigated each group's looking patterns separately for subject- and object-what questions.A repeated-measures analysis of variance (2 × 4 × 2) was conducted with group (ASD or TD) as the between-subjects variable, and visit  and trial type  as within-subjects variables. The results showed a main effect of trial  but no main effect of visit . The visit × trial interaction trended toward significance . The second ANOVA compared the object wh-questions and “where” questions, and revealed a main effect of trial , and no main effect of visit . This visit × trial interaction also trended toward significance .For the TD group, the first repeated-measures analysis of variance (4 × 2) was conducted with visits  and trial type (subject-what questions and where questions), revealed a main effect of trial  but no main effect of visit  and no visit × trial interaction . Similarly, the repeated-measures analysis of variance (4 × 2) for the object wh-questions with visit  and trial type (object-what questions and where questions) revealed a main effect of trial  but no main effect of visit  nor a significant visit × trial interaction .For the ASD group, the first repeated-measures analysis of variance (4 × 2), conducted by visit  and trial type  = 1.90, p = 0.038; visit 4: t(16) = 3.68, p = 0.001; visit 5: t(16) = 4.09, p < 0.001; visit 6: t(15) = 6.26, p < 0.001; see Figure t(16) = 1.27, p = 0.111; visit 4: t(16) = 3.75, p < 0.001; visit 5: t(16) = 3.57, p = 0.001; visit 6: t(15) = 8.52, p < 0.001; see Figure Overall, then, both groups demonstrated wh-question comprehension—they correctly looked t(13) = 3.39, p = 0.002, this effect disappeared at visit 4, t(13)= 0.998, p = 0.168 and visit 5, t(11) = 1.05, p = 0.157, then re-emerged at visit 6, t(11) = 2.07, p = 0.031; see Figure t(13) = 2.30, p = 0.019; visit 4: t(13) = 0.807, p = 0.217; visit 5: t(11) = 1.58, p = 0.07; visit 6: t(10) = 0.857, p = 0.206; see Figure The ASD group's performance was less consistent for object-what questions: while they appeared to show comprehension at visit 3, visit4: M = 43.31, SD = 11.95; ASDvisit6: M = 48.28, SD = 19.35; t(28) = −0.859, p > 0.05] or their expressive (EOWPVT) vocabulary  Thus, it appears that the TD and ASD groups achieved comprehension of wh-questions at similar language levels.In sum, TD children displayed evidence of wh-question comprehension by 32 months of age . The ASD group demonstrated significant comprehension at visit 3; however, the ASD group was unable to maintain this level of comprehension consistently for the rest of the visits (with re-emerging significant comprehension for object wh-questions at visit 6). When the two groups are compared by age and/or visit, there is a discrepancy in the point of wh-question comprehension attainment; however, it is important to compare the groups by language level as well. As Table 2 = 3.76, p = 0.05]; visit 4: , visit 5: ; and visit 6: }, indicate that in all the visits there were more Comprehenders than Non-comprehenders in the TD group. Within the ASD group, there were more Comprehenders than Non-comprehenders at visit 3 .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Metastatic non-small cell lung cancer (NSCLC) is a highly fatal and immunogenic malignancy. Although the immune system is known to recognize these tumor cells, one mechanism by which NSCLC can evade the immune system is via overexpression of programmed cell death ligand 1 (PD-L1). Recent clinical trials of PD-1 and PD-L1 inhibitors have returned promising clinical responses. Important for personalizing therapy, patients with higher intensity staining for PD-L1 on tumor biopsies responded better. Thus, there has been interest in using PD-L1 tumor expression as a criterion for patient selection. Currently available methods of screening involve invasive tumor biopsy, followed by histological grading of PD-L1 levels. Biopsies have a high risk of complications, and only allow sampling from limited tumor sections, which may not reflect overall tumor heterogeneity. Circulating tumor cell (CTC) PD-L1 levels could aid in screening patients, and could supplement tissue PD-L1 biopsy results by testing PD-L1 expression from disseminated tumor sites. Towards establishing CTCs as a screening tool, we developed a protocol to isolate CTCs at high purity and immunostain for PD-L1. Monitoring of PD-L1 expression on CTCs could be an additional biomarker for precision medicine that may help in determining response to immunotherapies. These APCs help activate cytotoxic T-cells, helper T-cells and natural killer cells. All of these components work in concert against tumor cells. However, many metastatic tumors such as in non-small cell lung cancer (NSCLC) have adopted methods to evade immune detection and/or clearance3. One of the recently discovered pathways that tumors use is the overexpression of programmed cell death ligand 1 (PD-L1). PD-L1 binds to PD-1 on T-cells and suppresses its activity5. Immunotherapy based on inhibition of PD-1 or PD-L1 represents a breakthrough in the treatment of advanced cancers, particularly lung, renal, and melanoma cancers. Although only a minority of patients have clinical response, those that do often have a durable and lasting response12.The immune response to cancer involves a complex network of cellular interactions. Antigen presenting cells (APCs) can recognize neoantigens from some immunogenic tumors13. Past studies of renal cancer show tumors with high PD-L1 levels respond, while those with low levels do not respond10. Based on these findings, a clinical trial for anti-PD1 (pembrolizumab) was conducted where NSCLC patients were screened based on expression levels of PD-L1 on the primary tumor14. Response rate, progression free survival and overall survival with PD-1 inhibitors were greater in tumors with high tumor PD-L1 expression11. Although current guidelines call for anti-PD1 therapies after failure of standard chemotherapy, these are rapidly changing and recent phase I trial data suggests that anti-PD1 therapies may be effective as first line therapy15. First line therapy is especially promising for patients with higher PD-L1 expression on their biopsies and is associated with improved responses7.Finding biomarker predictors of response to PD-L1 blockade has proven challenging. Some studies have suggested that the level of PD-L1 expression on the initial tumor tissue can predict positive response, while other studies suggest that the level of infiltrating CD8+ lymphocytes are the most significant predictors of response7. Biopsy of multiple sites or serial biopsies during treatment could address some of these issues, however it may not be feasible due to the invasiveness of the procedure and the potential risks to the patient. In this regard, PD-L1 expression on circulating tumor cells (CTCs) could aid in screening and monitoring patients16. CTCs are tumor cells that are shed from various locations of the primary and/or metastatic tumors19. As such, they may represent a greater portion of the spectrum of genetic and epigenetic variability within a patient’s tumors 22. Most of these previous studies utilized specific surface markers for CTC capture, either with CellSearch or with similar magnetic bead technology, and did not isolate cells in a manner that is unbiased to surface expression. There are still other knowledge gaps, particularly in how PD-L1 expression on CTCs correlates with expression on tumor biopsies, what method to use for quantifying PD-L1 expression, on both CTCs and tumor biopsies, and how PD-L1 expression on CTCs varies, both at time of initial treatment and as therapy continues.There have been few studies to explore PD-L1 expression on CTCs, either in breast cancer (15) or in bladder cancerHere, we evaluate the PD-L1 expression on 31 CTC-containing samples, obtained from 22 patients with metastatic NSCLC who were scheduled to receive or were receiving PD-1 or PD-L1 inhibitors, including 11 metastatic NSCLC patients scheduled to receive the anti-PD-1 treatment pembrolizumab (one patient ended up receiving erlotinib) Fig. , Table 1This study included 32 volunteers for blood donation, 22 metastatic NSCLC patients and 10 healthy donors . All patients provided informed consent for participation in this study. After obtaining informed consent, 6–10 cc of blood were collected from each patient in EDTA tubes. Collected samples were processed to isolate CTCs within four hours of collection. Blood samples from healthy volunteers (n = 10) of various ages were similarly processed. All methods were performed in accordance with the relevant UCLA IRB guidelines and regulations.23 , H1703 (squamous carcinoma) and H3255 (adenocarcinoma) were cultured in RPMI media supplemented with 10% FBS and 1% pen/strep. Hela cells were cultured with DMEM media supplemented with 10% FBS and 1% pen/strep. At 70% confluence, cells were harvested using 0.25% trypsin and fixed with 2% paraformaldehyde. White blood cells (WBC) were isolated from healthy blood using RBC lysis buffer (eBioscience). WBCs were similarly fixed with 2% paraformaldehyde. During each staining experiment, an aliquot of each of these fixed cell solutions was stained in the same well-plate alongside with the CTC samples for normalization.23. All CTC and WBC counts were checked by two independent reviewers  for 10 min, permeabilized with 0.4% v/v Triton X-100  for 7 min, blocked with 5% goat serum (Invitrogen) for 30 min. To identify CTCs, cells were labelled for 40 minutes at 37 °C with 4,6-diamidino-2-phenylindole (DAPI) (Life Technologies), anti CD45-phycoerythrin , and a cocktail of primary antibodies to identify cytokeratin (CK) positive cells . To identify PD-L1 levels, cells were also stained with anti-PDL1 antibody (ProSci Inc), A secondary antibody labeled with Alexa Fluor 647 was used as the fluorescent reporter for the PD-L1 antibody. One set of cells consisting of A549, H1703 and healthy white blood cells (WBCs) were stained along with each patient sample. These additional cells were necessary to normalize for the staining process, antibody batch, and microscope conditions over the length of the study and report fluorescent intensities that could be compared between CTCs from many samples. The staining protocol for PDL1 was optimized to positively stain lung cancer cell lines  or PD-L1 positive. The quantity of PD-L1 expression was further categorized as either low , medium (0.4–0.7), or high (>0.7) as defined in the cutoff values in Fig. In order to quantify PD-L1 expression on CTCs, we developed a semi-automated imaging algorithm using a custom MATLAB script. The script was used to quantify the cell sizes and normalized fluorescence levels for each cell from each patient sample . The intensity signal from each cell was categorized as negative for PD-L1 expression (intensity between 0–0.04) or as positive (0.04–1). Positive cells were further categorized into low (intensity between 0.04–0.1), medium (0.1–0.2) and high levels (0.2–1), as indicated by the cutoff values shown in Fig. 26. Patients were categorized by the overall best response to treatment . The association between CTC and PD-L1+ counts with progression free survival was performed using Cox proportional-hazards models. In order to quantify the effects of interest, hazard ratios (HR) along with 95% confidence intervals (CI) were estimated by the model. All statistical analyses were performed using R V3.1.2  and IBM SPSS V23 . P values less than 0.05 were considered significant.We categorized patients by number of CTCs (<1.32 CTCs/mL or ≥1.32 CTCs/mL) and obtained clinical information  and immune related response criteria (irRC) based on imaging scan data when availableWe first confirmed that CTCs could be enriched from metastatic NSCLC patients with Vortex technology. A total of 22 patients were enrolled in this study, with 31 samples collected, processed with Vortex technology and enumerated for CTCs. 30/31 (96.8%) samples had at least 1 CTC, 15/31 (48.4%) samples at least 10 CTCs, with CTC total counts ranging from 0.1 to 9.67 CTCs/mL of blood Table . BesidesWe then developed a method for quantifying PD-L1 expression on lung cancer cells using immunofluorescence staining. To identify the optimal primary and secondary antibody concentrations, we utilized HeLa cells as a positive control and RBCs as a negative staining control for PD-L1 (Lee) We tested three commercially available PD-L1 antibodies  and determined that the ProSci antibody had the most intense specific staining while maintaining the least non-specific staining , and H3255 (squamous cell carcinoma) to develop and validate the PD-L1 fluorescence immunostaining protocol and quantification algorithms. The H1703 line was found to have the highest overall expression of PD-L1 while H3255 had minimal to no PD-L1 expression . Tumor biopsies prior to treatment were only available for 4 patients from the 22 patients in this study. For these 4, thin sections of tumor were cut from the paraffin block, stained and the resulting PD-L1 levels quantified as described above. Sample images for negative, low, medium, and high PD-L1 staining are shown in Fig. As PD-L1 expression is detectable on CTCs, we wanted to determine whether its detection can be an adjunct predictor for response to PD-1/PD-L1 inhibition. We determined progression using the irRC criteria. To assess the impact of overall CTC count and PD-L1 expression, we counted all CTCs and the subset that were PD-L1 positive Table . We limiAlthough for this initial study, we did not serially collect blood in all patients, we did so for two patients  are correlated with patient response, there is still a need for other ancillary and non-invasive biomarkers that can be used to monitor response to ultimately help guide the treatment course. CTCs have recently gained momentum as a non-invasive liquid biopsy for monitoring of treatment response and as a source of genetic material to understand treatment failures. Although the analysis of circulating cell-free tumor DNA (ctDNA) is also being explored, especially in identifying the presence of known druggable mutations, such an approach is not suited for identifying phenotypic changes such as levels of PD-L1 on tumor cells. There are no known consistent genetic lesions associated with up-regulated expression of PD-L1. Also, a variety of other cell types express PD-L1 (for example macrophages13) and could release protein, extracellular vesicles, or mRNA into the blood stream. CTCs are ideally suited to characterize PD-L1 expression through a non-invasive liquid biopsy in that they are short-lived markers of the active invasive tumors, with holistic phenotypic and proteomic information.Although immunotherapy represents a breakthrough in the treatment of selected cancers, only a fraction of patients respond. In metastatic NSCLC, the overall response rate is approximately 20%This study represents one of the first to examine PD-L1 expression on NSCLC CTCs in the context of anti-PD1/PD-L1 treatment. Some of the newly approved checkpoint inhibitors require a companion diagnostic indicative of tumor positivity for PD-L1 prior to administration . So, in this pilot study, we wished to develop algorithms to quantify PD-L1 expression on CTCs and tumor biopsies respectively and secondly, explore whether CTC PD-L1 expression was correlated with tumor expression. Although PD-L1 expression alone was not predictive of progression free survival, we did note several potential trends in CTC count and PD-L1 expression. Even given a limited sample size, we did note that for patients with >1.32 CTCs/mL, those with >50% PD-L1 + had improved overall response (3 of 4 patients), though most of these patients were also PD-L1+ in their tumor biopsies. Heterogeneity of PD-L1 levels could indicate intra-tumoral or intra-patient heterogeneity, as each of these patients had multiple metastatic sites and the CTCs could break off from any or multiple of these sites.Nevertheless, the trends we find here are suggestive that PD-L1 status on CTCs may track that of tumor tissue and that this may be a useful correlate in helping to assess potential for response to immunotherapy. Our findings indicate that PD-L1 expression on CTCs can be quantified as an adjunct biomarker. This work also suggests that serial monitoring of patient overall CTC count and PD-L1 + CTC count may help to identify changes in response to treatment, and these cells may be a readily obtainable source to understand tumor evolution, work that needs to now be confirmed with larger prospective studies. Future work will examine the time course of CTCs and their PD-L1 levels during immunotherapy treatment as well as how changes in CTC count and PD-L1 expression may correlate with overall response.One limitation of this study or any CTC study in general is that it can be difficult to draw associations with few cells. In many cases, CTCs are present prior to treatment but their numbers can be limited, due in part to previous chemotherapeutic treatment or the fundamental patient-to-patient heterogeneity of tumor behavior. This fundamental issue is exacerbated by practical challenges with the limited number of patient samples that are available from patients on clinical trials for new therapies.27 can be tuned based on the cancer type known to be present.CTC PD-L1 quantified levels, when combined with the tumor biopsy results, could aid in identifying patients more likely to respond to therapy or likely to have become resistant to treatment when tracking levels over time. This study indicates that continued work is warranted to analyze and compare more CTC samples from patients on anti-PD-1 pathway treatment to more robustly determine how PD-L1 expression correlates to tumor levels, fluctuates in response to treatment, and is predictive of response. The methods we describe here are potentially applicable to any tumor type and to potentially any treatment course, as the size-based approach does not exclude cells on the basis of presence or absence of surface markers and the size selectivity criteriaSupplementary Figures


Charge density fluctuations can be measured with orders of magnitude higher precision than previously accomplished using the G-band shift with charge. The two 2D intrinsic peaks can be associated with the “inner” and “outer” Raman scattering processes, with the counterintuitive assignment of the phonon closer to the K point in the KM direction (outer process) as the higher energy peak. Even low charge screening lifts the phonon Kohn anomaly near the K point for graphene encapsulated in hBN, and shifts the dominant intensity from the lower to the higher energy peak.Pristine graphene encapsulated in hexagonal boron nitride has transport properties rivalling suspended graphene, while being protected from contamination and mechanical damage. For high quality devices, it is important to avoid and monitor accidental doping and charge fluctuations. The 2D Raman double peak in intrinsic graphene can be used to optically determine charge density, with decreasing peak split corresponding to increasing charge density. We find strong correlations between the  The 2D Raman mode is of particular interest, as it involves two D band phonons with opposite, non-zero momenta. Despite the higher order process and non-zero momentum phonons, the 2D mode is the strongest Raman mode in single layer graphene. The double resonance (DR) mechanism2 explains why this higher order phonon mode is so strong; resonant virtual electron and hole scattering picks phonon q vectors that satisfy momentum and energy conservation. The DR process together with the strong K-point Kohn anomaly is why the 2D line provides so much information about the electronic and phonon dispersions around the K and K’ points. The phonon Kohn anomaly near K softens the D band phonon energies conically near K, resulting in a discontinuity of the derivative of the dispersion at K3. The slope of the phonon dispersion is a direct measure of the electron-phonon coupling (EPC) strength3. The DR and phonon dispersion are responsible for the shift in the 2D band energy with changing laser excitation energy4. Similarly, the strong dependence of the 2D mode on dielectric screening is readily observable by the shift in the 2D position for different environments, e.g., suspended graphene, graphene on SiO2, graphene on top of hexagonal boron nitride (hBN) or encapsulated in hBN, etc5. Larger dielectric screening6 will reduce the Fermi velocity, vF, so that the DR conditions will select larger phonon energy. The screening affects vF since electron energies are much higher than expected from a single particle picture, with a significant self-energy correction in a 2D material, in particular for free standing graphene near the charge neutrality point (CNP)9. The self-energy is decreasing as ε−1, where ε is the effective dielectric constant of the environment6. Charging the graphene layer will also renormalize and reduce the self-energy and therefore vF. For example, a study of gated graphene on hexagonal boron nitride (hBN) examined with scanning tunnelling spectroscopy demonstrated a change of vF from 1.3 to 1.05 × 106 m/s as the doping increased one order of magnitude from 2 × 1011 cm−2 to 2 × 1012 cm−2 9. Ultraclean suspended graphene with mobility reaching 106 cm2/Vs have shown vF reaching 3 × 106 m/s, measured by Shubnikov-de Haas oscillations7. Screening will also have an effect on the phonon dispersion, as it may decrease the strength of the EPC, and thereby weakening the Kohn anomaly at K, resulting in higher phonon energies with increased screening11. The reduced vF and reduced Kohn anomaly with screening are both expected to lead to a higher energy 2D peak. Hence, changes to the dispersion of the Dirac cone and the phonon branch can be experimentally deduced from the 2D peaks.Raman spectroscopy is a versatile experimental technique for exploring fundamental properties of graphene and characterizing properties such as layer thickness, defect density, charge and strain13. The vectors are found by translating the iso-electronic energy contour determined by half the laser energy at K, with a vector that puts the K iso-electronic energy contour tangentially in contact with the slightly smaller iso-energy contour (EL/2 − hcωD) at K’, shown in Fig. q vectors associated with the 2D Raman peak. While the DR Raman scattering is a two-dimensional process, the strongest contributions come from the high symmetry direction ΓK and KM, so that we can consider the one-dimensional case where electron (hole) scattering from ΓK to K’Γ followed by hole (electron) scattering back from K’Γ to ΓK are referred to as the outer process and the similar process between KM and MK’ as the inner process13, as shown in Fig. K was cylindrically symmetric, the 2D peak would yield two very different phonon frequencies from the inner and outer process since qi > qo where q is measured from the K point with cylindrical coordinates, shown in Fig. 12. Indeed, early in the study of graphene, a symmetric 2D peak for graphene on SiO2/Si was identified as a signature of a single layer14. Later studies showed that the intrinsic 2D line shape of single layer suspended graphene is asymmetric17, but reverts to a symmetric line shape when charge is added by field doping to 2 × 1011 cm−2, indistinguishable in shape from graphene on SiO218. Hence, we can deduce that there is not a perfect compensation of the phonon trigonal warping at low doping condition, and that the two peaks can reveal the behaviour of the inner and outer scattering processes, respectively. In addition, the energy upshift of the 2D peak is indicative of screening due to changes in the Fermi velocity, which has been pointed out previously20, but also due to changes in the phonon velocities12, addressed here.The DR method can be used for mapping out a contour of participating phonon wave-vectors21. The accidental doping in these samples is approximately ~1011 cm−2 or less as deduced from transport measurements22, more than an order of magnitude lower than for graphene on SiO2. We find strong correlations between the 2D peak splitting and the relative peak intensities and linewidths. The decrease of the 2D2 peak energy with charge screening is opposite to expectations. It follows from a crossover, shown in Fig. 11. This is a consequence of screening the Kohn anomaly at K. One has to carefully consider the ratio of the inner and outer Fermi velocities with the ratio of the inner and outer phonon velocities before determining if 2D1 is an inner or outer process. Here we present evidence to suggest that the lower energy 2D1 peak originates from the inner process, based on the calculated stronger intensity from the inner process in the low doping regime12, and linewidth behaviour. The analysis below is based on the assumption that increasing the charge also increases the screening. This assumption is supported by the reduction in Fermi velocity with increasing charge level, especially close to the charge neutrality point, attributed to screening the self-energy correction of the electronic energy dispersion9.Here we are considering the effect of very low doping levels in ultra-clean graphene, dielectrically screened by encapsulation in hBN. Encapsulated graphene possesses many of the superior transport properties of suspended graphene with the added advantage of being mechanically more robust and easier to fabricate2D1 and 2D2 can be used as a measure of charge doping two orders of magnitude lower than is possible to measure using the G band response to doping24.On a practical level, the peak split between 2D1 (blue), and the higher energy peak is denoted 2D2 (black). The 2D peaks measured at different locations have different 2D1 and 2D2 peak separations, linewidths, energies and intensities as shown in Fig. Raman data is collected from a graphene sample encapsulated in hBN. Examples of 2D spectra (λ = 532 nm) are shown in Fig. 25. Figure 2D1 (blue symbols) and 2D2 (black symbols) versus the corresponding G peak position where the different data sets are boxed with their respective laser colour. The red laser selects shorter q vectors through the DR mechanism and therefore has a significantly lower 2D energy than the green laser, as expected. Both data sets exhibit strain lines, indicating some compression26. We first consider Fig. 2D1 peak has a well-defined strain-induced slope of 1.7 ± 0.13, very close to the calculated value for uniaxial strain/compression in a random lattice direction26. The strain variation across the sample is on the order of ±0.1% which is small enough not to cause strain-induced peak-splitting27. The presence of the small strain variation will increase the linewidth only marginally26. The higher energy peak 2D2 does not fall along a single, well defined line. Instead, the data falls mainly in two groups: a lower 2D2− vs G, and a higher 2D2+ vs G. Despite there being two 2D2 groups, there is only one 2D1 line. (Supplementary Information (SI) Fig. 2D2− vs G and 2D2+ vs G are 2.2 ± 0.3 and 3.0 ± 0.4 respectively. The variation and range of the strain slopes matches earlier studies of graphene/hBN sandwiches28. For reference, 2D1 and 2D2 values for λ = 532 nm for freely suspended graphene has been included in Fig. 18.In order to evaluate the range of 2D spectra across the sample, we plot all the data measured on the same sample for two laser lines, λ = 532 nm and λ = 633 nm, in a 2D versus G diagram which reveals the contribution to the peak position due to strain or charge densityper se, but rather the effect of low charge doping on the graphene Raman peaks. The charge present is much lower than can be evaluated with help of a 2D versus G plot25, or by the G band linewidth or peak position24  to the right. Figure 2D1 and 2D2 band versus peak splitting, and Fig. 2D1 and 2D2 evolve as the splitting decreases. Figure Here we are not interested in the strain 11 cm−2 based on the back-gate voltage at the CNP. The charge variation is 0.4 × 1011 cm−2 given the residual conductivity at the CNP (see supporting information section 2). Comparing with our Raman data, which has a mean and standard deviation of −8.1 and 3.6 cm−1 for the 2D peak splitting, we find that for each cm−1 of 2D peak splitting, the doping changes by 0.11 × 1011 cm−2 making the 2D peak splitting a very sensitive probe of local charge density. Extrapolating linearly, we also find from this relationship that the CNP corresponds to a 2D peak splitting of 20.4 cm−1 (see supporting information section 3). While we know the mean and standard deviations for the doping in the sample used for transport measurements with high precision, and we know the mean and standard deviations for the 2D peak splitting for the sample used for Raman measurements, there is sample-to-sample variation which introduces uncertainty in our values for the slope of the linear correspondence and 2D peak splitting at CNP. We estimate a roughly 5% sample-to-sample variation, which translates into uncertainty in charge per wavenumber of 0.11 × 1011 ± 0.02 × 1011 cm−2 per cm−1, and 20.4 ± 1.1 cm−1 for the 2D peak splitting at the CNP.We establish a quantitative relationship between 2D peak splitting and doping by assuming a linear relationship between split and charge, and comparing means and standard deviations between the two. Transport measurements on a similarly prepared sample show that the mean doping level is 1.4 × 102/Si produces optical excitations of defect transitions in hBN and charge transfer to the graphene. In fact, light exposure during gating quickly returns the graphene to the charge neutrality point, undoing the gate doping29. The sign of the charge is also undetermined, but recent measurements30 on ultraclean samples have shown a symmetric electron and hole blue-shift of the 2D peak (single peak fitting) for charge density up to 2 × 1012 cm−2. Hence, the 2D peak response for holes and electron doping is indistinguishable on the Raman spectra at such low charge density. Early gated Raman measurements31 on graphene on SiO2 showed a weak difference between electron vs hole doping below 1012 cm−2 in contrast to the newer easurements30. This illustrates the need for excellent charge homogeneity to observe the low charge density effects achieved on suspended or hBN encapsulated graphene.While it would be advantageous to use electrostatic field gating to confirm, attempts to control the charge level via back-gating samples for optical measurements are complicated due to light-induced charge screening of the gate voltage for graphene on hBN. This is because light illumination on a gated graphene/hBN sample on SiO15, see Fig. q-dependent scattering matrix elements of the phonon intensity have shown that the inner process dominates despite the stronger vector nesting for the outer process13. Our results show the 2D1 peak with the strongest intensity at the lowest doping level. A higher 2D1 intensity has also been observed experimentally previously on both suspended18 and hBN encapsulated17 graphene. Based on the observed and predicted higher intensity for the inner process for pristine graphene, we tentatively assign the 2D1 peak as originating from the inner process, and show below how this assignment is supported by the results. Figure qi is larger than the outer vector qo, but the inner KΓ phonon dispersion is flatter than the steeper outer KM phonon branch32, as illustrated schematically in Fig. 2D1 as the inner process.Generally, a high density of vector nesting creates a stronger Raman peak. This would indicate that the outer process, with flatter parts of the trigonal warping of the iso-energy contour matched by the phonon contour, has a stronger contribution to the Raman spectra than the inner process2D1 to 2D2 with increased charge screening may be an effect of changing wave vectors and phonon energies that will affect both the scattering matrix terms in the numerator, as well as the denominator of the Raman intensity scattering expression12, which will change the relative strengths of the 2D1 and 2D2 peak due to quantum interference. Regardless of the cause for the intensity behaviour of the two 2D peaks, it is clear that added charge to graphene in hBN changes the dominant intensity from the 2D1 peak (inner) to the 2D2 peak (outer) with increasing charge.Intensity behaviour as a function of charge has not yet been explored theoretically. We suggest that the observed switch in intensity from 2D1 and 2D2 components and their linewidths is the ratio of the phonon velocity vph to the Fermi velocity vF for the inner and outer process. Using the DR mechanism and the simplifying assumption of perfectly linear electronic and phonon dispersions around the K and K’ points (but with different slopes for the inner and outer directions)13, and using vph ≪ vF, we have−1) at the K point (q = 0), γ is the imaginary part of the electronic self energy (eV), h is Planck’s constant, and c is the speed of light. The subscripts i and o refer to the inner and outer phonons, respectively. Equations 33, ∂ω/∂EL, of the 2D1 and 2D2 band for “pristine” and charged graphene and use Eqn.  of the inner branch, as expected11.It is counterintuitive that the rge Fig. , since iqi Fig. . The red2D1 and 2D2 are not indicative of the phonon lifetime, but rather determined by the electronic lifetime (imaginary part of the self-energy) and ratio of the phonon to electron velocity. However equation δqi,o. Phonons that fall within the blurred contour make a significant contribution to the 2D peak, and we will regard them as participatory phonons, and the blurred line as the participatory contour. The middle of the participatory contour is mapped almost perfectly to the triangularly warped iso-energy phonon contour with the “corners” pointing towards Γ rather than M. The width of the participatory contour in the KΓ direction (inner), δqi, has been calculated12 to be broader than the width in KM direction (outer), δqo, which provides a method for assigning 2D peaks to inner and outer processes. Using equations  The electronic self-energy is momentum dependent, and the momenta that contribute to the 2D1 and 2D2 peaks are different. Our observations are then consistent with an electronic self-energy that increases more greatly with charge for momenta that contribute to the 2D2 peak than momenta that contribute to the 2D1 peak. 2) The Fermi velocity decreases with charge less dramatically along the direction of the 2D2 process than the 2D1 process. If this is the case, then the 2D2 phonon velocity reduces by smaller amount than the 2D1 phonon velocity, and will result in a 2D2 linewidth that increases more rapidly with charge.Charge affects the 2D linewidth through two primary mechanisms: scattering and phonon velocity renormalization. Eqn.  shows thses Fig. . It is g2D1, has the highest intensity. The screening of the K-point Kohn anomaly with increasing charge has not yet been addressed experimentally on suspended graphene. A significant difference is the substantial decrease in the peak split at the CNP for λ = 633 nm, not observed for suspended graphene where the 2D peak-split stays nearly constant (~13 ± 3 cm−1) over a laser energy range from 1.5–2.7 eV18. This indicates that the dielectrically screened graphene is less well fit with a linear model than suspended graphene: The peak split EL, Berciaud et al.18 dismissed the theory of the peak split originating from the difference in the inner and outer 2D phonons. The phonon linewidth is expected to decrease with lower laser energy, since γeh ∝ EL, and Berciaud et al.18 observed an increasing linewidth for laser line energies below ~1.7 eV for suspended graphene. We note that it is possible that the predicted increase in phonon dispersion slope closer to the K point11, probed with lower laser energies, dominates over the decrease in electronic linewidth, γeh.The results presented here on hBN encapsulated graphene have both similarities and differences to suspended graphene. Both types of samples exhibit a decrease in the 2D double peak split with increased charge, and the lower energy peak, io, Eqn. , which iK-point Kohn anomaly with screening observed here is a sign of the weakened EPC3. Excitation of D band phonons by electrons is a fundamental bottleneck for ballistic transport at high fields35. Hence, it is possible that encapsulated graphene with low and uniform charge screening could have better transport properties at high fields than suspended graphene if the screening sufficiently reduces the strength of the EPC of the D phonons, without introducing other scattering mechanisms.The lifting of the 2D1 and 2D2 peak separation removes influence of strain, and reveals strong correlations between charge, peak intensities, peak positions and linewidths. The peak split can be used to estimate the charge density with sensitivity ~ 1010 cm−2 per cm−1 2D peak split, an improvement of two orders of magnitude compared to use of the G band Kohn anomaly at the Γ point. Hence, the 2D peak split could be very informative for detecting low amounts of charge, with the caveat that the peak split with charge behaviour is known for the particular substrate screening and laser line used. The method also reveals information about the origin of the double peak and the effect of increased charge, for example the intensity shifts from the inner to the outer phonon with increased charge. We associate the lower energy 2D1 peak with the so called inner process, and the higher energy 2D2 peak with the outer process. Even the low amount of charge puddling found in these samples is enough to significantly alter the strength of the D band Kohn anomaly at the K point.In conclusion, we have used Raman spectroscopy of graphene encapsulated in hBN to explore the intrinsic double peaked 2D phonon behaviour under low charge screening. The analysis of the 2D data as a function of 21. This particular sample did not have functioning edge contacts, but similar samples have a room temperature mobility of ~3.2 × 104 cm2/Vs.The encapsulated graphene sample was fabricated at Columbia University by Carlos Forsythe with their pick-and-place method that avoids direct contact between the pick-up polymer and graphene layer or the accompanying hBN surfaces that contact the grapheneEL = 2.33 eV) and red laser with wavelength of 633 nm (EL = 1.96 eV), are applied at 1 µm by 1 µm spacing with a laser spot of approximately 1µm2 with typical spectra shown in Fig. The collected 2D Raman spectral map is fitted with two Voigt peak functions at all different locations on the hBN encapsulated graphene. Different incident laser photon energies, green laser with wavelength of 532 nm (The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Info