An efficient route to synthesize macroscopic amounts of graphene is highly desired and bulk characterization of such samples, in terms of the number of layers, is equally important. We present a Raman spectroscopy-based method to determine the typical upper limit of the number of graphene layers in chemically exfoliated graphene. We utilize a controlled vapour-phase potassium intercalation technique and identify a lightly doped stage, where the Raman modes of undoped and doped few-layer graphene flakes coexist. The spectra can be unambiguously distinguished from alkali doped graphite, and modeling with the typical upper limit of the layers yields an upper limit of flake thickness of five layers with a significant single-layer graphene content. Complementary statistical AFM measurements on individual few-layer graphene flakes find a consistent distribution of the layer numbers. Apart from individual graphene flakes, bulk graphene is exploited in numerous systems8. Notably, bulk single-layer and few-layer graphene (SLG and FLG) are proposed as applicable in efficient Li-ion batteries, components of photovoltaic cells, and are viable candidates for spintronics applications10. Thus, scalable methods are required both for high-yield production and characterization techniques. One of the major challenges, i.e., to establish mass-production techniques leading to high-quality SLG and FLG was overcome in recent years12. Amongst the numerous synthesis (mostly top-down) means towards mass graphene production, wet chemical exfoliation methods prevail in terms of material quality and synthesis facility16. Nevertheless, measurement methods suitable for large sample quantities are still lacking.Graphene, the latest discovered carbon allotrope19. It was demonstrated to be an ideal characterization tool not only in the laboratory but also at the mass-production level. In particular, investigations of single-layer graphene are facilitated by the lack of a band-gap in the band structure leading to resonant processes at all wavelengths20. Details of the Raman spectra can reveal the layer number of single graphene flakes or identify the edge of the flakes24, it also enables detailed studies of electrostatic gating, strain, or chemical modifications of graphene36.Raman spectroscopy evolved to be an essential probe for studies of carbon structures especially of nanocarbon38. Bulk analysis of the number of the layers of unmodified bulk graphene is not feasible by optical means. Raman spectra of SLG and FLG single flakes differ significantly in their 2D mode (the overtone of the defect-induced D mode), which provides a way to identify the number of graphene layers, N39. However, twisted multilayers might have 2D modes resembling SLG20. Furthermore, the methods reviewed in Ref. 40, i.e. the so-called C peak and the interlayer breathing modes43, the ZO’ modes. However, this cannot be applied for powder samples built up of a distribution of different thicknesses.Historically, Raman, phase contrast spectroscopy and AFM studies focused on the characterization of separated graphene flakesHere, we present a compelling analysis method based on Raman spectroscopy for analyzing the typical upper limit of the number of graphene layers in a powder sample. We utilize controlled vapour-phase potassium intercalation to distinguish SLG and FLG content through following a stepwise intercalation process. This technique enables us to track the evolution of doping in lightly doped stages, where the Raman modes of the undoped and the doped FLG flakes coexist. The applicability of this intercalation method is demonstrated on a few-layer restacked graphene, poly-dispersed powder prepared by wet chemical exfoliation. The method reveals that the studied samples are composed of graphene flakes with dominantly less than five layers. AFM statistical measurements on individual SLG and FLG flakes of the same material confirm the observed distribution.μm surface, revealing a distribution of flakes on the surface. Representative AFM images of the graphene flakes are presented in Fig. We performed a detailed AFM statistical analysis study on a large number of as-prepared ultrasound treated individual FLG flakes in a single batch chosen randomly. Figure Figure 45 that this approach may be misleading due to the improperly chosen measurement parameters, and complementary studies are required. In particular, partial restacking of the chemically exfoliated graphene flakes on the substrate may lead to bigger aggregates of multiple layers where the graphene sheets are misaligned. AFM, however, is unable to resolve this change of the flake morphology, and cannot identify the thickness of individual flakes.Although AFM-based thickness measurement of individual flakes is a standard method for graphene characterization, it was suggestedμm lateral and vertical resolution, which is large enough for a representative surface average without the biasing effects of nano-imaging.This diversity of the flakes highlights the need for bulk characterization methods, such as Raman spectroscopy. Micro-Raman spectroscopy in our case has about a 5 … 10 46. The corresponding Raman spectra (recorded at 514 nm) are depicted in Fig. 33. The 2D band of the starting material is best fitted with a single Lorentzian line, unlike the composed structure in graphite. Whereas the width of the Lorentzian hints that the material may be a mixture of flakes with different number of layers, the Raman response of the starting material is insufficient to determine the exact distribution of the thicknesses. Here, it is worth to note that FLG flakes are stable on their own (i.e. self/standing), no substrate was used during the Raman measurements.Starting from undoped FLG, we performed controlled temperature-gradient driven potassium doping experiments. Saturation doping was achieved in approximately 10 steps. We intentionally refer to “steps” in our experiments rather than “stages”, as the latter is reserved for the well-known intercalation stages of bulk graphiteinhomogeneous Step 3 and the homogeneous Step 5 reveals that only the intensity ratio of the G modes changes, the position of the 2D modes is unchanged. Step 5 is of particular importance, as this was found to be a stable phase. The highest doping level , we used each time twice longer intercalation times than at the previous Step. In the meantime, we kept a constant and large temperature gradient to homogenize the material at a small and fixed doping level. Comparison of the 8), where similar Raman bands appear upon intercalation. A detailed analysis is given in the Supplementary Materials, and it indicates that the position, the width (ΓFano), and the coupling strength of the electronic continuum, measured by the asymmetry parameter, q, differ. The Cz mode arises from from a vibration, where carbon atoms displace perpendicularly to the graphene sheets. Even though this mode exists at the M point of the Brillouin-zone, it is folded back to the Γ point due to a 2 × 2 doubling of the unit cell during intercalation50. The presence of the Cz mode is a clear indication of a 2 × 2 ordered potassium lattice present on the graphene sheets, or between the graphene layers50. As a consequence, this mode is naturally present in Stage I KC8 and is a clear indication of successful and high doping yield.It is intriguing to compare this spectrum with the Raman spectrum of Stage I potassium intercalated graphite  GIC. Nevertheless, the spectroscopic details are markedly different.The most surprising observation in Fig. 72. Although the doublet structure of the G mode appear similar, two details are different: (i) the G mode with the larger Raman shift lies with about a 6(1) cm−1 difference in the two types of materials, (ii) the 2D mode is markedly different in the two kinds of materials: the FLG contains a 2D mode component with a smaller Raman shift, which is absent in graphite. Albeit these difference may appear to be subtle, these enable us to qualitatively differentiate between the two types of materials. The figure also shows that Step 5 intercalated FLG can be resolved into a mixture of a Stage 3 GIC and the undoped material. This fitting procedure  is capable of explaining both the position of the G mode and the composite structure of the 2D mode. This indicates an interesting scenario for the Raman spectrum of the alkali intercalated FLG: it consists of a mixture of (1) entirely undoped pieces, whose Raman spectrum remains identical to that of the starting material, and (2) relatively highly doped phases .In Fig. 51. In this respect, charges in GIC and our stepwise doped FLG are both inhomogeneously distributed, however, the inhomogeneity is completely different. Intercalation in graphite proceeds from homogeneous and crystalline graphite and the charging inhomogeneity is due to the intercalation itself: it occurs due to the thermodynamic preference for fully doped alkali layers which are inevitably separated by uncharged graphene layers. However in our FLG material, the inhomogeneity is present a priori in the sample (in terms of the different layer numbers in the grains) and the inhomogeneous doping merely reflects this inhomogeneity as we show below.We emphasize that the origin of the doublet structure in GIC is related to the presence of charged and uncharged graphene layers; graphene layers in GIC, which are adjacent to an alkali layer are charged, whereas two which are further apart, remain neutral or uncharged1 around 1580 cm−1 and G2 around 1600 cm−1) are shown in Fig. c band) and uncharged layers (the Guc band), respectively51. Upon doping, the Gc band moves to lower Raman shift beyond experimental error  remains low and varies with stage numbers of the GIC. We note that the Guc band also shifts slightly between the different stages due to strain effect.To gain deeper insight into the composition of the multiple restacked FLG, we analyze the G- and 2D Raman-bands. Intercalation step dependence of the split G-bands  independent of the doping steps, ii) its Raman shift position lies between the position of charged G-band in KC24 and in KC36. This is a strong indication that the G2 band corresponds to graphene layers that appear to be doped as in Stage 2 or 3 graphite. It also means that in our FLG samples, no higher stages (or lower doping levels) can be achieved. Given the heterogeneous nature of the number of layers in the FLG sample, this reveals that our sample is free from flakes with more than 3–5 restacked graphene layers. This observation is in full agreement with our AFM statistical analysis.In FLG, the Gsee Fig.  unveils 1-line barely changes as a function of doping as long as a Lorentzian line fits best the Raman line. This exposes that the induced strain is not affected by the doping level, hence, the G1 line corresponds to a significant amount of undoped flakes. Presence of these undoped flakes along with appearance of Stage 3 doping in five-layer-thick flakes highlights the important contribution of undoped flakes with smaller thickness .Figure ωFano is the measured position of the G-line peak, ωA and ωNA are the calculated adiabatic and non-adiabatic phonon frequencies52. We approximate the latter two quantities with the ones calculated for KC8: ωA = 1223 cm−1 and ωNA = 1534 cm−1, as no exact calculation exists for FLG. This approximation was found to be valid in similar hexagonal carbon systems such as potassium doped multiwalled carbon nanotubes53.To further emphasize the differences between the FLG and the graphite powder, we extract a measure of the charge transfer, the electron-phonon coupling parameter (EPC). The electron-phonon scattering linewidth can be estimated from the positions of the Fano lineshape using the expressionFano is the linewidth of the Fano lineshape. In accordance with previous findings in GICs52, the γEPC of SGN18 and FLG follow the linewidth of the Fano lineshape linearly  doping yields a formation energy gain (ΔF) that decreases for lower stages up to Stage 2, and increases for Stage 1. This behavior is confirmed by calculations for all thicknesses with a layer number n ≥ 3. This staging phenomenon means that all flakes of the sample reach the same stage before a new stage is started to be formed, independently of the layer number. The same effect was found experimentally in bilayer graphene individual flakes, i.e., that the doping occurs first on one of the layers reaching a full Stage 2 doping  before accumulating in-between all layers59.This scenario is supported by first-principles studies2 peak has to be compared with that found in GICs. Secondly, the 2D peak has to be fitted to a combination of Lorentzian curves. The position of the lowest fitted Lorentzian curve must be correlated to the highest stage possible in the material.A practical protocol for the use of the present method is as follows. The chemically exfoliated graphene being studied needs to be intercalated with potassium in a stepwise manner along the protocol, which we present in the Methods Section. Clear evidence for a material, which consists of monolayer graphene only, is when the stepwise doping steps result in a fully intercalated material, without the presence of the intermediate stages (e.g. the G mode shifts continuously). When the intermediate steps are present, the typical upper limit of the layer number can be determined in three steps. First, the position of the GNote that for samples with flakes with a large fraction of 10 or more layers, our method is only capable to prove that the sample contains multilayer flakes.Here, we limited ourselves to determine the typical upper limit of the distribution of the number of layers in chemically exfoliated graphene powder samples. However, further development of our analysis of the Raman spectra could yield more information about the precise statistical distribution.61. Recently, multilayer graphene flakes with ABC stacking were successfully isolated exceeding 17 graphene layers62. This stacking could be identified using Raman spectroscopy as described in recent works62. Specifically, first-principles calculations demonstrated that the 2D band is broader, and it has several components for ABC stacking. In addition, a shoulder emerges at 2576 cm−1 for 1.96 eV laser energy. These Raman fingerprints would be all the more relevant to study due to the interesting arbitrary restacking previously observed in few-layer graphene flakes64.It would be of interest to investigate the stacking of the graphene layers using an extension of our Raman spectroscopy-based technique. Graphene multi-layers typically exhibit two structures of stacking: the more stable AB  stacking and the less stable rhombohedral, ABC stacking. The rhombohedral stacking could lead to promising correlated states displaying superconductivity and magnetismin-situ K doping of FLG samples. Our method uses the combination of the G-band position, its intensity, and the position of the 2D mode components to determine the typically thickest flakes in the sample and to confirm the presence of single-layer ones. The technique works well on FLG powder samples prepared using wet chemical exfoliation technique and was tested for three different mechanical processing routes. Statistical AFM shows that such samples consist of flakes with a non-uniform distribution of the number of graphene layers, and 90% of the flakes consist of mostly less than 5 layers. The Raman spectra of intermediately doped FLG samples can be best described as a sum of two components, corresponding to doped and undoped graphene flakes. The former was argued to arise from five-layer-thick flakes and the latter from flakes made of fewer numbers of graphene layers. Remarkable agreement of our AFM statistical analysis and our Raman data validates our method. Our method provides an efficient way to characterize graphene samples with an arbitrary distribution of the number of graphene layers, where other alternatives  fail.In conclusion, we presented a Raman spectroscopy-based technique to identify the maximal flake thickness in few-layer bulk graphene samples. The presented method is based on studying 66 and as shown in Fig. 67. Further details of the sample preparation are described in Ref. Few-layer graphene samples were prepared from saturation potassium doped SGN18 spherical graphite powder (Future Carbon) using DMSO solvent for the wet chemical exfoliation as described elsewhereμm surface of a Si/SiO2 wafer. Unlike the samples used for Raman studies, only partial restacking may occur due to drop-casting and presence of the substrate in this configuration used for AFM studies. These measurements were carried out using a Bruker Dimension Icon microscope in tapping mode. Bruker Scanasyst-Air silicon tips on nitride levers with a spring constant of 0.4 N/m were used to obtain images resolved by 512 × 512 pixels.The as-prepared bulk samples were characterized by atomic force microscopy (AFM) topographic measurements and images to determine the flake size and the typical number of graphene layers in the bulk material. For AFM statistical analysis, the samples were drop-casted on a 100 × 100 68. We first examined few, incompletely exfoliated nanosheets, which showed clear terraces. As the terrace step heights are multiples of the apparent thickness, we were able to identify the individual layer thickness to be ~1.2 nm.To overcome the difficulties of the determination of the individual layer thickness due to residual solvent, capillary forces, and adhesion, we took advantage of the frequently used step-height analysis procedurein situ under a vacuum of 4 × 10−8 mbar pressure. To perform the in situ intercalation process, we used a similar setup to the one presented and described in Fig. 1 of Ref. 46. Due to lack of substrate, significant restacking of individual graphene flakes occurs, leading to misaligned layers in the sample. The powder sample consists of sponge-like structures, as thermodynamic equilibrium is achieved to create a three-dimensional solid material. As a result of the described sample morphology, Raman measurements provide detailed information not only of single graphene flakes but a large ensemble of flakes that are restacked. As restacking creates only mechanical contact, electronic properties of individual flakes are preserved. Thus, the staging phenomenon necessary for our Raman-based technique occurs at the level of individual flakes.Potassium intercalation was carried out Raman measurements were performed at 458, 514.5, and at 568 nm laser excitation wavelengths. To facilitate our discussion, our manuscript focuses on the observations at 514.5 nm wavelength, only the statistical analysis of the G-band positions in Fig. 69. Potassium with a purity of 99.95% (Sigma-Aldrich MKBL0124V) was evaporated to the sample in several steps in a controlled in situ process. At the first step, the potassium was evaporated for about 2 minutes. In the following steps, we used each time twice longer intercalation times than at the previous Step with constant temperature gradient until homogeneous doping was concluded through Raman measurements of the intensity ratio of the G modes .A power of 0.5 mW was used to avoid laser-induced deintercalation71. The spectrometer was operated with a 1800 grooves/mm grating. A typical 0.5 mW laser power was used with a built-in microscope (Olympus LMPlan 50 × /0.50 inf./0/NN26.5).Following each intercalation step, Raman spectra were recorded on a modified broadband LabRAM spectrometer (Horiba Jobin-Yvon Inc.). The built-in interference filter was replaced by a broadband beam splitter plate with 30% reflection and 70% transmission. The principles of the broadband operation are described elsewhereSupplementary Material


A novel, ultrahigh-sensitivity wide-range resonant micro-accelerometer using two differential double-clamped monolayer graphene beams is designed and investigated by steady-state simulation via COMSOL Multiphysics software in this paper. Along with stiffness-enhanced optimized folded support beams, two symmetrical 3-GPa prestressed graphene nano-beams serve as resonant sensitive elements with a size of 10 μm × 1 μm (length × width) to increase the acceleration sensitivity while extending the measurement range. The simulation results show that the accelerometer with cascade-connected graphene and proof-mass assembly exhibits the ultrahigh sensitivity of 21,224 Hz/g and quality factor of 9773 in the range of 0–1000 g. This is remarkably superior to previously reported studies characterized by attaching proof mass to the graphene components directly. The proposed accelerometer shows great potential as an alternative to quartz and silicon-based resonant sensors in high-impact and highly sensitive inertial measurement applications. Due to their direct frequency signal output, stable performance, excellent repeatability and high sensitivity, resonant accelerometers as an important development of inertial area have been widely applied in aviation, aerospace and automation fields. The resonance frequency-dependent resonant sensitive element is a key part of resonant accelerometers. At present, resonant sensitive elements are mainly composed of precision transverse elastic alloy, monocrystalline silicon, fused quartz and piezoelectric quartz crystal. Resonant accelerometers using these materials generally demonstrate low sensitivity and large sizes ,2,3. TheQ), and then compared the results with the theoretical model of a vibrating beam under tension which indicated the feasibility of resonant graphene sensors [Graphene is a two-dimensional material with a single-layer thickness of 0.335 nm, which is the thinnest material known up to now . Since m sensors . Chen et sensors . Oshidar sensors . Recentl sensors ,16. Addi sensors .15 g level, which is improper for practical applications. Byun et al. also attached micro mass (4.0152 × 10−22–4.0152 × 10−20 g) to graphene nanoribbon to sense acceleration and examined the linear relationship between acceleration and resonance frequency on a log–log scale [2 [Due to excellent resonant characteristics, graphene resonators have been explored to measure the acceleration. Kang et al. investigated highly sensitive resonant accelerometers based on graphene nanoribbon via classical molecular dynamics simulation. They pointed out that the acceleration was closely related with structural parameters and built-in strain of graphene nanoribbon ; howeverog scale . Unfortuog scale . Note thscale [2 . NeverthQ of 9773 in the defined range of 0–1000 g, which is significantly higher than the previously reported research where Q values were confirmed as 1000 [Therefore, in this paper, we propose an architecture of differential resonant accelerometer by sensing graphene beams’ stretching applied by a proof mass. The COMSOL Multiphysics steady-state simulation of the proposal accelerometer without proof mass attached on graphene exhibits the acceleration sensitivity of 21,224 Hz/g and  as 1000 , 7000 [1 as 1000  and 9000 as 1000 , respecty-axis) of accelerometer, and two grooves for placing graphene resonant beams. As shown in y-axis direction. In other words, the fabricated graphene resonant beams operate under double-clamped boundary condition. In this case, the substrate and sensitive mass plate could be fabricated by thermal growth oxidation [xidation ,28, and xidation .In particular, the designed resonant sensing structure is symmetrical along the center of accelerometer, thereby making the whole sensitive structure highly symmetrical. Combined with high axial stiffness and low cross-sensitivity of folded support beams, the sensing structure can fundamentally eliminate the sensitive mass block’s movement caused by the acceleration in other directions and the rotation around other sensitive axes. More importantly, it can guarantee not only high acceleration induced displacement sensitivity and smaller off-axis crosstalk, but also high resonance frequency. Furthermore, the differential resonant structure can enhance the signal intensity and improve nonlinear characteristics of graphene accelerometer, which contributes to increasing the sensitivity and measurement accuracy, as well as better robustness to the interference resulting from conjugate suppression and compensation effect.y-axis) of the sensitive mass plate in vacuum environment. The measured acceleration imposed on sensitive mass plate is converted to a concentrated force by the folded support beams in the sensitive mass plate as shown in Referring to the model in mined as (2)K=2EhThe displacement caused by the folded support beams will result in a movement in one end of the DCGB and then lead to axial stress changes in the graphene resonant beams. The resonant frequency (ribed as (4)fR=n2y-axis) of accelerometer. The end of the DCGB connected to the sensitive mass block is free and the other end is clamped. Assuming the density of graphene (v) are 2208 kg/m3, 1.1 TPa and 0.41, respectively. In this case, we apply a concentrated force load to the sensitive mass block in axial direction (y-axis) to simulate the concentrated force induced by acceleration.The model is imported to COMSOL Multiphysics by utilizing “Livelink for Inventor” module, wherein the initial values of structural parameters of designed accelerometer are listed in Graphene beam is a key element of resonant accelerometer. In view of the aspect ratio of graphene, the module “Membrane Interface” is selected in COMOSL, which is exclusively oriented to prestressed-ultrathin membrane simulation. Then, the module “Swept Mesh” is established by regulating the number, size and distribution of mesh in the thickness direction of membrane, thereby ensuring the computing integrity and accuracy of simulation. It is worth noting that, currently the COMSOL simulations have been widely adopted to investigate mechanical, optical and thermal behaviors of graphene-based beam, coating and ribbon with single-layer, few-layer or multi-layer thickness ,32,33. FAccording to Equations (1) and (2), the dimension of folded support beam has an influence on the equivalent stiffness  for DCGB with zero bending stiffness is solved by using a density value ρ (ρ = 2208.96 kg/m3) larger than theoretical value grapheneρ (grapheneρ = 2208 kg/m3) in this paper. Therefore, there is a small deviation existing between the simulation and theoretical results. That is, the former is slightly higher than the latter, as shown in 10 N/m2 [7 N/m2 to 1010 N/m2. It should be mentioned that, as depicted in inset in 9 N/m2 (3 GPa). For ensuring the DCGB under compression to work in resonant status with high natural frequency (1.954 × 107 Hz) for the proposed accelerometer, the prestress of the designed graphene beam is confirmed as 3 × 109 N/m2 (3 GPa). The prestress of DCGB could be applied by improving the surface roughness of insulating layers and electrodes [Given the resonant frequency shift closely depends on the prestress of graphene beam, a proper prestress should be determined. We investigated the frequency variations with the prestress ranging from 10n et al. , the twoectrodes ,18 or emy-axis). The sensitive mass block in the center is driven into motion and causes the deformation of two support beams and two DCGBs, resulting in a distinct graphene beams’ resonance frequency change of 0.75 × 107 Hz compared to natural frequency (1.95 × 107 Hz).By utilizing the parameters as defined above, we further study the input-output performance of resonant accelerometer in response to measured acceleration. As shown in aforementioned Based on the established simulation model, we can achieve the resonance frequency variation of two DCGB's as a function of acceleration. From Q means stronger ability to resist external interference, better working stability and easier detection of resonance frequency shift. Q factor can be calculated by 7 Hz, which is consistent with the simulation result mentioned above. The corresponding calculated Q value is 9773, which is by far larger than the previous results obtained at room temperature, such as a Q factor of 1000 for the epitaxial graphene resonator in Reference [Q factor of 3000 for the drum graphene resonator in Reference [Q= 7000) in identical dimension order (μm) [Q factor accordingly [The quality factor is one of the important parameters for resonant sensors, which is strongly related to the energy loss speed in each oscillation cycle. For resonant accelerometer, higher eference  and Q faeference . It is ader (μm) . In thisder (μm) . In addider (μm) ,20,21, sordingly ,37.To sum up, the simulation results in this study demonstrate the excellent performance of the suggested differential resonant graphene accelerometer, such as ultrahigh sensitivity, high resonance frequency and wide acceleration range, owing to the excellent mechanical properties of graphene with high prestress, non-proof mass sensitive structure and optimized folded support beam design. It is worth noting that, although the simulation analyses in this paper are performed under non-damping and room temperature conditions, the results obtained herein could provide great potential in applications of high-g accelerometers wherein the thermal and damping effects would be considered.9 N/m2 (3 GPa) based on simulation analysis and theoretical model. The resonant response simulation of the designed accelerometer demonstrates an ultrahigh sensitivity of 21,224 Hz/g, which is significantly better than the results reported in previous studies. Furthermore, a superior Q factor of 9773 is achieved, which well represents the accelerometer’s excellent stability and anti-interference ability. Therefore, these effective efforts validate the advantages of resonant graphene accelerometer over conventional quartz and silicon devices in high-g measurement and provide potential applications in the field of NEMS inertia measurement.An ultrahigh-sensitivity, differential resonant graphene accelerometer which can offer high-g acceleration measurement is designed and investigated via COMSOL Multiphysics in this paper. The simulation results agree well with the theoretical resonance frequency in consideration of prestress in graphene beams, thus verifying the applications of the graphene accelerometer. In order to enable both of the two graphene beams in resonant condition, the prestress of graphene beams is set as 3 × 10


The negative colossal magnetoresistance (CMR) of manganite-cobaltite and positive one of graphene gives the possibility to increase the sensitivity to magnetic field of the hybrid sensor. The performed magnetoresistance (MR) measurements of individual few layer (n = 1–5) graphene structures revealed the highest MR values for three-layer graphene (3LG), whereas additional Co-doping increased the MR values of nanostructured manganite films. The connection of 3LG graphene and Co-doped magnanite film in a voltage divider configuration significantly increased the sensitivity of the hybrid sensor at low and intermediate magnetic fields (1–2 T): 70 mV/VT of hybrid sensor in comparison with 56 mV/VT for 3LG and 12 mV/VT for Co-doped magnanite film, respectively, and broadened the magnetic field operation range (0.1–20) T of the produced sensor prototype.The demand to increase the sensitivity to magnetic field in a broad magnetic field ranges has led to the research of novel materials for sensor applications. Therefore, the hybrid system consisting of two different magnetoresistive materials – nanostructured Co-doped manganite La MR) sensors are widely used in daily applications1, for example, in information storage, where the data are received from a magnetic hard disk with MR read sensor that is very sensitive to low magnetic fields3. A group of materials, so called mixed-valence manganites, Ln1−xAxMnO3 7 are the materials that have been intensively investigated for magnetic sensor applications due to the Colossal Magnetoresistance (CMR) phenomenon10. Indeed, manganite-based MR sensors, consisting of nanostructured thin film deposited on a polycrystalline substrate were already successfully developed11 measuring the magnitude of magnetic field independently of its direction 12. The demand for such MR sensors is increasing rapidly due to the development of advanced scientific and industrial devices and techniques such as non-destructive pulsed-field magnets, electromagnetic launchers, forming and welding, contactless high current measurement technique, etc. However, several existing problems concerning MR sensors have to be solved, like: (i) MR anisotropy (MRA) of nanostructured manganite films at fields <0.3 T (limited by demagnetization or ‘shape’ effect)13, (ii) sensitivity to magnetic field at temperatures higher than room temperature (limited by Curie temperature)14, and (iii) sensitivity at fields close to megagauss which is limited by MR saturation of manganites11. These problems are related to a very rich and complex physics of manganites (due to the electron-lattice and electron-electron interactions in these films) which then leads to the close relation of their structural, electronic and magnetotransport properties, therefore, the investigation of new manganite materials and innovative concepts for such sensors is of highest importance.Magnetoresistive  sensor based on combination of a single layer graphene (SLG) and La0.82Sr0.18MnO3 manganite film was recently proposed and investigated up to 2.3 T20. In the mentioned paper, the hybrid single layer graphene/manganite sensor showed the increased sensitivity in comparison to the individual graphene and manganite sensors. Therefore, further investigations concerning optimization of the magnetoresistive properties and the efforts to increase the sensitivity of these hybrid GM devices are of great interest.At this point, two-dimensional graphene has become a material of scientific and technological interest, due to remarkable electronic, mechanical, and thermal properties making graphene a highly attractive material for a large number of different applications1−xSrx(Mn1−yCoy)zO3 films with partial Co substitution for Mn, as well as the results of a novel hybrid manganite-cobaltite/three-layer graphene sensor prototype are reported.In this study, the comprehensive investigations in magnetic fields up to 21 T of individual graphene structures consisting of few graphene layers and nanostructured manganite La2O3 substrate by wet chemical etching procedure (see Methods). A subsequent transfer of single layers of graphene and placement on top of another graphene layer resulted in few-layer graphene samples. The resonance Raman spectroscopy provides rich structural information on graphene-based materials24. It was demonstrated that the intensity ratio of the 2D band to G (I2D/IG) can be used to probe the number of graphene layers27. Figure 2O3 substrate. The characteristic Raman feature for a single layer graphene is enhancement of I2D/IG ratio . For our studied samples the I2D/IG ratios were found to be 2.48, 1.44, and 0.47 for SLG, 3LG, 5LG, respectively. This is consistent with previously reported data28. In addition, slight G peak frequency downshift was observed with increasing number of graphene layers indicating that I2D/IG ratio trend is associated with changes in number of graphene layers26. The ratio ID/IG sensitively probes the defects in graphene23. For our studied samples the intensity of D band was found to be very low (ID/IG = 0.062 for single layer) indicating high structural quality of the graphene films.Firstly, the structural quality of the prepared single and few-layers graphene was examined. The commercially available CVD grown single layer graphene (SLG) was transferred from Cu foil on AlHowever, the XPS analysis revealed the possible organic contamination with the increase of graphene layers. The residuals of PMMA polymer, used as support layer for the transfer of graphene, could explain the organic contamination which was confirmed by surface sensitive XPS measurements and Auger C KVV spectra. The main component 1 in the C 1 s photoemission spectra see Fig.  at bindi29. Despite the D parameter change to graphitic value of 21.2 eV for 5LG sample, the dominant presence of graphene is determined. XPS and XAES results revealed the existence of polymer traces with the increase of graphene layers, probably due to incompletely removed PMMA, captured at the interface between graphene layers.The characteristic D parameter determined from XAES measurements was in the range of 13.0−13.9 eV see Fig.  for almovs. magnetic flux density for different number of graphene layers (1–5) is presented in Fig. MR behavior of graphene samples was investigated using Corbino disk configuration . This geometry eliminates any Hall voltage contribution and allows achieving the maximal values of longitudinal magnetoresistance (caused by Gauss effect) in graphene. The manetoresistance was determined from the following equation at a certain magnetic field (magnetic flux density B): MR(%) = [(R(B)-R(0))/R(0)] × 100%, where R(0) – resistance in zero magnetic field and R(B) – resistance in magnetic field. The observed MR values subsequently increased for the SLG, 2LG and 3LG and started to decrease for 4LG, 5LG samples, in the investigated magnetic field range  and depends strongly on the number of layers, on the stacking sequence 31 or stacking in rotational disorder between the placed graphene sheets, resulting in turbostratic graphite. The significant influence of graphene stacking sequence on the properties of the system was noticed in the field-effect transistor configuration which was used with changeable gate voltage31. Liao et al.32 observed the reduction of MR of 3LG in comparison with 2LG, with the current applied perpendicularly to graphene. The authors obtained inconsistent decrease of the MR with the increase of number of graphene layers (up to 10) and explained it by decrease of charge carrier mobility evaluated from quadratic magnetoresistance dependence on magnetic flux density MR~B2. In our study, in comparison to ref.32 the current flows in-plane. However, the change of carrier mobility could be also the most probable reason for the MR decrease in multi-layer graphene. We propose, that the grain boundaries/impurities (due to graphene growth and transfer processes) in graphene are acting like scattering centers for charge carriers and reduce their mobility. The vertical transport of charge carriers at graphene/graphene interface allows to “overcome” different inhomogeneities and leads to the reduced electrical resistance and increased magnetoresistance in the few-layer graphene structure. The carrier mobility (µ) values in graphene layers were extracted from the fitting the quadratic part of MR dependences of the experimental curves (MR~(μB)2) presented in Fig. 2/Vs for SLG; 1530 cm2/Vs for 2LG; 2420 cm2/Vs for 3LG; 1790 cm2/Vs for 4LG and 1780 cm2/Vs for 5LG. It is obvious, that three-layer graphene exhibits highest mobility values. High μ values could be explained by small number of defects which is confirmed by intensity of the D-band in Raman spectra  it is possible to induce a large non-saturating linear magnetoresistance, desirable property for practical applications32. Linear MR has been observed in multilayer graphene35, in exfoliated36 and in epitaxial bilayer graphene19 up to 62 T. Our study agrees well with the literature data since we also observe linear magnetoresistance dependence on magnetic flux density. The interesting point is that the region of quadratic (B2) dependence of magnetoresistance differs depending on the number of the stacked graphene layers. In our experiments, in the sequence of SLG, 2LG and 3LG, the quadratic part of MR decreases  and the linear part starts at lower and lower magnetic field. The reduction of quadratic MR could be explained by the interlayer tunneling of charge carriers in few layer graphene in comparison with SLG37. For the multi-layer graphene (4LG and 5LG) the quadratic parts increase again in comparison with 3LG up to 1 T, but still has linear dependence of the MR with higher values in comparison with SLG  magnetic field, the single- and three-layer graphene were further investigated in pulsed magnetic fields up to 21 T. The experiments revealed that room temperature MR of three-layer graphene increased by 2 times (up to 758%) at 21 T . Moreover, the 3LG showed the MR increase in all investigated range starting already from very low magnetic field of 0.1 T.According to the observed 1 T Fig.  in compaMR of 60% at 14 T magnetic field and at room temperature was reported32. In our study, the MR of 300%, at the same 14 T magnetic field, was achieved for transferred SLG. Nevertheless, one has to be aware by comparing the results of different groups since the MR values depend on the growth/production method of graphene, number of the graphene layers, supporting substrate and configuration of the measurement system15. In the case of 2LG graphene Kisslinger and others19 observed MR of about 800% at 21 T and 300 K, which is in a good agreement with our results. Additionally, our results demonstrate that magnetic field sensor consisting of 3LG (instead of SLG) could lead to higher sensitivity to the magnetic field in the range of [0.1−20 T].In comparison to the CVD grown few-layer graphene, the 39 and have tendency of saturation at high magnetic fields11. Therefore, the idea to combine two materials (graphene and manganite) having different magnetoresistance behavior was realized. In order to increase the MR of lanthanum manganite (La1−xSrxMnO3), the La1−xSrx(Mn1−yCoy)zO3 (LSMCO) films were grown by Pulsed-Injection Metal-Organic Chemical Vapor Deposition (PI MOCVD) technique on polycrystalline Al2O3 substrate. The films grew either by layer-then-island or by island growth mechanisms resulting in the polycrystalline structure with the crystallite size varying from 70 up to 120 nm (nanostructured film) and surface roughness of 18−19 nm  exhibit higher resistivity and magnetoresistance values at room temperature43 thus, the substitution of Co for Mn in manganite films could increase the sensitivity of the sensors to magnetic field. Figure MR(%) = [(R(B) − R(0))/R(0)] × 100%. Acording to our results, the certain content of Co (y = 0.14) increased the MR from 55% to 63% at 21 T magnetic field in comparison with undoped manganite film.Previously, it was demonstrated, that the lanthanum manganites exhibit high magnetoresistance values at relatively low magnetic fields nm Fig. -inset. TMR was observed in the whole range of investigated magnetic field, showing the increase of sensitivity to magnetic field for Co-doped manganites not only at high (up to 21), but also at low magnetic fields (lower thant 2 T). The dopping at so-called B-site (Co substitution for Mn), destroys the long range ferromagnetic ordering and Mn3+-O-Mn4+ network, resulting in the change of electric and magnetic properties. Thus, the Co substitution for Mn results in the decrease of the transition temperature from the paramagnetic to ferromagnetic phase and increase of the resistivity, resulting in the increase of room temperature magnetoresistance43. However, to compare MR values of manganite-cobaltite films and MR of graphene layers is complicated and not straightforward. As follows, in manganite-cobaltite case, the zero field resistance (R(0)) was the largest at zero magnetic field due to the negative MR, while it was smallest one for graphene, due to positive MR effect. Therefore, the MR values of manganite-cobaltite films were always less than 100% . In our case, the current was much higher and therefore no dependence of the MR on the flowing current magnitude was observed for various graphene layers having different resistances.In order to increase the sensitivity of graphene at low magnetic fields, the hybrid sensor was designed  and hybrid LSMCO/3LG sensor at magnetic fields up to 0.5 T is presented in Fig. ΔVr is normalized to the source voltage VS: ΔVr/VS = /VS, where Vr,B=0 – response voltage without magnetic field and Vr,B – at magnetic field, respectively. The individual elements (LSMCO and 3LG) were connected in series with the ballast resistors in the same voltage divider configuration, by replacing other element (LSMCO or 3LG) with ballast resistor, respectively, depending on the investigated element (3LG or LSMCO). In order to compare the response of individual elements with the hybrid device, the ballast resistors with the same values as zero field resistance of the other element in the hybrid structure, were used for the measurements. The measurements revealed, that in magnetic fields up to 0.2 T the response signal of individual 3LG was lower than signal of individual LSMCO sample: e. g. at magnetic field of B = 0.07 T it differs more than two times. This could be attributed to different MR behavior of both materials at low magnetic fields: quadratic dependence of graphene and almost linear of LSMCO. Therefore, the connection of 3LG and LSMCO in voltage divider geometry lead to increased sensitivity of the device, in particular, at the low magnetic field range. This effect could be explained by different signs of magnetoresistance - positive MR of graphene and negative MR of nanostructured manganite-cobaltite film /RLSMCO(0). It is obvious, that hybrid LSMCO/3LG sensor exhibits significantly higher response signal in all range of magnetic field in comparison with individual elements. However, depending on the ratio r, one can tune the highest response signal of the hybrid sensor and its sensitivity to the lower or higher magnetic field ranges. The normalized sensitivity of the hybrid sensor was evaluated as S = (δΔVres/δB)/VS. The sensitivity values of individual 3LG, LSMCO elements and the hybrid LSMCO/3LG sensor . These results demonstrate improvement of sensitivity of Co-doped manganite/3-layer graphene sensor in comparison with previously published results on manganite/single layer graphene sensor20 exhibiting normalized sensitivities of 16 mV/VT at 0.5 T and 29 mV/VT at 2 T. However, at 20 T the sensitivity of the LSMCO/3LG sensor drops to 7 mV/VT for r = 0.5, respectively. It has to be noted that in the case of r = 0.1 the sensitivity of hybrid sensor is lower at low fields (20 mV/VT at 0.5 T), however, it is high enough in a wide range of magnetic fields (40 mV/VT at 2.5–7 T and 17 mV/VT at 20 T) and no saturation of the response signal was observed up to 20 T  mV/VT46, however mentioned sensors were operating only in mT range of permanent magnetic field.Additionally, the response signals of hybrid sensor and individual samples in pulsed magnetic fields up to 20 T is presented in Fig. see Fig. . It is d12. Additionally, LSMCO/3LG sensor could be also used at lower operation temperatures since three-layer graphene exhibits even higher MR at cryogenic temperatures .In conclusion, the obtained results show the potential application of Co-doped manganite/3LG sensor for magnetic field measurement application at room temperature in very broad magnetic field range [0.1−20 T] with a certain sensitivity values depending on the magnitude of magnetic field. It has to be noted, that for the device fabrication the sensitivity dependence on the magnetic field can be solved by modern electronics with in advance stored calibration data, converting the measured signals into magnetic field values2O3) with formed Ag contacts in Corbino disk configuration for electric and magnetotransport measurements. PMMA polymer is used as supporting and protecting layer from folding and physical damages of graphene layer. The same transfer procedure was applied in order to get few-layer graphene samples, only the PMMA layer was removed each time with acetone and iso-propanol before placing the next graphene layer on the top of the previous one.The commercially available, high quality monolayer graphene grown by CVD method on Cu foil was used for the experiments. The single graphene layer grown on Cu foil was transferred to target substrate by applying wet chemical etching procedure. During this procedure Cu substrate was etched by ammonium persulfate solution from the bottom, leaving a floating graphene/PMMA flake on the surface of etching solution. Afterword, the SLG + PMMA flake was rinsed several times in deionized water and transferred to the target substrate  equipped with thermoelectrically cooled (−70 °C) CCD camera and microscope. Spectra were excited with continuous-wave diode-pumped solid state laser providing 532 nm radiation. The 50x/0.75 NA objective lens and 1800 lines/mm grating were used to record the spectra. The accumulation time was 50 s. To avoid damage of the sample, the laser power at the sample was restricted to 0.6 mW. The laser beam was focused to ~2 μm diameter spot on the sample. Raman scattering wavenumber axis was calibrated by the polystyrene Standard Raman spectrum. Parameters of the bands were determined by fitting the experimental spectra with Gaussian-Lorentzian shape components using GRAMS/A1 8.0 (Thermo Scientific) software.47 of carbon. Spectroscopic data were processed by the Avantage v.5 software (Thermo Fisher Scientific Ltd.).X-ray photoemission (XPS) and X-ray induced Auger (XAES) spectra were collected by using an Escalab 250Xi  spectrometer with a monochromatic Al Kα excitation source and six-channeltron detection system. The photoemission spectra were collected in standard mode of electromagnetic lenses from the area of about 1 mm in diameter at 40 eV pass energy of the analyzer. For Auger electron spectroscopy (AES) was employed an Escalab MkII spectrometer  with electron gun LEG200 operated at low energy (1–2 keV) and low beam current (few nA). All C KVV spectra (XAES and AES) were acquired at 100 eV pass energy and later smoothed by moving average algorithm for 11 times with a width of 1.8 eV. Afterwards, these spectra were differentiated by using a width of 7 data points for the determination of D parameter1−xSrx(Mn1−yCoy)zO3 (LSMCO) films were grown on polycrystalline Al2O3 substrates by pulsed-injection MOCVD technique with supplying the mixture of precursor solution in certain micro-doses48. The 360 nm thick LSMCO films were deposited at 750 °C temperature and 10 Torr pressure with a partial 3.5 Torr oxygen pressure. The Co doping level in the films (y = 0.14) was chosen in order to achieve higher magnetoresistance values of the films at room temperatures in comparison with manganite films without Co doping. Other deposition parameters  were controlled and optimized in order to get LSMCO films with a certain column-like nanostructure responsible for high magnetoresistance values in a wide temperature and magnetic field range.The nanostructured LaThe morphology of the films was investigated using Atomic Force Microscopy (Veeco Multi-Mode Nanoscope II) (AFM) and Scanning Electron Microscopy (Hitachi SU70) (SEM). The nanostructure of the films was studied using Transmission Electron Microscopy (Tecnai G2 F20 X-TWIN) (TEM).2 atmosphere. The distance between electrodes was constant and equal to 50 μm, whereas the width of the sample was changed to match the zero field resistance ratio between graphene and LSMCO samples.For the magnetoresistance measurements of manganite-cobaltite films the traditional photolithography and mesa etching was applied to form rectangular shaped LSMCO films. The Ag electrodes with a Cr sublayer were thermally deposited over the LSMCO films and post annealed at 450 °C for 1 h in OMR measurements of manganite-cobaltite and graphene samples were performed at 300 K in permanent magnetic field up to 2.35 T by using electromagnet. The mobile 43 kJ pulsed magnetic field generator was used for investigation of the samples in pulsed magnetic field up to 21 T. The generator was capable to generate pulsed magnetic fields of half-sine shape with amplitudes up to 40 T and pulse durations in the range of 0.3–2 ms depending on the chosen inductor (fabricated in the laboratory). The high pulsed magnetic field generator consists of high voltage power supply, capacitor bank, high power switch and a special multi-shot magnetic field coil (inductor). The high-field facilities were designed and are used for the investigations of properties of different materials in high magnetic fields at the Center for Physical Sciences and Technology in Vilnius, Lithuania49.The resistivity and MR measurements showed the linear dependence of magnetoresistance for all graphene structures reaching the highest MR values of 758% at 21 T for the three-layer graphene (two times higher as single one). The revealed highest response of 3LG to magnetic field lead to the development of hybrid magnetic sensor prototype consisting of Co-doped manganite/three-layer graphene system for the sensing applications at room temperature. The prototype sensor demonstrated that combination of materials with positive (graphene) and negative (Co-doped manganite) magnetoresistance effects leads to a significant increase of the sensitivity  and broadens the magnetic field operation range from 0.1 up to 20 T. Moreover, the possibility to tune the sensitivities depending on the magnetic field range during the fabrication stage, by changing the ratio of zero field resistances of 3LG and LSMCO, was demonstrated.In the present study, the quality and magnetoresistance properties of graphene layers (n = 1–5) were measured. The Raman measurements confirmed the good quality of graphene samples, whereas the


Sarcoidosis is a systemic granulomatous disease of unknown etiology. It may develop in response to an exposure or inflammatory trigger in the background of a genetically primed abnormal immune response. Thus, genetic studies are potentially important to our understanding of the pathogenesis of sarcoidosis. We developed a case-control study which explored the genetic variations between firefighters in the Fire Department of the City of New York (FDNY) with World Trade Center (WTC)-related sarcoidosis and those with WTC exposure, but without sarcoidosis. The loci of fifty-one candidate genes related to granuloma formation, inflammation, immune response, and/or sarcoidosis were sequenced at high density in enhancer/promoter, exonic, and 5’ untranslated regions. Seventeen allele variants of human leukocyte antigen (HLA) and non-HLA genes were found to be associated with sarcoidosis, and all were within chromosomes 1 and 6. Our results also suggest an association between extrathoracic involvement and allele variants of HLA and non-HLA genes found not only on chromosomes 1 and 6, but also on chromosomes 16 and 17. We found similarities between genetic variants with WTC-related sarcoidosis and those reported previously in sporadic sarcoidosis cases within the general population. In addition, we identified several allele variants never previously reported in association with sarcoidosis. If confirmed in larger studies with known environmental exposures, these novel findings may provide insight into the gene-environment interactions key to the development of sarcoidosis. Sarcoidosis is a multisystem granulomatous disease that presents with pulmonary involvement in 90% of cases and extrapulmonary manifestations, mostly skin, in 10% of patients . BetweenSince the disorder clusters in families, genetic factors are thought to play an important role in sarcoidosis pathogenesis, presumably through mechanisms of immunologic response to antigenic or other triggering exposures. A recent population-based study suggests a genetic risk factor for sarcoidosis in a large Swedish cohort, where a 3.7-fold increase in risk of sarcoidosis was observed in those with first-degree relatives with the disease. The risk further increased in those with relatives with Lofgren’s syndrome or with ≥1 relative with sarcoidosis . A case-We selected several candidate genes for genetic analysis based on their function encoding proteins involved in granuloma formation, innate and immune recognition, and inflammation. Several have been implicated in genetic studies of sarcoidosis such as the highly polymorphic human leukocyte antigen (HLA) genes. HLA genes encode the major histocompatibility complex (MHC) in humans and are involved in immunologic recognition and regulation. Within various racial groups, allele variations within HLA genes are associated with sarcoidosis. Examples include the HLA-DRB1*1101 allele in both Caucasians and African Americans , HLA-DRBThe current case-control study is the first to examine genetic features associated with WTC-related sarcoidosis. Compared to other genetics studies of sarcoidosis, our study uniquely includes a baseline sarcoidosis rate, a known time point of exposure (9/11), and extensive, systematized clinical follow-up for over 14 years. The objective of this study is to explore the genetic characteristics of WTC-associated sarcoidosis and understand its similarities to and differences from sporadic sarcoidosis cases without a clear environmental exposure. The source population for the cases and controls was WTC-exposed firefighters. Recruitment of sarcoidosis cases for this study was part of a larger study that explored the clinical course of firefighters with post-9/11 sarcoidosis between the time of diagnosis and 14 years after 9/11 . As outlControls were randomly selected from the source population and were matched to cases based on race, age on 9/11, smoking history, and WTC-exposure history. WTC-exposure history was determined by arrival time to the WTC site: arrival group 1 (arriving the morning of 9/11) being the group with the highest dust cloud exposure and arrival group 4 (arriving between 9/13–9/24) having the least dust cloud exposure . ControlThe final study population included 55 cases and 100 controls. All cases and controls had peripheral blood drawn in 2015–2016 and completed a questionnaire at the time of blood draw to aid with matching on clinical characteristics and WTC-exposure history. The study was approved by the Montefiore Medical Center/Albert Einstein Institutional Review Board (2014-4291). All study participants provided written informed consent.Peripheral blood mononuclear cells were obtained from cases and controls for sequencing analysis. A panel of 51 candidate genes were pre-selected based on their function encoding proteins involved in granuloma formation, innate and immune recognition, inflammation, and sarcoidosis. The loci of targeted candidate genes were sequenced using a custom designed sequencing panel for IonTorrent Ampliseq technology. The custom sequencing panel contained 1193 amplicons spanning all known exons, splice junctions and promoter regions of the genes. Promoter regions were defined as 2 kb upstream from the transcriptional start site. The panel overall coverage was over 90% for 422 exons . One excTM 1000 Spectrophotometer . The Qubit® dsDNA HS Assay Kit  was used to determine the concentration of each sample. Approximately 20 ng of DNA was used to generate libraries for sequencing. When not in use, samples were stored at −20 °C. For each sample, a PCR reaction was set up according to the manufacturer’s protocol using 20 ng of genomic DNA, two separate primer pools, and 16 amplification cycles. Following amplification, each sample was treated with FuPa reagent and ligated to a uniquely barcoded adapter to enable sample multiplexing. Libraries were then purified using the 1.5X Agencourt AMPure XP  kit. Amplification products from each primer pool were quantified individually using the KAPA Library Quantitation kit  and then pooled together. Template preparation was performed using the Ion OneTouch 2 system and the Ion PI Template OT2 200 kit v2 , according to the manufacturer’s protocol. All libraries were sequenced on the Ion Proton sequencer using the Ion PI chip and the Ion PI Sequencing 200 kit v2  to generate 200bp single ended sequencing.DNA was extracted using the QuickGene 610 system and the whole blood kit (Kurabo #DB-L), and quality verified and quantitated by the NanoDropTM  for filtering and annotation. For the identification and reporting of germline variants, we used a custom validated bioinformatic pipeline for the identification of SNPs as well as insertions and deletions (INDELs) included in the target panels ,21,22. Tp-value for Hardy-Weinberg Equilibrium Test > 0.0001. The variants were classified by their genomic locations to four categories: exon, intron (likely intron/exon boundaries), upstream, and 5’untranslated region (5’UTR). For those in an exonic region, they were further classified by their function as synonymous, nonsense, missense, or frameshift (We identified 909 variants (SNPs or INDELs) with minor allele frequency (MAF) ≥0.05 and ameshift . For eacameshift . To veriWe conducted two secondary analyses. First, we explored the interaction between each common variant and degree of WTC exposure, measured as arrival time to the WTC site. For each variant, we applied unconditional logistic regression under the co-dominant model with sarcoidosis as the outcome and the genotypes, WTC exposure group and their interaction term, as the explanatory variables. Likelihood ratio tests are applied to compare two models with and without the interaction term between the genotype and degree of WTC exposure. We also conducted a secondary analysis in which we associate the genetic variants with extrathoracic sarcoidosis. In these analyses, for each variant we applied unconditional logistic regression under the co-dominant model with extrathoracic sarcoidosis as the outcome and the genotypes as the explanatory variable.For variants whose MAF < 0.05, for each individual, the total number of rare alleles located in each gene were counted. We then compared the number of rare alleles between the cases and controls for each gene by each genomic location category  using two-sample T-tests. Furthermore, we counted rare variants with MAF < 0.01 across all target genes by the above-mentioned categories by genomic location and function, and then performed two-sample t-tests comparing the cases and controls for each of the categories.Final genetics analyses were completed on 55 cases and 100 controls. All but one case (54/55) and one control (99/100) self-identified as Caucasian, the remaining one case and one control self-identified as African American. The cases and controls were well matched, with at least one control for each case .Organ involvement for the 55 cases included 47/55 (85.5%) with biopsy proven sarcoidosis. The 8/55 (14.5%) without biopsy results had their imaging and organ involvement reviewed by two pulmonologists to confirm consistency with sarcoidosis . As outlp-value < 0.01  . Geneticp-value < 0.01 and all were within chromosomes 1 and 6. Our results also suggest an association between extrathoracic involvement and allele variants of HLA and non-HLA genes found not only on chromosomes 1 and 6, but also on chromosomes 16 and 17. Comparing our findings to published studies of sporadic sarcoidosis, we found similarities between genetic variants with WTC-related sarcoidosis and those reported in studies of sarcoidosis without known environmental exposures. In addition, we identified several allele variants not previously reported to be in association with sarcoidosis. If confirmed in larger studies with known environmental exposures, these novel findings may provide insight into the gene-environment interactions key to the development of sarcoidosis.Sarcoidosis has been identified in all WTC cohorts , the most extensively described being that from FDNY ,24,25,26For example, our data are consistent with a prior study showing an association between SNP rs20417, in a non-coding region of the PTGS2/COX2 gene on chromosome 1, and sarcoidosis in a UK and Austrian Caucasian cohort without known environmental exposure . This alExcept as noted above, the genetic variants associated with sarcoidosis in our WTC-exposed cohort have not previously been reported. Several of the SNPs identified in our cohort as associated with WTC-related sarcoidosis have been associated with other diseases in cohorts without known environmental exposures. Perhaps the most frequently investigated is rs20417 in PTGS2/COX2. Studies have identified its association with carotid-calcified plaque in those with diabetes , protectThe main strength of this study was its reliance on a rigorously characterized prospective cohort, with a clearly defined time of exposure (9/11). One limitation of this study is that it was focused on candidate genes already identified in sarcoidosis studies from the general population without WTC exposure. Additionally, the identification of novel SNPs may be partially due to our high-density sequencing which evaluated almost every base pair of these candidate genes. The main limitation, however, is that this study includes a relatively small sample size. Our statistical power was limited to detect only sizable effects. At significance level 0.01, we had 0.8 power to detect an odds ratio of 2.3 at variants with MAF = 0.5 and an odds ratio of 4 at variants with MAF = 0.05; we had 0.2 power to detect an odds ratio of 1.5 at variants with MAF = 0.5 and an odds ratio of 2 at variants with MAF = 0.05. Additional power would be obtained if this study was expanded to include all of the various WTC health program cohorts to allow for both replication of the main effects observed, and to allow for gene-environment analyses. This could potentially allow for more robust causal inferences to be made regarding the etiology of sarcoidosis, and whether there are unique differences in those with extrathoracic disease, particularly cardiac and joint involvement.The incidence of sarcoidosis was increased after the collapse of the WTC towers and this is best demonstrated in the FDNY cohort where there was clear evidence that these cases were new in onset after 9/11 ,16. Our


FOXC2 and PITX2 variants in CG. Variants of the proximal promoter and transcribed sequence of these two genes were analyzed by Sanger sequencing in a cohort of 133 CG families. To investigate possible oligogenic inheritance involving FOXC2 or PITX2 and CYP1B1, we also analyzed FOXC2 and PITX2 variants in a group of 25 CG cases who were known to carry CYP1B1 glaucoma-associated genotypes. The functional effect of three identified variants was assessed by transactivation luciferase reporter assays, protein stability and subcellular localization analyses. We found eight probands (6.0%) who carried four rare FOXC2 variants in the heterozygous state. In addition, we found an elevated frequency (8%) of heterozygous and rare PITX2 variants in the group of CG cases who were known to carry CYP1B1 glaucoma-associated genotypes, and one of these PITX2 variants arose de novo. To the best of our knowledge, two of the identified variants ; and PITX2: c.535C>A, p.(P179T)) have not been previously identified. Examination of the genotype-phenotype correlation in this group suggests that the presence of the infrequent PITX2 variants increase the severity of the phenotype. Transactivation reporter analyses showed partial functional alteration of three identified amino acid substitutions (FOXC2: p.(C498R) and p.(H395N); PITX2: p.(P179T)). In summary, the increased frequency in PCG patients of rare FOXC2 and PITX2 variants with mild functional alterations, suggests they play a role as putative modifier factors in this disease further supporting that CG is not a simple monogenic disease and provides novel insights into the complex pathological mechanisms that underlie CG.Congenital glaucoma (CG) is a severe and inherited childhood optical neuropathy that leads to irreversible visual loss and blindness in children. CG pathogenesis remains largely unexplained in most patients. Herein we have extended our previous studies to evaluate the role of  CYTOCHROME P450, SUBFAMILY I, POLYPEPTIDE 1  mutations [CYP1B1 [LATENT TRANSFORMING GROWTH FACTOR-β-BINDING PROTEIN 2  [TEK  underlie PCG with variable expressivity [G-PATCH DOMAIN-CONTAINING PROTEIN 3  variants are present in a some congenital glaucoma (CG) cases [CYP1B1 mutations are the predominant known genetic cause of this type of glaucoma in different worldwide populations [LTBP2 gene alterations have been identified only in a few families [MYOCILIN  [FORKHEAD BOX C1  alterations [CYP1B1 genotypes [FOXC1 variants with moderate functional dysregulation as either causative of modifier factors in CG [Primary congenital glaucoma  is a severe and irreversible neonatal or infantile optic neuropathy of uncertain pathogenesis. The immature iridocorneal angle appearance observed clinically indicates that it results from arrested maturation of tissues derived from cranial neural crest cells. This alteration leads to increased aqueous humor (AH) outflow resistance, elevated intraocular pressure (IOP) and optic nerve degeneration. The diagnosis is based in finding isolated trabeculodysgenesis with no other ocular anomalies , 5. PCG  602091)  as the cG) cases . Null CYulations –13, and families , 15. MYOerations , 19 havers in CG –20.FOXC2, MIM# 602402) and PAIRED-LIKE HOMEODOMAIN TRANSCRIPTION FACTOR 2 , are genes encoding transcription factors that control the expression of other genes and form part of a complex regulatory network involved in ocular development and disease [FOXC2 is structurally and functionally closely related to FOXC1. Their chromosomal localization and genomic organization suggest a common origin from an ancestral gene through chromosomal duplications [FOXC2 function cause lymphedema-distichiasis syndrome, an autosomal dominant disease that affects the formation of the lymphatic vasculature system and is also characterized by a double row of eyelashes (distichiasis) [FORKHEAD BOX C2  .PAIRED-LIKE HOMEODOMAIN TRANSCRIPTION FACTOR 2  gene encodes a homeodomain-containing transcription factor. It presents a pattern of expression similar to that of FOXC1 and FOXC2, and it is also present in the periocular mesenchyme. Disruption of PITX2 is one of the major causes of Axenfeld-Rieger syndrome (ARS) [PITX2, like FOXC1, is dose sensitive and mutations that alter the level of functional PITX2 protein (either increased or decreased) by different mechanisms are pathogenic [PITX2 gene is composed of six exons, two alternative promoters located upstream of exons 1 and 4, and two alternative transcription start sites located in exons 2 and 4 [The me (ARS) . ARS is me (ARS) . Systemithogenic . The PIT 2 and 4 . The imm 2 and 4 .FOXC2 and PITX2 variants in CG patients, providing further evidence for the role of rare variants of these two genes involved in ocular anterior segment development as putative modifier factors.In this study we show an increased frequency of rare and functionally altered One hundred and thirty-three unrelated families affected by PCG participated in this study. The study and informed written consent procedures were approved by the Ethics Committee for Human Research of the Hospital Clínico San Carlos, Madrid (Spain), approval number 13/388-E) and followed the tenets of the Declaration of Helsinki. The clinical examination and diagnosis of patients were performed as previously described . GlaucomQIAamp DNA Blood Mini Kit . The DNA sequence variation analyses were carried out by automatic Sanger sequencing. The promoter region (nucleotides -1 to -1500), the coding region and both the 5'- and 3’-untranslated regions (UTRs) of FOXC2 were amplified by PCR, using the primers and conditions described in PITX2 were amplified as previously described [FOXC2 variants were screened for LTBP2 mutations using a customized gene panel [The genomic DNA was extracted from peripheral blood, using the escribed . The varne panel . LibraryFOXC2 variants p.(C498R) (rs61753346) and p.(H395N) were directly amplified and cloned from the genomic DNA of carriers using the following primers, which incorporated the EcoRI or BamHI restriction sites at the 5’ end (indicated in bold): FOXC2-Up-EcoRI, GAATTCCGCGCTCTCTCGCTCTCAGG-3'5'-GG and FOXC2-Dw-BamHI, GGATCCCCGTATTTCGTGCAGTCGTAGG-3'5'-GG. The PCR products were cloned into a modified version of the EcoRI/BamHI restriction sites of the pcDNA3.1(-) vector  [FOXC2 coding sequence from DNA of a control subject and the cloned DNA was completely sequenced to rule out the presence of rare FOXC2 variants. The PITX2 coding variants p.(P179T) was obtained by site-directed mutagenesis using the QuickChange Site-Directed Mutagenesis Kit , with the following primers (mutant nucleotides are indicated in bold): F, ACAGGCTATTCCTACAAC-3’5’-CCTACGACGACATGTAC and R, TGTACATGTCGTCGTAGG-3’5’-GTTGTAGGAATAGCCTG. A cDNA encoding the wild-type PITX2C (NP_000316) isoform, cloned into the pcMV6 vector , was used as a template for the site-directed mutagenesis reaction. The presence of the mutation in the site-directed mutagenesis product was confirmed by Sanger sequencing. The mutagenized cDNA was subcloned into the SgfI and MluI restriction sites of pcMV6 to avoid undesirable mutations in the vector.The R) rs617546 and p.CXCR4 luciferase reporter vector (50 ng), and the pMirTarget vector (50 ng), which expresses the red fluorescent protein (RFP) as a transfection control. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics , at 37°C in a fully humidified 5% CO2 atmosphere. Forty-eight hours after transfection the cells were harvested and assayed for firefly luciferase activity using the Luciferase Assay System (Promega) according to the manufacturer’s instructions. FOXC2 and PITX2 proteins were detected by Western blot as expression control and transactivation activity was normalized for these proteins. RFP and endogenous lactate dehydrogenase (LDH) were also assessed by Western blot as transfection and loading controls, respectively.Transactivation assays were performed using the Luciferase Assay System (Promega) following the instructions of the manufacturer. HEK-293T cells were transfected with the different recombinant versions of FOXC2 and PITX2C, cloned in the expression vector pcMV6 (500 ng each), along with the recombinant pGL3-basic-The protein stability was studied via western blot of transfected cells treated with cycloheximide (300 μg/ml) at different times as previously reported . The FOXFor Western blot analyses the cell lysates were prepared and fractionated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using the Mini-PROTEAN III Gel Electrophoresis System . A commercial mouse monoclonal anti-myc  was used as the primary antibody, diluted at 1:1000. Horseradish peroxidase-conjugated antibodies against mouse IgG  were diluted to 1:1000–1:4000. Chemiluminiscence detection and the densitometry for protein band quantification was performed as previously described . As an ahttp://genetics.bwh.harvard.edu/pph2/) programs. The MicroSNiPer software was used to identify putative miRNA target sequences (http://cbdb.nimh.nih.gov/microsniper) . Both nucleotide and amino acid sequence alignments were carried out with Clustalw . The novel FOXC2 and PITX2 variants were named using directions from Mutalyzer , according to RefSeq NM_005251.2 and NM_000325.5, respectively. The novel variants have been submitted to dbSNP (http://www.ncbi.nlm.nih.gov/SNP/) and to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/). Structure prediction of mutant and wild-type proteins were evaluated by QUARK (https://zhanglab.ccmb.med.umich.edu/QUARK2/) [The deleterious effect of mutations was predicted online with the Sorting Intolerant From Tolerant (SIFT)  and PolyPhen-2 (QUARK2/)  and furtt-test or the one-way analysis of variance (ANOVA). A Bonferroni correction was applied to adjust tests for multiple comparisons. The data were statistically processed by the SigmaStat 2.0 software .The statistical comparisons between groups were performed using either the FOXC1 variants [FOXC2 and PITX2 variants in CG using the same group of patients. The main clinical and demographic features of these patients have previously been reported [MYOC and CYP1B1. Eight of the patients (6.0%) carried rare FOXC2 variants in the heterozygous state , which is annotated in Ensemble and GeneCards databases as a promoter covering the entire FOXC2 sequence and most of the antisense lncRNA FOXC2-AS1 gene (FOXC2-AS1 intron 1 (n.145+174G>A) and the regulatory feature (promoter) (FOXC2 and a modifier outcome on both FOXC2-AS1 and the overlapping promoter. The nucleotide change c.*38T>G was located in the 3’UTR of the gene (FOXC2 nucleotide change was predicted to cause a novel amino acid change (p.(H395N)) located in the second transactivation domain of the protein  , and it the gene  and was the gene . This vathe gene  in the tthe gene , andacco protein  with inf protein . The str protein . In addipromoter . The patpromoter .FOXC2 and PITX2 defects on patients with CYP1B1 glaucoma-associated genetic backgrounds. To that end, a group of 25 CG cases who were known to carry CYP1B1 pathogenic genotypes, were selected based on DNA availability from a previously reported cohort of patients [PITX2 variants were identified in two (8%) of these patients (PCG-133 and PCG-139). As far as we know, one of these variants , CYP1B1 variants none of them presented this PITX2 nucleotide change (de novo. The patient who carried this variant (PCG-133) was diagnosed with bilateral PCG at the age of three months and presented advanced optic nerve excavations and was subjected to six surgical operations per eye to control IOP , that resulted in an inferred missense mutation (p.(A188T)), ),  also locA188T)), . This nuA188T)), . The varA188T)), . The carFOXC2 and PITX2 wild-type amino acids or nucleotides were highly conserved , could be cloned for functional evaluation. It showed approximately 130% increased transactivation compared with the wild type  stability was not significantly different from that of FOXC2 wild-type protein  and c.*38T>G (non coding 3’ UTR) also mapped at a promoter, which overlapped with FOXC2 and FOXC2-AS1 genes. FOXC2-AS1 encodes a lncRNA transcribed from the negative strand of FOXC2. Both molecules have been located in the cytoplasm and are concordantly upregulated in doxorubicin-resistant osteosarcoma cell lines and tissues [FOXC2-AS1 stabilizes FOXC2 mRNA by forming an RNA-RNA duplex, which subsequently upregulated steady-state FOXC2 expression at both the transcription and post-transcription levels [FOXC2 activity through FOXC2-AS1 dysregulation. Further studies are required to confirm these ideas. Finally, the high evolutionary conservation of wild type FOXC2 and PITX2 nucleotide or amino acid positions involved in the variants identified in this study, further indicate that their substitution by infrequent residues might modify their function.The two missense FOXC2 variants (p.(H395N) and (p.(C498R)) and one of the PITX2 amino acid substitutions (p.(P179T)) were inferred to cause a moderate functional effect at least by one bioinformatic analysis and, experimentally, they were found to be associated with moderately disrupted transactivation. The functional impact of the second PITX2 amino acid substitution, p.(A188T), could not be functionally evaluated due to DNA cloning difficulties. In fact, the two FOXC2 amino acid changes were found to be hypomorphic whereas the PITX2 amino acid substitution (p.(P179T)) behaved experimentally as a hypermorphic variant. Additional structural and functional analysis indicated that p.(H395N) alters polypeptide chain conformation and decreases protein stability, which can explain the associated reduced transactivation. However, our data showed that the transactivation effect of p.(C498R) is not due either to altered protein stability, protein structure or subcellular localization, suggesting that the substitution of a basic (arginine) for a polar (cysteine) amino acid may disrupt protein interactions in the second inhibitory domain of the protein, leading to reduced protein activity. Similarly, our results indicate that increased transactivation associated with the p.P179T PITX2 mutation is not related with altered protein stability, protein conformation or subcellular localization. Proline possesses a hydrophobic side chain, whereas threonine side chain has both hydrophilic and hydrophobic functions. Therefore, this amino acid replacement may affect protein interactions taking place in the transcriptional inhibitory domain where it is located, leading to increased PITX2 activity. In this line, an increased side chain polarity associated with amino acid substitution p.(A188T) could also interfere protein interactions involving the first PITX2 transcriptional inhibitory domain, leading to a functional alteration. Additional studies are required to evaluate these hypotheses. Interestingly, according to Ensembl Regulatory Build,  tissues . It has n levels . TherefoFOXC1 and PITX2A. In fact, it has been reported that PITX2A protein can act as a negative regulator of FOXC1 through formation of PITX2A-FOXC1 heterocomplexes [PITX2/CYP1B1, PITX2/FOXC1 [FOXC1/CYP1B1 [CYP1B1/GJA1 [CYP1B1 and MYOC has been reported in early-onset congenital glaucoma [PITX2 and CYP1B1 variants and showed very early glaucoma onset and severe disease, characterised by a large number of surgical operations required to control IOP. These data suggest a modifier role of rare PITX2 variants on age at onset and severity on CYP1B1 associated congenital glaucoma. Our data also revealed the presence of one PITX2 de novo mutation in one PCG patient, indicating that this type of genetic variants might also be involved in congenital glaucoma, adding additional genetic complexity to the genetic basis of this disease. We should keep in mind that reduced penetrance, low frequency of these variants and limited number of available patients due to low prevalence of the disease, will also complicate studies to determine the role of this type of genetic variation in CG. Combination of genome, transcriptome and even proteome analysis of individual patients, along with functional studies in animal models will be required to gain new insights into the genetic complexity of congenital glaucoma.Modifier genes are increasingly recognized as an important source of phenotypic variation that may explain the relationship of phenotype to genotype. Individually, genetic modifiers have small effect size on the phenotypic outcome, leading to reduced penetrance, i.e., not all individuals harbouring a pathogenic variant will exhibit clinical signs of the associated disorder . Normal omplexes . Therefoomplexes , 46, andomplexes , 47, 48.X2/FOXC1 , FOXC1/C1/CYP1B1  and CYP11B1/GJA1 ) provideglaucoma  and PCG glaucoma . LikewisIn summary, this study further supports the concept that mild functional alterations associated with variants in genes involved in ocular development may act as modifier factors in non-Mendelian CG cases, providing novel insights to understand the genetic complexity of this disease.S1 FigArrows over the peaks indicate the location of mutations. The mutant nucleotides are indicated in red and those in heterozygosis are shown below the corresponding wild type position. The sequences of the different variants were obtained from the corresponding probands in each family and the wild type sequences from control subjects.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.