TDP-43 protein plays an important role in regulating transcriptional repression, RNA metabolism, and splicing. Typically it shuttles between the nucleus and the cytoplasm to perform its functions, while abnormal cytoplasmic aggregation of TDP-43 has been associated with neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). For the purpose of this study we selected a set of proteins that were misregulated following silencing of TDP-43 and analysed their expression in a TDP-43-aggregation model cell line HEK293 Flp-in Flag-TDP-43-12x-Q/N F4L. Following TDP-43 sequestration in insoluble aggregates, we observed higher nuclear levels of EIF4A3, and POLDIP3β, whereas nuclear levels of DNMT3A, HNRNPA3, PABPC1 and POLDIP3α dropped, and cytoplasmic levels of RANBP1 dropped. In addition, immunofluorescence signal intensity quantifications showed increased nuclear expression of HNRNPL and YARS, and downregulation of cytoplasmic DPCD. Furthermore, cytoplasmic levels of predominantly nuclear protein ALYREF increased. In conclusion, by identifying a common set of proteins that are differentially expressed in a similar manner in these two different conditions, we show that TDP-43 aggregation has a comparable effect to TDP-43 knockdown. TARDBP gene, plays an important role in regulation of several processes, including microRNA processing, apoptosis, cell division, transcription, translation, splicing, axonal transport, and neurite outgrowth1467910TDP-43 protein, encoded by the TARDBP in familial and sporadic ALS cases proved the essential role of abnormal TDP-43 in disease1512171921232Abnormal cytoplasmic and occasional intranuclear aggregation of TDP-43 has been associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP)12In this study, however, we have specifically investigated whether the aggregation/sequestration of TDP-43 correlated with its loss of function by comparing the expression changes of selected proteins responsive to silencing of TDP-43 in an aggregation and sequestration model cell line HEK293 Flp-in Flag-TDP-43-12x-Q/N F4L. Our results show that differential expression of proteins in TDP-12xQ/N-F4L cells correlated with proteomic results of TDP-43 knockdown in SH-SY5Y, revealing a common set of proteins whose expression is influenced via TDP-43 aggregation or knockdown.TARDBP gene expression.For the purpose of this study, we selected 13 proteins  whose ex6In HEK293 Flp-in Flag-TDP-43-12x-Q/N F4L cells (referred to as HEK TDP-12xQ/N-F4L hereafter) TDP-43 aggregation was induced by addition of doxycycline (DOX) to the growth medium . The aggPOLDIP3 (SKAR) and RANBP1 splicing is under control of TDP-43226POLDIP3 (SKAR) exon 3 and inclusion of RANBP1 exon 5 in both SH-SY5Y and HEK TDP-12xQ/N-F4L in the same pattern  staining  and 4. SIn addition, some discrepancies between the proteomic data obtained for HEK TDP-12xQ/N-F4L in comparison to RNAi in SH-SY5Y may be on account of the difference in experimental model used (knockdown vs. aggregation), the cell line (HEK vs. SH-SY5Y), the experimental conditions , which in turn reflect a moderate difference in the regulation of expression of proteins in question. There is also some variation to be expected from the antibodies used, as some perform better in denaturing conditions of western blot, while others recognise better the proteins in cells (IF). The recognition of the latter can also be influenced by method of fixation of cells  or the accessibility of the epitope against which the antibody was raised due to specific protein conformation.In this study, using a cellular model of TDP-43 sequestration, we have examined expression variations of a series of proteins previously shown to be differentially expressed following TDP-43 knockdownPOLDIP3/SKAR) has been previously noted as a result of the depletion of TDP-4322632POLDIP3 gene has been reported to favour the synthesis of the β isoform in respect to the main α isoform3233POLDIP3 mRNA variant was also observed in ALS patients’ spinal motor neuronsTDP-43 is an RNA/DNA binding protein with multiple functions. For this reason, its downregulation influences a large number of RNA and protein targets. Decreased TDP-43 cellular levels affected splicing of 158 exons in neuroblastoma cells and altered 965 splicing events in adult mouse brain226Our comparative analysis of siRNA-mediated knockdown versus sequestration and aggregation of TDP-43 has allowed us to confirm two possible connections between loss of TDP-43 functional activity and disease. First of all, our analyses add further support to the possible link between TDP-43 and nonsense-mediated decay. In fact, POLDIP3 was also reported to associate with the exon junction complex (EJC), which is recruited to exon junctions during splicing37Drosophila study, ALYREF was singled out as potential modifier of G4C2 expansion related toxicity4C2)n hexanucleotide repeat expansion mutation in the C9orf72 gene, which can span from several hundred to several thousand repeats, is the major genetic cause of ALS and FTLD leading to TDP-43 proteinopathy40414C2 RNA are sequestered in nuclear foci, ALYREF may act as part of a control mechanism that retains unspliced or faulty RNAs in the nucleusSecondly, in a C9orf72 expanded repeats46Thirdly, TDP-43 sequestration impacts several HNRNP proteins by either increasing their concentration in the nucleus, or by nuclear clearance, thus altering RNA metabolism and potentially leading to ALS and FTLD. Following TDP-43 aggregation we observed altered levels of HNRNPA3 and HNRNPL. Previously, HNRNPA3 was found to be aggregated in cytosolic TDP-43 negative inclusions in the brains of patients with RANBP1 transcript, in turn decreasing the level of protein.Finally, we have previously shown in SH-SY5Y that depletion of TDP-43 influences intracellular transport through downregulation of RANBP1 and herein confirmed the expression drop in HEK TDP-12xQ/N-F4L modelin vivo still needs to be determined, our cellular analysis has added further support that aggregation and sequestration model overlaps TDP-43 LOF following its knockdown by siRNA. As a consequence, the major hits from this comparison might represent good candidates to be followed up in further studies that aim to elucidate the causes of ALS/FTLD pathology.In conclusion, although the pathological relevance of these processes HEK293 Flp-in Flag-TDP-43-12x-Q/N F4L cell line was established as previously describedCells were grown in 6-well plates and were harvested in cold CLB buffer , 0.25% Triton X-100, cocktail of protease inhibitors (Roche)) and centrifuged for 5 min at 3000 g at 4 °C. Supernatants were transferred to fresh tubes and re-centrifuged at 16100 g at 4 °C for 10 min. Obtained supernatants were used as cytoplasmic fractions. The first pellets were washed three times in cold CLB, resuspended in 1X SDS loading buffer without bromophenol blue, sonicated, boiled for 5 min and re-centrifuged. Resulting supernatants were saved as nuclear fractions. The protein concentration in the fractions were determined by Bio-Rad DC Protein Assay.Reducing SDS-PAGE was run on 4–12% SDS precast gels (C.B.S. Scientific) loaded with 10–20 μg of protein samples in 1X SDS loading buffer with 100 mM dithiothreitol at 175 V. Wet transfer onto nitrocellulose membrane  was carried out at 200 mA for 90 min. Membranes were blocked with 5% non-fat dry milk in TBS with 0, 05% Tween-20  at room temperature for 1 hour. Primary antibodies  diluted Cells were grown on poly-L-lysine (Sigma) coated glass coverslips and were washed once with PBS and fixed with 4% paraformaldehyde in PBS for 15 min. Then they were permeabilised with 0.1% Trition X-100 (Sigma) in PBS for 5 min. Blocking was performed with 3% BSA (Sigma) in PBS for 1 hour. Cells were incubated with primary antibodies  diluted ImageJ was used to quantify immunofluorescence signal intensities as follows. Several regions of interest (ROI) were selected in the appropriate cellular location, and the mean fluorescence of detected protein was measured, along with several adjacent background readings. The total corrected cellular fluorescence, (CTCF = integrated density−(area of selected cell × mean fluorescence of background readings), was calculated for each ROI. The averages and s.e.m. were determined for several images for each marker in induced cells compared to non-induced, where expression was set to 100%. Statistical analysis (2-sided unpaired Student’s t-tests) was performed.p-value of <0.05 was considered significant.Quantitative values of protein bands in western blots were normalized to GAPDH in cytoplasmic fractions and to fibrillarin (FBL) in nuclear fractions. Relative protein expression levels in TDP-43 aggregating HEK TDP-12xQ/N-F4L were calculated towards non-induced controls. For immunofluorescence quantification, immunofluorescence signal intensities of cells with induced TDP-43 aggregation and non-induced controls were compared and relative protein expression levels were calculated. Statistical significance of differential expression of proteins according to western blot and immunofluorescence was determined with unpaired Student’s t-test analysis. Student’s t-test was performed in Microsoft Excel Professional Plus 2013. A How to cite this article: Prpar Mihevc S. et al. TDP-43 aggregation mirrors TDP-43 knockdown, affecting the expression levels of a common set of proteins. Sci. Rep.6, 33996; doi: 10.1038/srep33996 (2016).


The result suggests carriers in the doped graphene are no longer massless, and an effective mass as small as one percent of free electron mass is sufficient to confine carriers within a 2D hetero space with energy 10 times larger than the room-temperature thermal energy. The interlayer excitons arise within 1 ps. Their formation effectively blocks charge recombination and improves charge separation efficiency for more than one order of magnitude.Phase transitions of electron–hole pairs on semiconductor/conductor interfaces determine fundamental properties of optoelectronics. To investigate interfacial dynamical transitions of charged quasiparticles, however, remains a grand challenge. By employing ultrafast mid-infrared microspectroscopic probes to detect excitonic internal quantum transitions and two-dimensional atomic device fabrications, we are able to directly monitor the interplay between free carriers and insulating interlayer excitons between two atomic layers. Our observations reveal unexpected ultrafast formation of tightly bound interlayer excitons between conducting graphene and semiconducting MoSe 2.The investigation into the dynamical transitions of charged quasiparticles on interfaces remains technically challenging. Here, the authors use ultrafast, mid-infrared micro-spectroscopy to unveil the formation of tightly bound interlayer excitons between conducting graphene and semiconducting MoSe The electrostatic interaction between the depleted valence band states (holes) and excited electrons can lead to the formation of long-lived insulating excitons4. The evolution of these electron/hole states on interfaces in the devices, which arises from interactions among phonons, photons, and charged quasiparticles, determine their fundamental properties, e.g., luminescence, heat generation, charge separation and transport, redox reaction kinetics, and outcomes7. In the past, the interfacial dynamics of excited optoelectronic materials were often studied on bulk or nano-sized samples that contain both interfacial and bulk components with absorption, photoluminescence, and visible pump/probe experiments close to the bandgap9. Despite providing a wealth of important information, these techniques are sensitive to creation and destruction of excitons and thus only probe the quasiparticles with negligible center-of-mass momentum because of the small photon momentum. However, the majority of excitons can assume finite momenta as a result of scattering and is optically dark in these measurements11.In optoelectronic and optoelectro-chemical devices composed of semiconductors and conductors, photo excitation can generate transient conducting free carriers by promoting electrons from the occupied valence band into the empty conduction band of the semiconductors14, photons with lower energy, e.g., the near- and mid-infrared (IR) or THz, can probe photo-generated excitons at center-of-mass momenta well outside the optically accessible range  heterostructures composed of only two atomic layers17 provides an ideal laboratory for the studies of pure interfacial dynamical transitions of charged quasiparticles. However, limited by spatial resolution, it remains a challenge for regular IR setups to probe electron/hole pairs in 2D heterostructures of which the sizes are typically smaller than the IR beam size. In this work, combining microscope, ultrafast visible/near-infrared/mid-infrared frequency-mixed spectroscopy  materials18 promise ideal combinations for atomically thin optoelectronic devices in which 2D TMDC functions as photon absorbers and graphene layers serve as transparent electrodes17.In our experiments, photo-excited electrons/holes are quantum confined within the 2D space of two atomic layers. We investigate heterostructures composed of one monolayer of MoSeene Fig. . The mon2/graphene heterostructure is illustrated in Fig. 2 is around 2.0 eV26. The Dirac point of graphene is 0.9 eV below CBM of MoSe2. The Fermi level of graphene in heterostructure is -0.17 eV, 0.02 eV higher than the monolayer graphene because of charge transfer from the n-doped MoSe2. The photoluminescence  and 400 nm (3.1 eV), respectively. The optical density change  with a Lorentzian width of 280 cm−1 remains in heterostructure that lasts for more than 20 ps , and e is the elementary charge, Γ is the broadening of the interband transitions, whereas τe is the relaxation time due to intraband carrier scattering. Quantitative analyses of the graphene signal according to Eqs. 31.The huge difference in signal reveals distinct evolutions of electrons/holes in graphene and heterostructure. The excitation photon energy 1.03 eV is lower than the excitonic transition energy (1.59 eV) of MoSe32. A similar 1s-2p transition peak was also observed in the mid-IR range for monolayer WSe2 on a diamond11. In this work, different from the two previous experiments, the graphene/MoSe2 heterostructure sample is not a pure substance, but a combination of two monolayers. Thus, the nature of the exciton that give rise to the resonant peak at 2185 cm−1 can have several possible origins: 1, the internal neutral and charged excitons, e.g., regular excitons and trions33 in graphene; 2, the internal neutral and charged excitons in MoSe2; and 3, the interlayer neutral and charged excitons.In the heterostructure, the detection frequency dependent signals and dynamics Fig.  resemble11. In brief, the binding energy of trions is only about 30 meV33. The binding energy of excitons with more particles is even smaller. At room temperature, where our experiments were carried out, most of the trions had dissociated due to thermal motions34, which has been verified with temperature-dependent photoluminescence34. Second, the intralayer exciton in graphene is not likely. Although the carriers in doped graphene are no longer massless due to the Fermi level shift from the Dirac point and a slight symmetry breaking35, tightly bound excitons cannot form because the bandgap of graphene in the heterostructure is too small  are not likely either. The combination of the experiments with 3.1 and 1.03 eV excitations in Fig. 2. Excitation with it cannot produce detectible amounts of free carriers or excitons in MoSe2 4 than the free carriers created with 3.1 eV excitation in the monolayer MoSe2. However, experimentally, the free-carrier dynamics detected at 1860 cm−1 of the heterostructure excited with 1.03 eV is significantly faster than that inside the monolayer MoSe2, and essentially the same as graphene  of the two monolayers. It is conceivable that the formation of interlayer excitons need carriers to diffuse in both the layers and the faster partner dominates.In the following, combining experimental data and literature, neutral interlayer excitons are concluded to be the dominant species that produce the resonant signals. First of all, the contribution of the charged excitons (including trions and other excitons composed of more than three carriers) is very minor in room temperature optical/IR pump/probe experiments. This has been completely addressed in the SI of the previous work −1 Fig.  was due ene Fig. . The detene Fig.  is inter−1 (0.27 eV) seem to be unlikely and completely unexpected if estimated with conventional hydrogen-like 3D model. It is well known that pristine graphene has massless carriers, which cannot form excitons36. In most CVD grown graphene, which is effectively doped, like the one used in our experiments, the Fermi level is not at the Dirac point. Because of the Fermi level shift and the slight symmetry breaking due to the formation of heterostructure35, the carriers are no longer massless , similar to 0.012 m0 reported for an epitaxy-grown graphene37. Estimated with this effective mass and the conventional 3D hydrogen-like model in which the binding energy is linearly proportional to the reduced mass, the binding energy of graphene/MoSe2 would be only 1/60 of the MoSe2 intralayer excitons. The binding energy of MoSe2 intralayer excitons has been computed to be between 0.4 and 0.6 eV19. Our experiments  , the 980 nm photoexcitation, which excites only graphene but not MoSe2, produces external photocurrent flowing from the portion of graphene that is on the top of Si (with a thin layer of SiO2) to that on the top of MoSe2, which is a clear signal that the quasi-equilibrium of MoSe2 (hole)/graphene (electron) inside the heterostructure is reached by the 980 nm photoexcitation. The results have a very interesting indication: under the experimental condition, graphene functions as a photon absorber and MoSe2 serves as a charge acceptor, rather than their traditional roles, in which graphene is the transparent electrode and MoSe2 is the chromophore.In summary, the excitation of MoSeeV) Fig. . As showons Fig. . Kinetic2. Figure 2 and MoSe2/graphene heterostructure detected in three different IR ranges at 16 ps after excitation with 3.1 eV photons. In the low-frequency range (1280–1380 cm−1), signals of both graphene and heterostructure are zero. Between 1900 and 2230 cm−1, the spectrum of graphene is very close to zero, whereas the spectrum of the heterostructure is at peak. In the high frequency range (2500–2800 cm−1), the signal of graphene remains zero, but that of heterostructure is a flat spectrum with an amplitude of about 30–40% of the peak value at 2156 cm−1. Similar to those following 1.03 eV excitation  is significantly higher than that of graphene (4.14%) at 3.1 eV42, overall the interlayer charge transfers result in higher electronic temperature in graphene. Excluding the free carriers remaining in MoSe2, the effect is around equal to 200% excitation flux increase for graphene monolayer, assuming semi-instantaneous interlayer charge transfers27 and electronic thermalization22. Our calculations show that the temperature increase with additional 200% flux slows the graphene electronic dynamics for about 30%, from 130 to 170 fs  interlayer charge transfers27 send both electrons and holes into graphene45, where they recombine within a couple of hundred fs25. The commonly accepted but oversimplified pictures, however, cannot account for the complex many-body dynamics in real systems and our experiments. Quasiparticles that excitons are composed of cannot be treated separately. In a photo-excited electron/hole gas, the ultrafast Coulomb interactions result in fundamentally new properties through pairing and higher-order correlations46. Some of these can be intuitively visualized by processes such as a pair of free electron and hole forming an exciton by transferring their energy and momenta to other free carriers or redistributions among pairs through collisions  keep the charged quasiparticles on different layers for more than 20 times longer. The greatly improved intrinsic charge separation efficiency is important for many applications that utilize the conversion of photo energy into electronic energy or electricity, e.g., solar cells, photo-detections, and photo-electrochemistry. We anticipate that the microspectroscopic IR response uncovered here will enable realtime and precise studies of the new non-equilibrium states on atomic interfaces that are critical for future developments and applications of atomically thin and other optoelectronic devices, but cannot be studied with other means.The transition of conducting free carriers into insulating interlayer excitons observed in the semiconductor/semimetallic 2D heterostructure reveals the significance of many-body interactions in mediating and thus producing sophisticated electronic dynamics and states on interfaces. The unexpected formation of the tightly bound interlayer excitons suggest that carriers in the doped graphene of the heterostructure is no longer massless, and an effective mass as small as one or two percent of 2 in our prior publication47 using Selenium pellets and Molybdenum oxide as sources.We follow the CVD method of growing high-quality monolayer MoSe2/Ar was used as a carrier gas and the pressure was kept around 1 torr during the whole process.CVD method was used to grow graphene on an electropolished copper foil with methane as a precursor. The copper foil was annealed at 1000 ℃ for 20 min, followed by graphene growth for 9 min using 3.5 sccm of methane under the same temperature. 15% H2 are transferred onto the CaF2 substrates for transmission mode laser experiments. The process is: at first, a thin layer of PMMA (PMMA-B4 4%wt) is spin-coated onto the sample on SiO2 substrate twice at the speed of 4000 rpm, and then the sample is carefully placed in a Buffer HF (1:5) and allowed to float. After 20 h, the PMMA sheets are separated from the SiO2 substrate and fished with glass plate, and then moved to Di-water for rinsing for a few times. The pre-cleaned CaF2 windows are used to fish the PMMA sheets. The CaF2 windows with PMMA and samples stay in room temperature and vacuum for 24 h. Finally, acetone is used to remove the PMMA. The MoSe2 triangles on the CaF2 substrate are confirmed by optical microscope. Graphene on the Cu foils spun with PMMA is dissolved in Cu2Fe3, and the PMMA sheet is fished and transferred like the same method above onto the MoSe2 monolayers. PMMA is removed with acetone. Finally, the sample is annealed in vacuum at 300 °C for 3 h.The as-grown MoSeRaman and PL spectroscopy were carried out using a Horiba Jobin Yvon LabRAM HR-Evolution Raman microscope. The excitation light is a 532 nm laser, with an estimated laser spot size of 1 μm and the laser power of 1 mW.4 of the ultrafast visible-NIR/infrared microspectroscopy is illustrated in Fig. 49 or a near-IR-2 pulse. In ultrafast experiments, the visible or NIR-1 pulse is the pump light with the central wavelength and excitation power adjusted based on need. The interaction spot on the samples varies from 120 to 250 micron. The mid-IR super-continuum pulse or NIR-2 pulse acts as the probe light, which is focused at the sample by the reflective objective lens  to reduce the spot size to the level of the sample area (<40 μm). A 300-megapixel microscope digital camera is used to align the pump/probe beam to proper sample area. The probe light is detected by a liquid-nitrogen-cooled mercury-cadmium-telluride (MCT) array detector or InSb detector after frequency resolved by a spectrograph with a resolution of 1–3 cm−1, which is dependent on the central frequency. The time delay between the pump light and probe light is controlled by a motorized delay stage.The experimental setupThe data that support the findings of this study are available from the corresponding author upon request.Supplementary InformationPeer Review File


Using square wave voltammetry, we show an increase in the electrochemical detection of trinitrotoluene (TNT) with a working electrode constructed from plasma modified graphene on a SiC surface vs. unmodified graphene. The graphene surface was chemically modified using electron beam generated plasmas produced in oxygen or nitrogen containing backgrounds to introduce oxygen or nitrogen moieties. The use of this chemical modification route enabled enhancement of the electrochemical signal for TNT, with the oxygen treatment showing a more pronounced detection than the nitrogen treatment. For graphene modified with oxygen, the electrochemical response to TNT can be fit to a two-site Langmuir isotherm suggesting different sites on the graphene surface with different affinities for TNT. We estimate a limit of detection for TNT equal to 20 ppb based on the analytical standard S/N ratio of 3. In addition, this approach to sensor fabrication is inherently a high-throughput, high-volume process amenable to industrial applications. High quality epitaxial graphene is easily grown over large area SiC substrates, while plasma processing is a rapid approach to large area substrate processing. This combination facilitates low cost, mass production of sensors. Being all surface, graphene is an excellent material to sense adsorbates as these can alter the charge carrier concentration, leading to measurable changes in conductivity. Schedin et al. , exploitCarbon nanomaterials have recently received attention in electroanalytical sensing applications with examples in implantable sensors , ion senPrevious work on graphene-based electrochemical detection of nitroaromatic substances used a variety of preparation methods utilizing graphene or reduced graphene-oxide as a working electrochemical sensor and reports that limits of detection (LOD) for TNT range from 0.2 ppb to 100 ppb ,26,27,28In this manuscript, we show that plasma modified  epitaxia2 etched to prepare the SiC surface for epitaxial graphene growth by removing any polishing scratches created during the manufacturing of the SiC substrate and forming bilayer stepped morphology. After growth, the sample was cooled in Ar to 800 °C, at which point the reaction tube was evacuated. The thickness of the epitaxial graphene layers was ≈2 monolayers as determined by X-ray photoelectron spectroscopy (XPS) using a Thermo Scientific K-Alpha spectrometer with a monochromatic Al-Kα source using a 400 µm spot size. Chemical analysis was performed using Avantage.The epitaxial graphene was synthesized by means of Si sublimation from semi-insulating (SI), Si-face, on-axis, 6H-silicon carbide (SiC) substrates . The syn2) in a ground anode, and terminates at a second grounded anode located further downstream (50 cm). The slotted anode defines the beam cross section and the volume between these two anodes defines the processing region. The electron beam is magnetically confined to minimize spreading via collisions with the background gas, producing a sheet-like plasma in the processing region. Substrates (graphene) are placed on a processing stage located 2.5 cm from the electron-beam axis. After evacuating the processing reactor to base pressure (~1 × 10−6 Torr), reactive gases, either O2 or N2 depending on the desired functionalities, are introduced at 5% of the total flow rate with argon providing the balance to achieve a target operating pressure. To vary the functional group density on the surface of epitaxial graphene [The epitaxial graphene was chemically modified using electron-beam generated plasmas, which are well-suited for large area plasma processing of atomically thin materials . A detaigraphene , operatiEx-situ surface diagnostics were performed before and immediately after plasma processing to determine the starting material quality and chemistry and the changes resulting from plasma modification. Chemical changes and the resulting bonding characteristics in the graphene due to plasma processing were tracked by XPS. Chemical analysis was performed using Avantage and Unifit softwares.2. Since the current signal generated in electrochemical measurements is proportional to the area of the working electrode, it was advantageous to restrict the active electrode to a well-defined area. When working electrodes are manufactured from rigid substrates, it is practical to form a cell on top of the electrode using an open-ended container and a gasket to prevent leaks. In order to form such a cell and to allow for the electrical connection to the graphene/SiC substrate, a custom cell was designed. The cell contains an integrated gasket, a broad base for clamping, and a cut-out to allow maximum access to the working electrode. The reference and counter electrodes were suspended in the upper funnel-shaped well which also holds the electrolyte.Electrochemical measurements were performed using a potentiostat model #660 from CH Instruments. An electrochemical cell was composed of three electrodes , and reference (Ag/AgCl)) all immersed in (or in contact with) an aqueous electrolyte solution. The working electrode was the epitaxial graphene, as received or chemically-modified with an approximately projected area of 1.6 mm2/Ar mixtures resulted in the introduction of oxygen functionalities on the surface of graphene. Shown in 2 bonding is labeled as EG, the interfacial layer is labeled as IR, and the silicon carbide signal is referred to as SiC. The spectra presented in 2/Ar plasma [Electron beam generated plasmas produced in Or plasma .2/Ar mixture at various operating pressures as seen in 2, and interfacial layer, respectively. The peaks located at higher binding energies (>286 eV) are attributed to nitrogen and oxygen functionalities. Notably C–N and C–O a ≈286.1 eV, C=N at ≈287.4 eV, C=O and N–C=O at ≈288.3 eV, and O–C=O at ≈289.4 eV. The N1s spectra indicates that nitrogen functionalities are present primarily in the amide and pyrolic configurations.Similarly, nitrogen functionalities can be introduced in a controlled manner using electron beam generated plasmas produced in an NpcE. The current is then measured at different points of the potential waveform to minimize capacitive charging at the solution electrode interface. Since non-faradic current decays faster than faradic current [pcI, is determined (measured with respect to the background current taken without TNT) at pcE.The electrochemical response of the reduction of TNT at the graphene working electrode was characterized using square-wave voltammetry, which is a common electroanalytical technique for analytical applications since it diminishes non-faradaic charging currents that develop at the solution electrode interface when employing potential sweeping techniques . In squa current , a squarEpc = −0.8 V vs. Ag/AgCl) is shifted to significantly more negative voltages compared to oxygen functionalized graphene (Epc = −0.58 V vs. Ag/AgCl) and nitrogen functionalized graphene (Epc = −0.5 V vs. Ag/AgCl) consistent with a similar report [pcE and pcI are a reflection of both thermodynamic and kinetic properties of the electrochemical system, hence these oxygen functionalized working electrode results indicate that functionalization either increases the active surface area of the graphene or increases the rate of reduction of the TNT or both. The more negative pcE would decrease the resistance to interference with oxygen.To record the square wave voltammograms, the modified graphene samples were mounted in a custom built electrochemical cell and background measurements were recorded before the addition of TNT. Representative examples of the square wave voltammetry for the reduction of 10 ppm of TNT at these modified graphene samples are shown in r report . In compr report . In squaIpc and corresponding Epc are shown for a 10 ppm sample of TNT in PBS vs. nitrogen or oxygen functionalized content of the graphene samples. Results for unfunctionalized graphene, are given for 0% content. pcI with increasing content, however, pcE becomes less negative with increasing nitrogen content and begins to saturate at 13 atomic %  the S/N ratio is 6. From this, we estimate a LOD of 20 ppb based on the analytical standard S/N ratio of 3.In Equation (1), a is the proportion of current due to the reduction of TNT at sites a, KFor carbon electrodes, surface preparation is often critical for good electrochemical response of analytes . This haWe have shown a 6-fold increase in sensitivity towards the lower detection limit of TNT using chemically-modified graphene on a SiC surface compared to untreated graphene. The graphene surface was chemically modified using electron beam generated plasmas produced in oxygen- and nitrogen-containing backgrounds to introduce oxygen or nitrogen moieties. While both treatments enabled the enhancement of the electrochemical signal for TNT, oxygen-functionalized epitaxial graphene provided a more pronounced response. The oxygen functionalized graphene revealed a two-site Langmuir isotherm response extending over 2 orders of magnitude with a LOD of 20 ppb for TNT in PBS. The approach described here to fabricate graphene sensors sensitive to TNT is attractive for low cost, high-volume industrial production. This is because high quality epitaxial graphene is easily grown over large area SiC substrates and the plasma processing approach to oxygen functionalization is amenable to large area substrate processing.


Functionalized and fully characterized graphene-based lubricant additives are potential 2D materials for energy-efficient tribological applications in machine elements, especially at macroscopic contacts. Two different reduced graphene oxide (rGO) derivatives, terminated by hydroxyl and epoxy-hydroxyl groups, were prepared and blended with two different molecular weights of polyethylene glycol (PEG) for tribological investigation. Epoxy-hydroxyl-terminated rGO dispersed in PEG showed significantly smaller values of the friction coefficient. In this condition, PEG chains intercalate between the functionalized graphene sheets, and shear can take place between the PEG and rGO sheets. However, the friction coefficient was unaffected when hydroxyl-terminated rGO was coupled with PEG. This can be explained by the strong coupling between graphene sheets through hydroxyl units, causing the interaction of PEG with the rGO to be non- effective for lubrication. On the other hand, antiwear properties of hydroxyl-terminated rGO were significantly enhanced compared to epoxy-hydroxyl functionalized rGO due to the integrity of graphene sheet clusters. Lubricants reduce friction between contacting surfaces and thus increase the energy efficiency of moving machine components. Several remarkable properties of graphene-oxide/reduced graphene-oxide (GO/rGO) make it an effective solid additive for liquid lubricants1234513612The properties of the liquid lubricant, including molecular weight and polarity, are also decisive for lubrication1214151 and rGO2, respectively. The synthesized rGO was subjected to various microscopic and spectroscopic characterization techniques for detailed morphological, structural and chemical characterizations. The friction and wear properties of the rGO additives were investigated in two different molecular weights of polyethylene glycol (PEG). PEG is nonhazardous and it chemically interacts with rGO. Both rGO and PEG contain polar groups which provide effective dispersion stability through hydrogen bonding. Moreover, PEG is widely used in biomedical applications for reducing the friction and wear of knee joints18In this work, hydroxyl- and epoxy-hydroxyl-functionalized rGO samples were chemically prepared by controlled oxidation of bulk graphite powder followed by reduction. For simplicity, the hydroxyl- and epoxy-hydroxyl-functionalized rGO samples were designated as rGO1 and rGO2 at 2θ of 21.5° and 24.8°, respectively, indicating lattice spacing of 0.4 and 0.36 nm in rGO1 and rGO2, respectively  plane were observed in rGOectively . In crysThe folded and corrugated morphology of the rGO sheets is shown in the low resolution TEM images . The sur62 than in rGO1 samples, indicating a higher content of COOH complexes. This functional group is typically located at the edges of the graphene sheets, while the epoxy group evolves at the basal plane of the graphene sheets creating the in-plane defects and disorderIn XPS, the C 1 s peak of both rGO samples was de-convoluted into three chemically shifted segments, designated CX, CY and CZ at binding energies of 284.3, 285.8 and 288.2 eV, respectively 2023. Seg202 (0.72) than in rGO1 (0.84), indicating higher content of oxygen functionalities in rGO2. This is presumably associated with the higher concentration of the oxidizing agent KMnO4 used for the oxidation reaction of rGO2. The phenolic functional groups in rGO were investigated by de-convolution of the singlet O 1 s peaks into three individual segments, designated as OX, OY and OZ at 532, 533.4 and 535.4 eV, respectively  compared to rGO1 (4.23 and 7.2), indicating more carboxyl and carbonyl groups in the rGO2 sample. This result suggests denser epoxy network at the basal plane and a larger content of carbonyl linked at the edges of the graphene sheets in rGO2 sample compared to rGO1, associated with the harsher oxidation reaction in rGO220The ratio of the CX peak intensity to the sum of the (CY + CZ) peak intensities was smaller in rGOectively . Segment–1 and 1587 cm–1, respectively  and 2923 cm–1 (asymmetric) stretching modes, assigned to alkyl moieties (as a result of the reduction of COOH to -CH3 segment)27FTIR spectroscopy further revealed differences in functional groups between both types of rGO . In rGO1–1 was related to the C–OH group and it was stronger in rGO1 sample, indicating a larger content of hydroxyl groups–1 and 1220 cm–1, respectively, were weaker in rGO1 sample. This is expected when concentration of oxidizing substances such as KMnO4 is not sufficient to break the strong covalently bonded carbon symmetry at the basal plane. In this condition, most of the oxygen groups will functionalize the edge of the graphene planes without producing lattice distortions and defects at the basal planes. The peak at 1650 cm–1 was attributed to the bending of O–H or C = C stretching, indicating stability of the graphene structure271 and rGO2 sample and this is in agreement with the XPS data, indicating structural integrity. The shift of this peak to lower wave numbers in rGO2 sample may indicate stronger hydrogen bond coordination between the graphene-oxide sheets. This might be possible when hydroxyl-hydroxyl segments at the edge of the graphene sheets interact through hydrogen bonding1 and rGO2 spectra were dominated by hydroxyl and epoxy-hydroxyl groups, respectively. Therefore, the coupling of the graphene-oxide sheets in rGO1 was expected to be facilitated via hydroxyl-hydroxyl interactions, mostly at the edges of the graphene sheets, as schematically shown in 2 sample, coupling of graphene sheets is expected to be predominantly through epoxy-hydroxyl interaction forming hydrogen bonding between the basal plane and edges of the graphene sheets  units of the PEG backbone. The band at 3410 cm−1 was ascribed to the stretching mode of C–OH in PEG200, which shifted to 3470 cm−1 and became weaker in PEG600 due to weak inter-molecular hydrogen bonding and smaller amount of hydroxyl units28292/O–H stretching mode peak intensities in PEG600, indicate dispersive interactions between backbones of –CH2 units. However, in PEG200 intermolecular interactions are dominated by hydrogen bonding through hydroxyl-hydroxyl units. The wetting characteristics qualitatively describe the polarity of the molecules. This was studied by contact angle measurement, which showed a higher value (82°) for PEG600 than for PEG200 (65°) on steel substrates . The absorption peak of C–OH was stronger in rGO1 functionalized by PEG200. In this sample, hydroxyl functionality dominated, and the coupling of graphene sheet and PEG molecules was expected through the hydroxyl and oxyethylene unit of graphene-oxide sheet and PEG2 sample showed a weak absorption band of the hydroxyl unit, but the absorption band of the epoxy group was stronger (i&di), as expected from hydrogen bonding between the epoxy unit of rGO2 and the hydroxyl moieties of the PEG molecules. This relation is schematically drawn in 2 is characteristic of PEG600. The peak at 1721 cm–1 was ascribed to the C = O stretching vibration mode in the carboxylic functional group (–COOH), indicating possible hydrogen bonding between PEG and graphene oxide27312 sample blended with PEG200 and PEG600 due to large amount of carbonyl and carboxyl complexes, in agreement with the XPS analysis /I(G) ratio  compared samples . This cls sample . Further1, PEG molecules interact only with the graphene plane edges via hydrogen bonding with the hydroxyl groups there. However, when blended with rGO2, PEG molecules intercalation between the graphene planes was indicated, mediated by the on-plane epoxide groups, in addition to hydrogen bonding with hydroxyl groups.We conclude that the analysis indicates weaker interactions of PEG600 with both rGO types compared to PEG200 due to the smaller amount of hydroxyl content and larger inter-chain dispersive interactions in PEG600. For similar reasons, PEG200 shows better wetting of steel surface due to stronger interaction with polar groups on the steel surface. When blended with rGOIn-situ detailed friction data are presented in 35The results of load dependent friction behavior of neat PEG200 and PEG600 are presented in et al.1238394041The friction coefficient of PEG600 was found to be smaller than that of PEG200 throughout the entire measured load range. Intermolecular shear resistance in liquid generally increases with the viscosity, as expected for PEG600 (see above)261 and rGO2 samples as a function of the additive concentration in PEG200 and PEG600 at 1 N and 5 N are shown in in-situ friction data is given in The friction results of the rGO1 and rGO2 in PEG600 were lower than those of blends with PEG200, in accordance with the neat PEG properties discussed above. rGO1 blended in PEG200 and PEG600 showed higher values of the friction coefficient than rGO2 under both loading conditions . The principal interaction mechanisms in the rGO1 sample include electrostatic attraction between the oxygen atom in one layer and the carbon atom of the C–O moieties in an adjacent layer2 sample was the epoxy-hydroxyl interaction via hydrogen bonding  sample is schematically shown in 2 was predominantly functionalized by the epoxy groups, allowing coupling of hydroxyl units of the PEG with oxygen of the epoxy units at the basal plane in rGO2 sample. Inter-layer shear of the graphene sheets was enabled by the intercalated PEG molecules, explaining reduction of the shear resistance. This is schematically presented in 1 and rGO2 blends the friction coefficients were almost concentration independent  that forms a high strength boundary film, minimizing the metal-to-metal contact which enhanced the antiwear properties . Selecti1 N load , where t1 N load . Howevernditions . Furthernditions , similarnditions . Therefonditions . This isnditions . This mi1-compared to rGO2- blended in both PEG200 and PEG600. This is associated with intrinsic characteristics of hydroxyl terminated rGO sample which effectively protect against wear. It was noticed that antiwear performance was extremely good in the rGO1 sample at 1 N load  hydrophilic properties of PEG200, which inherently protects the surface against wear forming boundary film. This is independent of the chemical properties of rGO1 and rGO2, and (b) rGO1 is hydroxyl functionalized and the compressive strength of such a functionalized structure is higher than that of rGO2. This is because rGO1 functionalization creates few defects on the plane of the graphene sheets as compared to epoxy functionalization of rGO2221 and rGO2 sample without PEG are presented in 1 sample, as expected. It can therefore be concluded that PEG200 and rGO1 blends provide the best anti-wear properties due to favorable combination of the constituents’ properties.Moreover, results showed smaller wear track dimensions in the case of rGO1 N load , but the1 N load  and 8. Isistance . Calcula1 sample . This ca2 sample  derivatives were prepared for tribological investigation and blended with two different molecular weights of polyethylene glycol (PEG). Chemical and structural characterization indicated that rGO2-containing blends showed significantly lower friction coefficients compared to hydroxyl-terminated rGO1 additives blended in both types of PEGs. This was explained by intercalation of PEG molecules between the basal planes of the graphene sheets through epoxy-hydroxyl interactions between rGO2 and PEG. This decreased shear resistance of the sliding sheets and further reduced the frictional energy at the steel-steel interfaces. An extremely small concentration of rGO2, e.g. 0.02 mg/mL, was found to be sufficient to effectively lubricate the steel-steel sliding contact.The friction and wear properties of PEG with two different molecular weights, namely PEG200 and PEG600, were compared in steel-steel contact interfaces. The higher molecular weight of PEG600 showed a lower friction coefficient, explained by the longer methylene-chain unit and enhanced anti-wetting properties, indicating effective boundary film lubrication. PEG200 showed higher values of the friction coefficient due to a strong wetting of the sliding interfaces. These results were compared with the tribological properties of PEG blends with the two different rGO additives functionalized by hydroxyl and epoxy-hydroxyl groups. Epoxy-hydroxyl functionalized rGO1. This was explained by a smaller defect density induced by the functional groups in the planes of the graphene sheets, which preserved the mechanical integrity of the graphene clusters to sustain the load bearing capacity of graphene-oxide boundary film. This kind of lubricant additives is easy to prepare and may be suitable for various potential applications. We summarize that rGO1 reduces wear but increases friction, while rGO2 reduces friction but does not effectively reduce wear.The mechanism of friction is well explained by adsorption of graphene/graphene-oxide sheets in the tribo-contact region which act as low shear strength boundary film, as demonstrated by XPS analysis performed in the various locations of the wear track. The antiwear properties were superior in hydroxyl-terminated rGO3 (10 g) were mixed in 500 mL of H2SO4 (98%) at 5 °C with continuous stirring for 2 h, and KMnO4 (30 g) was gradually added to the suspension at a controlled reaction temperature lower than 15 °C. The ice bath was then removed, and the mixture was stirred at 35 °C until it became pasty brownish; stirring continued for two days, after which the mixture was diluted with a slow addition of 300 ml of water. The reaction temperature was rapidly increased to 98 °C, and the color changed to dark brown. This solution was further diluted by adding an additional 400 ml of water and stirring continuously. The solution was finally treated with 150 ml of H2O2 to terminate the reaction, indicated by the appearance of a yellow color. For purification, the mixture was washed by rinsing and centrifugation with 10% HCl and then with deionized water (DI) several times. After filtration and drying under a vacuum at room temperature, the GO1 was obtained as powder. Reduction was performed to reduce the functional group density, towards decreasing the inter-plane spacing and the flake dimensions and improving the friction properties. Reduction was done via treating 100 mg of GO1 in 30 mL of water with 3 mL of hydrazine and refluxing at 95 °C for 24 h, followed by filtering, washing and drying at 60 °C under vacuum to obtain the rGO1 powder.The sample was prepared by Hummer’s method443 (10 g) were mixed in 600 mL of H2SO4 (98%) at 5 °C with continuous stirring for 6 h, and KMnO4 (60 g) was gradually added to the suspension while maintaining the temperature at 15 °C. The mixture was diluted with a slow addition of 400 mL of water and with stirring maintained for 2 h. The ice bath was then removed, and the mixture was stirred at 35 °C for 2 h and then refluxed at 98 °C for 30 min. The solution was finally treated with 200 ml of H2O2 and the color changed to bright yellow. The resulting mixture was washed by centrifugation with 10% HCl and then with DI water several times until it became gel-like. After centrifugation, the gel was vacuum dried at 80 °C for 8 h to obtain the GO2 powder. Reduction was performed similarly to rGO1.Graphite flakes (10 g) and NaNO1 and rGO2.Two different molecular weights of PEG200 and PEG600 (from Sigma Aldrich) were used as synthetic lubricating base oils for tribology evaluation of the rGO additives. The average number of oxyethylene units in PEG200 is approximately 4 and its viscosity is 8.5 cSt. Viscosity of PEG600 is 18 cSt, and the average number of oxyethylene units is 13. The higher viscosity is caused by stronger intermolecular dispersive forces. Both rGO samples were dispersed and ultrasonicated for 3 h with a concentration of 1 mg/mL in PEG200 and PEG600. The samples were centrifuged, thoroughly washed in ethanol and DI water, and heat treated at 130 °C for 8 h to remove physisorbed PEG, ethanol and water molecules. Chemical analysis was applied to the dry powder samples of PEG200 and PEG600 functionalized by rGO−1, was used for the analysis of functional groups. Contact angle measurements of PEG200 and PEG600 on a steel surface were carried out using a contact angle meter  equipped with a CCD camera. The volume of the droplet used for the contact angle measurement was approximately 1 μL.The surface topography of the rGO samples were analyzed by atomic force microscopy . For AFM analysis, the rGO samples were ultrasonicated in ethanol for 2 h, resulting in partial exfoliation. The flakes were deposited on silicon substrates that were kept inside the ethanol bath during ultrasonoication. Elimination of the oxygen functionalities from the rGO was the main reason for exfoliation. The microstructure and crystallography were further investigated by high resolution transmission electron microscopy (HR-TEM) and XRD pattern was obtained using a PAN-alyticalX’pert Pro dual goniometer consisting of a CuKα radiation source (λ = 0.15418 nm). The chemical composition was investigated by XPS (ULVAC-Phi ESCA1800), using a monochromatic MgKα source  and by a micro-Raman spectrometer (Ranishaw) operating at a laser wavelength of 514.5 nm, which was also used for tribofilm analysis. An FTIR spectrometer (Bruker Optics), operating in transmission mode with a spectral resolution 4 cmBefore the tribology test, the rGO was thoroughly dispersed in PEG by ultrasonication, and its dispersion stability was monitored. The tribological properties of the rGO dispersed in PEG were studied by measuring friction and wear at various loads, and rGO concentrations were measured using a ball-on-disc standard tribometer , operating in a linear reciprocating mode. A 100Cr6 spherical steel ball with a diameter of 6 mm was used to slide against a lubricated steel 316 LN) disc at a sliding speed of 4 cm/s. The surface roughness values of the ball and disc were approximately 32 and 17 nm, respectively. In each test, two drops of the ultrasonicated PEG blended with the rGO lubricant were used to thoroughly lubricate the sliding interfaces. A more detailed tribological experimental procedure is described elsewhere16 LN disHow to cite this article: Gupta, B. et al. Role of oxygen functional groups in reduced graphene oxide for lubrication. Sci. Rep.7, 45030; doi: 10.1038/srep45030 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Using a solid electrolyte to tune the carrier density in thin-film materials is an emerging technique that has potential applications in both basic and applied research. Until now, only materials containing small ions, such as protons and lithium ions, have been used to demonstrate the gating effect. Here, we report the study of a lab-synthesised sodium-ion-based solid electrolyte, which shows a much stronger capability to tune the carrier density in graphene than previously reported lithium-ion-based solid electrolyte. Our findings may stimulate the search for solid electrolytes better suited for gating applications, taking benefit of many existing materials developed for battery research. Therefore, in the past, only the electronic transport of semiconducting materials could be effectively controlled with this technique. It is difficult to induce a considerable conductivity change in metals and superconductors. In the last decade, a new gating technique based on liquid electrolytes was developed. The liquid gating technique relies on the accumulation of ions near the interface between a liquid electrolyte and solid sample and can induce a carrier density change larger than 1.0 × 1015 cm−2, which is high enough to induce a considerable electrical conductivity change in gold films2. However, the coverage of liquid electrolytes on a sample surface makes this technique not compatible with many surface analysis techniques. The liquid nature of the electrolytes also impedes its application in making practical field-effect devices. To overcome the drawbacks of liquid electrolytes, solid electrolyte gating could be a solution. It has been demonstrated that protons embedded in microporous silicon dioxide could effectively control electronic transport in ITO3. However, the proton-based technique is quite sensitive to humidity in air, which limits its application4. Last year, a few groups successfully demonstrated that lithium-ion-based solid electrolytes can be used to tune the electronic properties of magnetic thin films5, graphene6 and iron-based superconductors7. These works have proven the feasibility of the solid electrolyte gating technique. Until now, only solid electrolytes based on small ions, such as protons or lithium ions, have been used. A natural question, then, is whether solid electrolytes based on larger ions can also demonstrate carrier density tuning capability. If the answer is yes, many solid electrolytes developed for battery research can be explored to improve the performance of the solid electrolyte gating technique. Another motivation to search for new solid electrolytes is to gain better control of the electro-chemical doping effect. Electrolyte gating is not a pure electrostatic doping process9. In both liquid and solid electrolyte gating processes, the mobile ions in the electrolytes may intercalate into the solid samples under high gate voltage, leading to the electro-chemical doping effect, which is not always a favourable factor. For example, lithium ions can intercalate into certain iron-superconductors to increase the superconducting transition temperature10, but their intercalation into NbSe2 leads to the opposite effect11. Because the intercalation of ions depends strongly on the ionic radius and the stability of ions in the lattice of the parent material, it is highly desirable to have more solid electrolytes based on different fast-moving ions to meet the growing needs to tune the electronic properties of a rich variety of thin-film materials. In this letter we report the use of a Nasicon-type sodium-ion-based solid electrolyte (Na+-SE) to tune the carrier density in single-layer graphene. We show that it can induce a symmetric carrier density change in graphene, and its carrier density tuning capability is nearly one order of magnitude stronger than that of a previously reported Lithium-ion Conductive Glass-Ceramic (LICGC) solid electrolyte6. The double-layer capacitance of the solid electrolyte and the quantum capacitance of graphene were also extracted from the transport measurements data.Using an electric field to control electronic properties is an often-used method in materials science. It is also the key technique in the modern semiconducting industry. Traditionally, this technique relies on oxide-dielectric materials such as silicon dioxide. As an all-solid-state technique, it is easy to use and can be applied even at cryogenic temperatures. The main drawback of the oxide-dielectric-based technique is its weak capability to tune carrier density, which is usually limited to less than 1.0 × 10+-SE substrate is a white-looking material. The raw material is in cylinder form, as shown in the inset of Fig. +-SE substrates used in following experiments was hand-polished (Results using fine-polished electrolytes are shown in the supplementary information). Figure The Na+-SE substrate. The resistance measurements were performed at 300 K in Helium atmosphere. Initially, the back-gate voltage (Vg) was swept towards the positive direction. The resistance peak appeared near 0.4 V. The voltage sweeping was interrupted at each selected positive voltage point to perform the Hall measurement. The discontinuity in the R vs. Vg plot was caused by the delayed accumulation of sodium ions at the electrolyte/graphene interface during each Hall measurement6. After reaching the maximum gate voltage of 2.4 V, the gate voltage was swept back to zero in a continuous manner. There was a slight shift of the resistance peak position that was also caused by the delayed motion of sodium ions. We waited approximately eight hours at zero gate voltage to let the sample resistance relax to its initial value, as indicated by the dashed line in Fig. Figure +-SE substrate can effectively tune the Fermi level in graphene. To quantify the carrier tuning capability of the electrolyte, we deduced the carrier density and mobility from Hall measurement results. Figure 14 cm−2 at 2.4 V; on the negative voltage side, the hole density reached 3.0 × 1014 cm−2 at −1.2 V. Such high carrier density tuning capability is much stronger than that reported for LICGC solid electrolyte6. Indeed most of our samples can reach an electron density larger than 3.0 × 1014 cm−2 at a gate voltage near 2.5 V. Such performance is truly comparable and even stronger than that of a few commonly used liquid electrolytes such as DEME-TFSI and ABIM-TFSI12. We noticed a drop in the hole density near −1.4 V. Such behaviour is common in liquid/solid electrolyte gating experiments. Because graphene is a chemically stable, it was probably solid electrolyte/surface contaminants that decomposed at high voltage and thus changed the electro-chemical doping of the graphene sheet. The details of the electro-chemical reaction at the graphene/electrolyte interface are still under investigation.The ambipolar behaviour in the resistance measurement is preliminary evidence that the Na2V−1s−1. This value is nearly one order of magnitude lower than that of our single-layer graphene sheet transferred onto other substrates such as SiO2. Figure 14: 5 cm2 V−1 s−1, which is much larger than the measured Hall mobility. We can therefore neglect its contribution. The Coulomb scattering is independent of the carrier density: μc ~ constant. The short-range scattering is inversely proportional to the carrier density: e = 2.5 × 10−3 cm−2 V s and Figure F is the Fermi energy measured from the Dirac point. The total capacitance −2, which is one order of magnitude larger than the value found for the ionic liquid electrolyte BMIM-PF615. We also deduced from the fitting a reduced Fermi velocity ~16. A similar low Fermi velocity was previously reported in graphene irradiated with Ar+ ions17. The Fermi velocity in graphene follows 18. The impurities at the electrolyte/graphene interface may reduce the amplitude of the nearest-neighbour hopping of electrons in graphene, which leads to a decrease in the Fermi velocity.Tuning of the carrier density in graphene can be viewed as a process of charging capacitors in series. The inset of Fig. 15. The non-zero value of the quantum capacitance is due to the additional carrier density After finding the double-layer capacitance from the fitting of the carrier density, we can deduce the quantum capacitance of the single-layer graphene sheet. The total capacitance is defined as +-SE over lithium-ion-based solid electrolytes such as previously reported LICGC. First, Na+-SE provides a much stronger electrostatic carrier density tuning capability. For a single-layer graphene sheet, Na+-SE can induce an average electron density change larger than 6+-SE is even better than most commonly used liquid electrolytes.We summarise the advantages of Na+ (102 pm) is much larger than that of Li+ (76 pm)19. Intuitively speaking, it will be more difficult for sodium ions to intercalate into solid samples compared with lithium ions. This will be a favourable factor if one wants to reduce the influence of the electro-chemical doping effect. For example, it is known that Na+ is chemically inert and does not adhere to specific locations on graphene15. Sodium is also difficult to intercalate into graphite19. Therefore sodium-ion-based solid electrolyte can be an ideal material to realise electrostatic doping in single/multi-layer graphene. As shown in Fig. +-SE primarily tunes the carrier density in single-layer graphene in an electrostatic manner within the above mentioned gate voltage window.Second, the ion radii of Na+-SE may also induce electro-chemical doping in certain thin-film materials, which is also an attractive factor for future study because it provides us a method beyond the traditional material synthesis technique to induce exotic electron behaviour in sodium-containing materials. In conclusion, the successful demonstration of the carrier density tuning capability of Na+-SE further extends the research of solid electrolyte gating technique, and it suggests that the existing solid electrolytes are indeed a treasure waiting to be explored.Of course, the electro-chemical intercalation process is not solely determined by the ion radius. Na3.1Zr1.95Mg0.05Si2PO12 was synthesised through a solid-state reaction combined with mechano-chemical synthesis20. First, a Zr1.95Mg0.05O3.95 solid solution was prepared through mechano-chemical reaction in a high-energy ball mill for 2 hours (SPEX SamplePrep 8000 M Mixer). A mixture of ZrO2  and MgO was milled by alternating 30 minutes of milling with 30 minutes in standby mode to avoid excessive heating. The solid electrolyte with the formula Na3.1Zr1.95Mg0.05Si2PO12 was synthesised through a solid-state reaction by mixing stoichiometric amounts of Na2CO3 , SiO2 , NH4H2PO4 , and Zr1.95Mg0.05O3.95, and ball-milling with zirconium oxide balls for 2 hours. The precursors were decomposed at 900 °C for 12 hours in alumina crucibles, with repeated ball-milling for 2 hours. The calcined powders were then cold pressed and sintered at 1260 °C for 16 hours covered with the raw powders to avoid sodium loss. The obtained bulk solid electrolytes were hand polished using aluminum oxide sandpaper (P1200). Instead of water, ethanol was used during polishing to prevent water damage.Na3 solution. The floating graphene/PMMA layer was first transferred onto a Si/SiO2 substrate and rinsed successively in deionised water and isopropanol (IPA). It was then transferred to the solid electrolyte substrate in IPA. The PMMA capping layer was removed by acetone. The samples were then annealed in a hydrogen–argon atmosphere for 7 hours. The electrical contacts to graphene were made by pressing indium pellets onto graphene. The solid electrolyte substrate was attached to a chip-carrier using conductive glue. The gate voltage was applied to the backside of the substrate using conductive glue.Single-layer graphene samples were initially grown on copper substrates by the chemical vapour deposition (CVD) technique. A single-layer graphene sheet was transferred onto the solid electrolyte using a wet transfer method. A 250-nm-thick Poly(methyl methacrylate) (PMMA) layer was first spin-coated on top of the graphene. The graphene/PMMA layer was detached from the copper substrate using a 10 wt. % FeClThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.supplementary material


Corticostriatal circuits are widely implicated in the top-down control of attention including inhibitory control and behavioural flexibility. However, recent neurophysiological evidence also suggests a role for thalamic inputs to striatum in behaviours related to salient, reward-paired cues.Here, we used designer receptors exclusively activated by designer drugs (DREADDs) to investigate the role of parafascicular (Pf) thalamic inputs to the dorsomedial striatum (DMS) using the five-choice serial reaction time task (5CSRTT) in rats.The 5CSRTT requires sustained attention in order to detect spatially and temporally distributed visual cues and provides measures of inhibitory control related to impulsivity (premature responses) and compulsivity (perseverative responses). Rats underwent bilateral Pf injections of the DREADD vector, AAV2-CaMKIIa-HA-hM4D(Gi)-IRES-mCitrine. The DREADD agonist, clozapine N-oxide  or vehicle, was injected into DMS 1 h before behavioural testing. Task parameters were manipulated to increase attention load or reduce stimulus predictability respectively.We found that inhibition of the Pf-DMS projection significantly increased perseverative responses when stimulus predictability was reduced but had no effect on premature responses or response accuracy, even under increased attentional load. Control experiments showed no effects on locomotor activity in an open field.These results complement previous lesion work in which the DMS and orbitofrontal cortex were similarly implicated in perseverative responses and suggest a specific role for thalamostriatal inputs in inhibitory control. Adapting to changing environments requires the allocation of attentional resources including inhibition of inappropriate responding to allow efficient goal-directed behaviour. These functions have traditionally been associated with the corticostriatal network; however, emerging evidence suggests that corticostriatal inputs are subject to thalamic modulation in the striatum  produce deficits in inhibitory control measured in the five-choice serial reaction time task (5CSRTT), which allows the assessment of both attentional and inhibitory control Robbins . DMS lesi-coupled DREADD allowed the inhibition of Pf-DMS projections which is thought to be due to synaptic silencing  with constant temperature (22 °C) and humidity (50%). Ad libitum food was restricted to 1–2 h/day on weekdays and was freely available on weekends. Weights were monitored and remained at ∼90% of free-feeding weight. Behavioural testing was carried on weekdays during the dark phase. All experimental and surgical procedures were done in compliance with the Animals (Scientific procedures) Act 1986, UK.Training was carried out in four operant chambers  placed in sound-attenuated, ventilated cubicles. One side of the chambers comprised the five-choice panel, curved concavely with five apertures with stimulus lights at the back (prod. no. ENV-115A-07V). The other side contained a recessed food magazine, a magazine light and a house light. Entries into all apertures including the food magazine were monitored by a photocell infrared beam. A data acquisition board  interfaced with the operant chambers and custom-written software were used for experimental control and behavioural data acquisition . Dustless precision pellets were used as rewards .The 5CSRTT, as previously described by Robbins , involveInitial training was carried out with longer stimulus durations and limited holds and shorter ITIs which were gradually adjusted based on individual performances to reach target parameters which were stimulus duration (SD), 1.5 s; limited hold (LH), 5 s; ITI, 5 s and timeout 5 s. Virus injection and cannula implantation were carried out once animals achieved 80% accuracy .Two weeks following surgery, rats were retrained to criterion and tested on two challenges in the paradigm. The attentional challenge consisted of presenting trials with shorter SDs  in order to increase attentional load. LH and ITI were fixed at 5 s. A second challenge to test inhibitory control consisted of a varied ITI duration within each session to reduce stimulus predictability . Omissions were calculated as the percentage of omitted trials out of the total number of trials. Premature and perseverative responses were used as measures of inhibitory control. Premature responses were counted as responses made during the ITI and calculated as a percentage out of all trials. Perseverative responses were defined as nose-pokes into one of the five apertures after a correct response was made but before collection of the reward as has been reported previously  in O2 and maintained between 2 and 3%. The animal was mounted in a stereotaxic frame, and the head was adjusted so that lambda and bregma were aligned on the same horizontal plane. To prevent corneal desiccation, Lacri-Lube Eye Ointment  was applied to the eyes. A homoeothermic heat pad  was used to maintain body temperature between 36 and 37 °C. Glucose  was given via an infusion pump  for the duration of the surgery. Small craniotomies were made directly above the virus injection sites; Pf target coordinates were −4.2 mm AP, ±1.8 mm ML and −5.5 mm DV (Paxinos and Watson Rats were anaesthetised with 4% A 1-μl Hamilton syringe (Neuros 7000 Series) was mounted on an infusion pump  and filled with 0.8 μl of virus, AAV2-CaMKIIa-HA-hM4D(Gi)-IRES-mCitrine (University of North Carolina – Vector Core). The virus was injected at a rate of 50 nl/min into Pf, and the injection needle was left in place for a further 10 min to allow diffusion. This injection was done bilaterally with the syringe cleaned and reloaded with virus between injections . Stainless steel stylets were placed in the cannulas to prevent blockage.A non-steroidal anti-inflammatory  was administered 1–2 h before recovery and twice a day for 5 days post-surgery. An antibiotic  was given immediately after recovery and twice daily for 5 days after surgery.One hour prior to testing, 1 μl of 3 μM CNO or vehicle  was injected through the DMS cannula guides using a 10-μl Hamilton precision syringe connected to polyethylene tubing with a 30-gauge steel injector on the end. The injector protruded beyond the cannula length by 1 mm. CNO was injected at a rate of 0.5 μl/min, and the injector was left in place for another minute to allow for drug diffusion. The CNO dose used here was similar to published work  using a digital camera and the ANY-MAZE tracking software . Each session consisted of 40 min, and all animals completed two sessions  on two separate days. One hour prior to testing, the animals were injected with 1 μl of either CNO or artificial CSF.w/v in saline) and kept at 4 °C until sectioning. Coronal sections of the striatum and the thalamus were cut at 50-μm thickness using a vibratome . Sections were incubated in a blocking solution consisting of 10% normal donkey serum (NDS) in PBS containing 1% Triton X-100 for a minimum of 1 h. Sections were then washed three times in PBS and incubated with antibody against green florescent protein  in 1% normal donkey serum (NDS), 0.03% Triton X-100 in PBS. Sections were mounted in slices and protected with Vectashield. Z-Stack fluorescent images were obtained with a confocal microscope  using the 504-nm filter for Alexa Fluor-488 at two different magnifications, ×4  and ×20 , at 2048 × 2048 resolution and a digital camera . The brightness and contrast of the images were subsequently adjusted in ImageJ. Virus diffusion area and injector tracks were reported on a single projection slice.DREADD expression in Pf cell bodies and axon terminals in the DMS was verified using labelling with green fluorescent protein. Rats were terminally anaesthetised with pentobarbital  and perfused with 5% formaldehyde. Brains were extracted and kept in saline for 2 days at 4 °C and subsequently transferred to 0.05% sodium azide (http://pcl.missouri.edu/bayesfactor). In all cases, the scale of r was set up to 1.0 which serves as a natural benchmark  with drug (CNO vs. vehicle) and parameter value (ITI or SD duration) as the within-subject variables. Statistical analyses were performed using SPSS . In several cases, drug treatment was critically found to have no effect on omissions or in locomotor activity. In these cases, we computed Bayes factors for use in supporting the null hypothesis using a freely available Bayes factor calculator  = 0.08, p = 0.788 and F = 0.25, p = 0.617, respectively].Prior to testing, animals reached an average accuracy of 86.5 ± 1.5% (mean ± SEM) and an average of 20.0 ± 4.2% omissions. CNO did not have an effect on the total number of trials that the rats completed on the varied ITI or the shorter SD challenges  and an increase in premature responding   where ITI duration was varied within-session: 4.5, 6, 7.5 or 9 s. This experiment was analysed using a 4 × 2 × 2 (ITI duration x drug x repetition) within-subjects ANOVA. The variable repetition was added since two CNO and two vehicle days were recorded. Due to a violation of the sphericity assumption, the Greenhouse-Geisser correction was applied. Reduced stimulus predictability affected response measures as expected: we saw an increase in omissions , omissions  or premature responses   = 7.13, p = 0.032]  = 0.72, p = 0.550]. CNO treatment did not affect response latency or latency to collect rewards  . CNO or vehicle was injected on 2 days , and the experiment was analysed using a 2 × 2 × 2 (stimulus duration × drug × repetition) within-subjects ANOVA. The variable repetition was added since two CNO and two vehicle days were recorded.F = 12.83, p = 0.009], and percentage omissions were higher . Also, as expected response latencies were shorter for 1-s stimuli than for 2-s stimuli . Stimulus duration did not affect perseverative responses .As expected, accuracy was lower for 1 vs. 2 s stimuli , percentage of omitted trials , premature responses , perseverative responses , response latency  or latency to collect rewards . There were no CNO × SD interactions on either of the performance measures. A Bayesian analysis conducted on omission trials found that these data are 3.87 times more likely to be observed under the null hypothesis predicting no difference between CNO and vehicle.CNO did not appear to affect response parameters in this challenge of the task: CNO had no significant effect on accuracy .Finally, our analysis of perseverative responses followed previous work on response inhibition  = 1.17, p = 0.314 and F = 1.17, p = 0.339, respectively]. A Bayesian analysis found that these data are 2.34 times more likely to be observed under the null hypothesis predicting no difference between CNO and vehicle. As expected, locomotor activity showed significant habituation over time  . Distance travelled (m) was binned into eight 5-min time windows. A 2 × 8 (drug × bin) within-subject ANOVA revealed no significant effect of drug or drug × bin interaction [The present study investigated the role of the Pf-DMS pathway in inhibitor control and attention. We used chemogenetic inhibition to temporarily and specifically inactivate the Pf-DMS pathway and assessed performance on behavioural measures in the 5CSRTT. Viral expression in Pf cell bodies and processes in DMS was confirmed immunohistochemically. Task parameters were systematically manipulated to increase attentional load or reduce stimulus predictability, respectively. The main finding of the study is that inhibition of the Pf-DMS projection significantly increases perseverative responses (a failure of inhibitory control related to compulsivity) compared to vehicle when stimulus predictability was reduced but had no effect on accuracy, even under increased attentional load.It is unlikely that non-specific effects of CNO injections into DMS could explain the observed pattern of results. Firstly, gross locomotor effects are unlikely because we did not observe an effect on the total number of trials completed, response latencies, reward retrieval latencies and ambulatory activity in an open field. This conclusion was supported by Bayesian analyses which suggested that the null hypothesis (i.e. no effect) was more strongly supported by the data. While our sample size was similar to previous reports of within-subject pharmacological manipulations in the five-choice serial reaction time task , they were unable to update the action-outcome association and thus failed to reduce responding to the degraded lever  (Brown et al. Finally, we have to acknowledge that the current study did not specify the type of Pf cells targeted with CNO. Studies suggest that Pf striatal projections are mainly glutamatergic (Smith et al. In summary, the present study examined the role of thalamic input to the dorsomedial striatum in attention and behavioural flexibility using the 5CSRTT. We manipulated task parameters to either increase attentional load or reduce stimulus predictability to challenge behavioural flexibility; we exploited the selectivity afforded by the DREADD approach to specifically interrogate the contribution of the Pf-DMS pathway to these processes and found that thalamic projections are implicated in perseverative responses, indicating a role of this projection in inhibitory control. Our results suggest that the behavioural role of dorsomedial striatum must be considered in the context of thalamostriatal projections in addition to previously described prefrontal inputs.


The abrupt boundaries of nanowires create electronic junctions via spatial biomolecular doping of graphene and manifest themselves as a self-assembled electronic network. Furthermore, designed peptides form nanowires on single-layer MoS2 modifying both its electric conductivity and photoluminescence. The biomolecular doping of nanosheets defined by peptide nanostructures may represent the crucial first step in integrating biology with nano-electronics towards realizing fully self-assembled bionanoelectronic devices.Self-assembly of biological molecules on solid materials is central to the “bottom-up” approach to directly integrate biology with electronics. Inspired by biology, exquisite biomolecular nanoarchitectures have been formed on solid surfaces. We demonstrate that a combinatorially-selected dodecapeptide and its variants self-assemble into peptide nanowires on two-dimensional nanosheets, single-layer graphene and MoS We show that peptides assemble into organized or disorganized structures distinctively modifying electrical and optical properties of single-layer graphene and MoS2, respectively. The ability to regulate electronic states of atomic single layer materials defined by peptide sequences establishes the first step in a unique route to integrate biology with 2D nanomaterials towards realizing fully self-organized biomolecular-electronics.Interface between electronic materials and biomolecules is essential in integrating human-made devices with biological systems178e.g., amyloids, also form ordered patterns on mica and graphite72, WSe2, etc.) are new members of 2D semiconductors with unique electronic202223Proteins regulate biomineral formation in organisms through specific molecular interactions leading to self-assembly of hard tissues  with exceptional physical functions101213via chemical doping25262 with its photoluminescenceElectronic characteristics of graphene are highly sensitive to adsorbed molecules 2, WSe2, WS2, MoSe2, and hBN. Atomic force microscopy (AFM) images show peptide organized structures with six-fold symmetry  of graphene and MoS2.In our previous work, graphite-binding dodecapeptides (GrBPs) were combinatorially selected using a phage display librarysymmetry . A simpl2, indicating they only specifically bind to graphene.First, we demonstrate that GrBP5-WT also spontaneously forms ordered structures even on single-layer graphene in aqueous solution at room temperature . AFM ima2  and Glutamic acid (E) residues were replaced with Alanine (A), a charge-neutral amino acid. Similarly, M8 was designed with “YRRY”, containing positively-charged Arginine (R) within the binding domain.Instead of nanowires on graphene, the WT peptide forms confluent disorganized film on single-layer MoS2 . To mainsequence . First, 2 and high aspect ratio structures even on the other layered materials: MoSe2, WS2, WSe2, and BN  worked remarkably well in the formation of nanowires on MoS, and BN . Similarnd BN  upon peptide assembly . While pi.e., “random islands”, without the symmetry of the underlying solid lattice . Here, Tyrosines (Y), with phenyl group in the WT’s binding domain, are replaced by Tryptophans (W) containing indole moiety. Possibly due to strong binding affinity of Tryptophan on graphene, M2 forms disorganized nanostructures,  lattice . Corresps to 96% . The abs2/Vs (as-prepared GFETs) and stays constant over the full range of coverage, the mobility modified by WT decreases to 300 cm2/Vs at 20% coverage and gradually recovers up to 2800 cm2/Vs as coverage increases and peptides assemble into more ordered nanowires. Similar to the reduction in mobility observed by chemical adsorbates on graphene30To investigate the observed effects further, we quantitatively analyze graphene conductivity by fitting with a well-accepted equation, representing series resistance of two independent regions in a GFET  see Met. The fitTo gain better insight into possible effect of peptide molecular folding and subsequent ordering, we carried out DFT  simulationvs. disorganized peptide nanostructures on graphene conductivity, we next examine the same in single-layer MoS2. Mechanically exfoliated single-layer MoS2 is used in FET configuration to quantify conductivity modulated by the peptides  spectra obtained from peptide-functionalized MoS2 display a similar tendency as the conductivity measurements. While M6 shows a large peak shift of 9 meV in the PL spectrum, M8 exhibits no shift  or optical (MoS2) characteristics. The work opens up potential utility of 2D nanomaterials in biology and medical implementationsin vivo integration of the worlds of biology and solid-state devices of the future.We show here that peptides can form organized biomolecular nanostructures on single-layer graphene and MoS2/Si wafer. We employed this resist-free process, instead of a conventional lithography, because of the difficulty to completely remove residues from the resists that disrupt peptide self-assembly into ordered structures. Second, we directly place a droplet of peptide aqueous solution on GFETs to prevent contamination. After the incubation, samples are quickly dried by nitrogen blow. In the solution process, water molecules are known to significantly change the conductivity of the graphene on SiO2 in the presence of oxygen. To prevent the water effect, we utilize deoxygenated DI water prepared by bubbling with nitrogen or argon gases. All the conductivity measurements, therefore, have been performed in argon atmosphere to eliminate the water effect. In the conductivity measurement, gate-voltage was cyclically swept over a range from 0 V to maximum, from maximum to minimum, and back to 0 V.Special care was taken for reliable characterization of graphene conductivity induced by the ordered peptides. First, GFETs are fabricated by direct soldering of indium contacts to an exfoliated graphene on a SiOWe use a modification of the original equation  for the R is total resistance of graphene, Rc is contact resistance, e is electron charge, μ1,2 is charge mobility, no1, no2 are residual density of electron-hole puddles on SiO2 that screen the electric potential from the bottom gate, C is capacitance of the GFET, V is gate voltage, θ is the coverage of the peptides on graphene, and V1,2 is CNP.where, How to cite this article: Hayamizu, Y. et al. Bioelectronic interfaces by spontaneously organized peptides on 2D atomic single layer materials. Sci. Rep.6, 33778; doi: 10.1038/srep33778 (2016).


Sarcoidosis is an idiopathic, noncaseating granulomatous disorder with wide systemic involvement. It is encountered widely around the world and it affects both sexes, all the races in all age groups. Lungs, eyes, and skin are the organs most commonly affected. Constitutional features such as weight loss, fatigue, and myalgia are the most common symptoms. Bone involvement, which is very rare, was reported as present in 3 to 13% of effected cases, and it is most commonly seen in hands and feet, compared with long bone involvement, which is extremely rare. We hereby present a case with a diagnosis of sarcoidosis and multiple bone involvement emphasizing the importance of differential diagnosis. Sarcoidosis is an autoimmune disorder of unknown etiology with noncaseating granulomatous inflammation and multiple organ and system involvement. It is more common in individuals younger than the age of 50 years with a slight predominance in females.In this report, we aimed to present a case with sarcoidosis and bone involvement and emphasize on the importance of differential diagnosis because sarcoidosis can mimic any granulomatous disease, primary or metastatic cancers to the bone.A 44-year-old female patient was admitted to our outpatient clinic due to complaint of right ankle pain. The patient, who described the pain as continuous during the day unrelated to physical activity, informed us about the past medical history of sarcoidosis. She had applied to a health care facility due to pale and nodular lesions present on trunk, upper and lower extremities 8 years ago . We fouSarcoidosis is a disease characterized by multisystemic involvement of noncaseating granulomas of unknown etiology. It is commonly encountered with lung, skin, and lymph node involvements. Total 80% of cases diagnosed with sarcoidosis are females.An acute form of sarcoidosis is a course defined with arthritis, erythema nodosum, and bilateral hilar lymphadenopathy, called Löfgren's syndrome.Although the etiology of sarcoidosis is not known definitely, genetic (human leukocyte antigens) and acquired factors  play an important role.Sarcoidosis generally involves skull, vertebrae, nasal bones, as well as bones in hands and feet. Lytic and sclerotic lesions have been observed in cases of vertebral sarcoidosis. Thoracic spine is the most frequently affected site in vertebral sarcoidosis.Radiologically, it may manifest as cystic or osteolytic lesions or as cortical defects and reticularizations of cortical bones as well as sclerotic and destructive lesions or similar to periostitis.Other granulomatous diseases of the bone, such as Langerhans cell histiocytosis, tuberculosis with bone involvement, fungal infections, viral infections such as Epstein–Barr virus and cytomegalovirus, and Ewing sarcoma, as well as primary and secondary metastatic bone tumors must be included in differential diagnosis.In bone biopsies, granulomas in medullary cavity and destruction in surrounding bone tissue are characteristic.There are different drug alternatives in the treatment of sarcoidosis. There are different drug alternatives depending on the site of involvement in the body .Consequently, the definitive diagnosis should be made with biopsy followed by pathologic investigation. The importance of other systemic examinations and anamnesis should be emphasized to suspect of sarcoidosis. Certainly, diagnosis of sarcoidosis should be consistent systemic finding with biopsy.


These results suggest that TA microscopy is a valid tool for the study of graphene based materials.We report a transient absorption (TA) imaging method for fast visualization and quantitative layer analysis of graphene and GO. Forward and backward imaging of graphene on various substrates under ambient condition was imaged with a speed of 2 μs per pixel. The TA intensity linearly increased with the layer number of graphene. Real-time TA imaging of GO  Graphene and graphene oxide (GO) have attracted great interest and been considered as promising materials for, for example, future electronics1357in vitro imaging of graphene, but this contrast is not sensitive to GOTo image graphene with layer sensitivity, various microscopic and spectroscopic techniques have been developed. Atomic force microscopy2111013151714GO with abundant oxygen-containing groups can be readily modified with targeting ligands to facilitate drug delivery72425728253032Here, we report a label-free highly sensitive imaging method for fast visualization and quantitative layer analysis of graphene and graphene oxide based on the transient absorption (TA) process. TA imaging has been developed for visualizing single nanomaterials, such as gold nanoparticles343739404244In this work, we demonstrate TA imaging with single layer sensitivity. We used megahertz modulation that effectively avoids the low-frequency laser noise and employed a resonant circuit that electronically amplified the heterodyne-detected signal. On this imaging platform, we achieved high speed (2 μs/pixel) imaging of graphene on various substrates  under ambient condition and of graphene oxide in living cells and animals. The intensity of TA images is found to linearly increase with the layer number of graphene. It takes a few seconds to acquire a TA image of graphene samples, which is much faster than Raman mapping. More importantly, our method is able to image graphene and GO in biological environment with capability of quantitative analysis of intracellular concentration of well-dispersed GO functionalized with polyethylene glycol (PEG).I is quantized and linearly proportional to the number of graphene layers, n, with I = nIs where Is is the signal intensity from single layer graphene area. To further confirm the layer resolved by the image, we measured Raman spectra from different domains using the Raman capability on our transient absorption microscopy platform−1) to 2D band (~2620 cm−1), together with the intensity of G band and the peak shape of 2D band, was taken in consideration to confirm the layer number. The Raman spectrum  induced by the presence of pump. This signal can originate from different optical processes, including optical bleach, stimulated emission, excited state absorption, and multiphoton absorptionprobe/Iprobe > 0) or negative (ΔIprobe/Iprobe < 0) depending on different processes. For example, signals from optical bleach and stimulated emission are positive, while signal from absorption is negative. Here, we observed a positive TA signal from graphene, indicating an increase of transmitting probe induced by pump. We also measured the dependence of TA signal level on pump and probe laser intensities  at density of 1 × 105 cells/plate. GO-PEG solution was added to the cell culture medium 24 h after plating at a final concentration of 10 μg/ml. Cells were incubated at 37 °C for 0~3 h. Then the dishes were placed under optical microscope for imaging. For in vitro TA imaging, a femto-second coherent laser was used. The pump beam was at 1090 nm with average power of 20 mW at sample, and the probe beam was at 850 nm with average power of 5 mW at sample. At this frequency, the SRS signal from C-H or other bands in cells can be avoided. The scanning speed for all experiments is 2 μs/pixel.Chinese hamster ovary (CHO) cells were used for imaging. CHO cells were cultured in DMEM/F-12 medium supplemented with 10% FBS at 37 °C incubator with 5% COin vitro imaging process.All protocols were approved by the Purdue University Animal Care and Use Committee and were performed in accordance with guidelines. Adult Long-Evans rats (~300 g) were anesthetized by isoflurane. 500 μl of 2 mg/ml GO-PEG solution in PBS was injected through the jugular vein. 30 min after the injection, a small piece of rat ear was cut off and placed under the microscope for TA imaging. The imaging parameters were the same as those used in How to cite this article: Li, J. et al. Highly sensitive transient absorption imaging of graphene and graphene oxide in living cells and circulating blood. Sci. Rep.5, 12394; doi: 10.1038/srep12394 (2015).