Long Interspersed Nuclear Element-1 (L1) is an oncogenic mammalian retroelement silenced early in development via tightly controlled epigenetic mechanisms. We have previously shown that the regulatory region of human and murine L1s interact with retinoblastoma (RB) proteins to effect retroelement silencing. The present studies were conducted to identify the corepressor complex responsible for RB-mediated silencing of L1.Chromatin immunoprecipitation and silencing RNA technology were used to identify the repressor complex that silences L1 in human and murine cells.Components of the Nucleosomal and Remodeling Deacetylase (NuRD) multiprotein complex specifically enriched the L1 5′-untranslated DNA sequence in human and murine cells. Genetic ablation of RB proteins in murine cells destabilized interactions within the NuRD macromolecular complex and mediated nuclear rearrangement of Mi2-β, an ATP-dependent helicase subunit with nucleosome remodeling activity. Depletion of Mi2-β, RbAP46 and HDAC2 reduced the repressor activity of the NuRD complex and reactivated a synthetic L1 reporter in human cells. Epigenetic reactivation of L1 in RB-null cells by DNA damage was markedly enhanced compared to wild type cells.RB proteins stabilize interactions of the NuRD corepressor complex within the L1 promoter to effect L1 silencing. L1 retroelements may serve as a scaffold on which RB builds heterochromatic regions that regulate chromatin function. Human L1s are non-LTR mammalian retrotransposons that consist of an internal bidirectional promoter, two open reading frames encoding for ORF1p (RNA-binding protein) and ORF2p (reverse transcriptase and endonuclease activities), a 3′- untranslated region (UTR), and a polyA tail [We have previously shown that recruitment of E2F/RB (E2F-retinoblastoma) to the L1 promoter, along with histone deacetylases 1 and 2  , 5 and mMCF7 cells, HeLa cells, primary mouse embryo fibroblasts (MEFs) and RB null MEFs lacking all three family members  (TKO) were cultured in Dulbecco’s Modified Eagle’s medium  supplemented with 10 % fetal calf serum (FCS), 200 mg/ml streptomycin, and 200 U/ml penicillin G at 5 % CO2 and humidified air at 37 °C.Total RNA was extracted and 1 μg used for cDNA synthesis (Invitrogen Superscript II). For each reaction, 25 μL of 2X SYBR green (Biorad) was mixed with forward and reverse primers to give a final concentration of 10 μm. One μL of cDNA mixture was brought up to 50 μL using DEPC water. Cycling conditions included an initial denaturation at 95 °C for 3 min followed by 50 cycles at 95 °C for 30 s, 55 °C for 30 s and 72 °C for 45 s. The homogeneity of PCR products was confirmed using a real time PCR melting curve.Cells were grown in 150 mm petri dishes to 90 % confluence, fixed in 1 % formaldehyde for 7 min and rinsed 3X with cold PBS. Chromatin was sheared to fragments below 1 Kb and 20 μg immunoprecipitated using selected antibodies. After purification, 0.5 to 2.0 μL of DNA were used for PCR. DNA from non-precipitated chromatin samples was extracted and used as input for PCR. Controls included isotype-matched IgG or Mock reactions which included all reagents except chromatin.Cells were grown on glass coverslips to 30 % confluence, rinsed 2X with PBS , fixed in 3 % paraformaldehyde for 15 min at 4 °C and rinsed twice with PBS. Fixed cells were permeabilized with 0.1 % Triton-X100 for 5 min, blocked in 2 % dry milk for 15 min, rinsed with PBS twice and incubated with primary antibody  in PBS containing 1 % dry milk and 0.1 % Triton-X100 overnight. Secondary antibody, mouse mAB-Alexa-488 conjugate, was incubated for 1 h at room temperature. Cells were washed 3X with PBS and incubated in 1 μg/ml Hoescht nuclear dye for 20 min and then washed twice. Cells were visualized in a CARL ZEISS AXIOVERT 200 inverted microscope at a magnification of 63X.Cells were lysed with buffer and centrifuged at 4000 rpm for 2 min. Immunoprecipitation using anti-Mi-2β and anti-RbAp46 were completed as described previously [Cells were transfected with an EGFP-tagged L1RP vector. After the disappearance of EGFP fluorescence by epigenetic silencing, cells were replated and treated at 30-50 % confluence with 4 nM siRNA for 48 h followed by direct measurements of fluorescence.Cells were challenged with 3 μM BaP or 0.06 % dimethyl sulfoxide (DMSO) as vehicle control for 16 h to reactivate L1.No ethics approval was required for the experimental work performed in this study.RP 5′-UTR was compared to Snail and glucose 6-phosphate dehydrogenase (G6PD) gene promoters as positive and negative control sequences, respectively. All NuRD subunits specifically enriched the sample for the human L1RP 5′-UTR DNA sequence, but not the G6PD sequence , RB-associated proteins 46 and 48 (RbAp46 and RbAp48), MBD2 and MBD3, and metastasis-associated proteins 2 and 3 (MTA2 and MTA3) –11. Valince Fig. . The preSubsequent experiments were conducted to examine the role of RB proteins in NuRD complex recruitment comparing wild type MEFs to TKO MEFs in which all three member of the RB family were genetically ablated . In thesSince basal L1 mRNA levels are markedly upregulated in TKO MEFs , we sougRP carrying a chimeric ORF1p-GFP fusion protein. The backbone vector, pGL4.13, lacked an episomal sequence and is known to undergo silencing upon integration, as evidenced by the loss of fluorescence pyrene (BaP), a genotoxic carcinogen that reactivates L1 via epigenetic mechanisms . BaP indThe NuRD complex represents a multiprotein complex that mediates formation of heterochromatin and localized domains of repressive chromatin. Its repressor activity involves assembly of a histone deacetylase macromolecular complex that couples histone modifications with nucleosome-stimulated ATPase activity. Mi2-β, RbAp46, MBD3, MTA 2 and MTA3 have been identified as critical NuRD multiprotein complex components. Mi2-β functions as an ATP-dependent helicase , RbAp46 The epigenetic silencing profile executed by the NuRD complex includes histone deacetylation, histone H3 lysine K9, H3 lysine 27, and H4 lysine 20 trimethylation, and DNA methylation, which together contribute to the recruitment of non-coding RNAs and repressors and corepressors to induce facultative or constitutive heterochromatin formation . Genes sRepetitive L1 sequences in the mammalian genome are extensively silenced via DNA methylation, and L1 methylation status is frequently used as a proxy of global cellular methylation , 25. ThiOur previous findings are consistent with the new data showing that L1 silencing by RB proteins is effected through the NuRD multiprotein complex. All major components of the NuRD complex are recruited to the silenced L1 promoter, and recruitment exhibits promoter-specificity. RB appears to orchestrate the recruitment of NuRD proteins and loss of RB function is associated with delocalization of Mi2-β from the nuclear to the cytosolic compartment. A critical role for RB in regulation of L1 retrotransposon was established in studies showing that genetic ablation of RB family members is associated with highly unregulated overexpression of L1 in MEFs. Recent studies using non-transformed human bronchial epithelial cells have shown that L1 reactivation by BaP is associated with disruption of NuRD complex assembly and function . Similar studies using cancer cell lines are confounded by differences in RB mutant status and RB-related functions. Such differences notwithstanding, comparative analyses of constitutive L1 expression have shown that human cells exhibit remarkably lower expression than murine cells , a findiCollectively, our findings identify RB as a critical regulator of the NuRD multiprotein repressor complex assembly within the regulatory region of L1. NuRD recruitment is in turn responsible for effective silencing of L1 transcriptional activity. Genetic ablation of RB proteins destabilizes protein-protein interactions within the NuRD macromolecular complex to mediate nuclear rearrangement and delocalization of Mi2-β from the nuclear to cytoplasmic compartment. These findings suggest that Mi2-β is critical to transcriptional repression of L1. These findings are highly relevant to our understanding of oncogenesis and malignant progression given that NuRD is a key determinant of cellular differentiation, and that inappropriate recruitment of NuRD to specific loci contributes to tumorigenesis . Given tOur findings indicate that the L1 silencing by RB is effected through the NuRD macromolecular complex and implicate these molecular interactions in the formation of L1 heterochromatin structures. Full-length L1s in the mammalian genome are therefore potential targets for NuRD-mediated silencing and recruitment of silencing proteins such as the polycomb family of proteins. As such, unregulated silencing of L1 may play a central role in the initiation and progression of cancer.


Using nano angle resolved photoemission (nanoARPES) complemented by transport studies and Raman spectroscopy as well as density functional theory (DFT) calculations, we report the direct observation of massless Dirac particles in monolayer graphene, providing a comprehensive mapping of their low-hole doped Dirac electron bands. The micrometric graphene flakes are oriented along two predominant directions rotated by 30° with respect to each other. The growth mode is attributed to the mechanism when small graphene “molecules” nucleate on the Ge(001) surface and it is found that hydrogen plays a significant role in this process.The practical difficulties to use graphene in microelectronics and optoelectronics is that the available methods to grow graphene are not easily integrated in the mainstream technologies. A growth method that could overcome at least some of these problems is chemical vapour deposition (CVD) of graphene directly on semiconducting (Si or Ge) substrates. Here we report on the comparison of the CVD and molecular beam epitaxy (MBE) growth of graphene on the technologically relevant Ge(001)/Si(001) substrate from ethene (C Graphene is widely supposed to be a material on the way to enable new development of many modern technologies13436791112131516171819202122232421μm227Graphene films grown on Ge may be used in two ways. First, if large area graphene (like 300–450 mm in diameter) is obtained, the film may be transferred to the target wafer at any point of the fabrication process  without the hazard that the production tools become contaminated. In practice this means that the growth must be performed on Ge(001)/Si(001) wafers, because Ge wafers of this size are not commercially available and it would most probably be prohibitively expensive to develop and introduce a Ge wafer growth technology solely for this purpose. The second way to use graphene films grown on Ge may be open when graphene can be grown directly on device-size Ge(001) islands pre-deposited on the Si(001) substrate. For example, the width of the active region of a graphene base transistor may be smaller than a micron, and its whole area may be of the order of 4-based CVD2H4 precursor with the Ge(001) surface is then analyzed in the framework of ab initio density functional theory (DFT) calculations and conclusions for the microscopic mechanisms of the growth process are drawn. These mechanisms are then compared to those expected for the growth of graphene on Ge(001) from atomic carbon.Graphene growth on the technologically relevant Ge(001) and Ge(001)/Si(001) wafers has been reported for a CHEF in the CVD sample vanishes there (see discussion below).Morphology of the CVD- and MBE-grown graphene on Ge/Si(001) was studied by means of scanning electron microscopy (SEM) . The graEF the Fermi level (EF) of the CVD-produced graphene flakes on Ge(001)/Si(001) substrate was studied in the micro and nanoARPES experiments with 120 μm and 100 nm spot size resolution, respectively2930ED) coincides with EF. In the vicinity of ED, the energy of graphene electronic states depends linearly on the wave-vector k . Here, EB is the binding energy and kx, ky are two in-plane components of the wave vector of electron. When a neutral graphene has two domains rotated with respect to each other by 30° and the same area of each of them is illuminated by the photon beam, the Fermi surface in the ARPES experiment consists of 12 equivalent spots centred at the 12 equivalent K-points around the BZ centre, Γ. When a neutral graphene is fully disordered or polycrystalline, the Fermi surface forms a circle centred at Γ and similar to the one observed for highly oriented pyrolytic graphite (HOPG).The electronic structure in the vicinity of vector k . These evector k . In caseI measured for graphene/Ge(001)/Si(001) at hν = 100 eV (spot size 120 μm) as a function of two emission angles, polar θ (detector angle) and azimuth ϕ (in-plane sample rotation), and then converted into I. One can clearly see that the photoemission at the Fermi level has a circle-like shape centred at Γ and having equidistant intensity maxima. The radius of the circle in the reciprocal space is 1.7 Å−1 and the angular distance between the maxima is 30°. This indicates that the observed photoemission intensity originates from graphene flakes with orientation distribution that has probability maxima in the directions corresponding to graphene hexagons being parallel and/or perpendicular to the surface dimer rows of the Ge(001) surface (see discussion below).hν = 30 eV along the Γ − K direction for one of the preferential domains is given in A detailed map of photoemission intensity measured with circularly polarized light of EF is located by 0.185 eV below ED, which corresponds to the hole concentration of 2.3 × 1012 cm−2. The linear fit of the Dirac cone in the ARPES intensity map in 6 m/s. This can be compared with the graphene/Ge/SiC(0001) system, where p-doping of graphene was obtained via intercalation of Ge atoms in graphene/SiC at 720 °C and the hole doping of 4.1 × 1012 cm−2 was measured−1.These data allow us to estimate the energy distance between the Fermi level and the Dirac point, and hence the doping level of graphene: s and Ge 3d core levels are marked. The photoemission intensity at the K-point for one of the preferential graphene domains and in the vicinity of the Fermi level , which in turn is determined by the doping level; therefore VFB measures the doping level. Comparison of the VFB value for CVD and MBE prepared films indicates that former films are more strongly p-doped. The measurements in the FET configuration allow to estimate the carrier mobility of the graphene channel. For this purpose, one assumes that the film area controlled by the gate tip corresponds to a square and one measures the sample-tip distance d by approaching the tip towards the sample until mechanical contact is observed. The measured carrier mobilities were lower than for high-quality graphene and amounted to 1000 ± 500 cm2/V ⋅ s at room temperature for CVD and MBE prepared graphene/Ge(001). 2/V ⋅ s are reportedResults of a electrical measurements in the field-effect transistor (FET) configuration in 4-point STM  are consents cf. : the Fer samples . The gatet al.−112 cm−2 for the MBE graphene and around 3 × 1013 cm−2 for the CVD sample. The presence of data points with G-mode positions on the left side of the zero-doping line can be partially explained with the anomalous phonon softening of the G mode for Fermi-level shifts close to the Dirac point353712 cm−2.In accordance with the electrical 4-point STM measurements, the doping level of the CVD graphene film is higher than that of the MBE-grown film as deduced from the Raman spectra measurements. A rough estimate yields the average doping below 1 × 102), whereby a mobile Ge adatom is released. The process is reversible: a Ge adatom may eject the C atom from a C-Ge surface dimer. The mobile and highly reactive C interstitially produced in this process can diffuse under the surface. The mobile species controlling the MBE growth mode of graphene on Ge(001) are C atoms, short chains consisting of few C atoms, and Ge adatoms; the latter are attracted to the edge of growing graphene. Carbon dimers (C2) are trapped by Ge dimer vacancies (DV) to become graphene seeds; a similar role is attributed to longer C chains (like C8) trapped between dimer rows. In contrast to that, results of our calculations suggest that nucleation of CVD graphene on a flat Ge(001) surface takes place predominantly on the DV defects (see Although carbon and Ge form no stable carbide phase (GeC), the chemical interaction between the Ge(001) surface and C atoms is strong. This is particularly pronounced when atomic carbon is deposited2H4 precursor molecules do not give all H atoms away to the substrate. Calculations indicate that the major unpolymerized diffusing species may be, apart from adsorbed H, C2H3 molecules, with small admixture of C2H and C2 (see 2H2 (acetylene) produced during C2H4 decomposition desorb before they decompose further. The surface reactions do not lead to significant production of Ge adatoms. Moreover, although there are Ge adatoms on the surface (due to high temperatures used in these experiments), they are repelled from the edge of a hydrogen-terminated graphene. This is in strong contrast to the MBE process: adsorption of Ge adatoms at edge sites of hydrogen-free graphene is energetically preferred to adsorption at surface steps.The vast majority of Csp3-like bond DV, trapped inside Ge dimer vacancy defects DV is produced also by atomic C in two kick-out eventsGe + Ge(bulk) −0.13 eV, and C(diff) + CGe → (C2)DV + Ge(bulk) + 0.31 eV, where C(diff) is the mobile atomic carbon diffusing under the surface and Ge(bulk) is a Ge atom having the same energy as in the Ge bulk. The total energy gain of 0.2 eV is in this case much smaller than for trapping on a DV (1.0 eV), because a new DV site must be produced, by subsequent ejection of two Ge atoms. The difference between these two energies (0.8 eV) should thus be equal to the DV formation energy (0.6 eV). The discrepancy (0.2 eV) comes from inaccuracies in the calculation of atomic relaxations, caused by the presence of nearly degenerate metastable configurations of defects and by finite size effects. This inaccuracy amounts to at least 0.1 eV per defect.] Dimer vacancies collect carbon; e. g., when the second C2 is trapped by a DV already occupied by C2, the energy decreases by 0.9 eV, which is similar to the energy gained by trapping of the first C2. Trapping of carbon by dimer vacancies provides a means to align the orientation of graphene with that of the substrate  dimers by GeC bonds. But if a polyarene has grown large enough to lean towards the horizontal position as the result of van der Waals attraction, the GeC bonds become weakened and are now vulnerable to attack by H atoms. The latter leave the Ge(001) dimers and stick to C. We estimate that, at the high temperatures needed for the CVD growth, even when the H originates only from C2H4 decomposition and when the partial pressure of C2H4 is only 2 × 10−2 mbar (as in our experiments), the whole edge of such a polyarene becomes saturated with H  to ethylene, DVs have no influence: at the growth conditions, they do not trap Cd with H  and the periment .2H molecules. First, the process of C4H2 formation is significantly more favorable inside a DV (4H2 (DV) traps C4H4; the resulting C8H6 molecule is likely to remain inside the dimer vacancy /Si(001). According to the mechanism proposed above, the domain size is associated with the abundance of Ge dimer vacancies (on which graphene nucleates), and with the presence of surface steps on the substrate surface . When the surface is well oriented and flat , the average step-step distance (or terrace width) equals to as is the surface lattice constant of Ge(001). This is in accordance with the observation that both orientations occur with the same frequency on the area covered by the X-ray spot. The quality of graphene increases with the substrate temperature because the concentration of dimer vacancies NDV  ≈ 0.6 eV) increases with temperature slower than is the decrease of the fraction pocc of DVs occupied with hydrocarbons x. For example at thermal equilibrium, pocc is thermally activated with Etrap(C4H2) ≈ 1.7 eV. In other words, the concentration s~poccNDV of the growing nuclei decreases with temperature.ARPES measurements strongly indicate that the orientational domains are so small that many of them fit into the area sampled by the X-ray beam spot, i. e. into space of about 100 × 100 nmμm range.Small domain size is responsible also for the curved shape of CVD graphene edges . The shaDoping of the MBE film may originate solely from molecules from the ambient air that are adsorbed on top of the graphene. Higher doping of the CVD film may be caused by Ge atoms−6 K−1) and Ge (6 × 10−6 K−1). Cooling from 930 °C to 25 °C would produce strain of about −0.8%, which by its absolute value is higher than the −0.3% estimated under the assumption of biaxial strain (The compressive strain in the MBE film (−0.3%) may possibly be caused by thermal expansion coefficient difference between graphene  technology compatible Ge(001)/Si(001) substrates from CThe major experimental result is that the graphene film on Ge(001)/Si(001) consists of two orientational domains rotated by 30° with respect to each other, and that the grain size is comparable to step-step separation of the substrate. Poor crystallographic quality (high Raman D mode peak) and poor electrical quality (low carrier mobility) of the CVD graphene produced in this process are attributed to the small domain size. Arguments linking the grain size with the size of surface terraces separated by Ge(001) monatomic steps are presented. It was argued that the dimer vacancies omnipresent on the surface act as seeds on which graphene can nucleate and that the orientation of a domain is determined by the orientation of Ge dimers on the surface terrace in which the domain nucleated. Small graphene nuclei released from the dimer vacancy sites by hydrogen atoms are volatile; this contributes to the reduction of graphene growth rate by increased surface concentration of hydrogen and to the increase of the average domain size. For completeness and illustration, the CVD growth mode and mechanism was compared to those for the growth from atomic carbon in MBE chamber.−10 mbar range. The substrate was a Ge(001) film (2 μm) grown by CVD on a Si(001) wafer and then overgrown in situ by epitaxial Ge buffer (about 4 nm). The substrate temperature during graphene growth was close to but below the melting point of bulk Ge (it was around 930 °C). Growth time was varied between 90 min and 200 min in both cases. The substrate was heated from the backside by a graphite filament and the temperature was calibrated by pyrometer. The graphene CVD growth was performed by using ethene (C2H4) as a precursor in a UHV-CVD chamber (Riber). The pressure was in the range of 10−2 mbar during deposition and the flow of ethylene was 5 sccm. In addition, graphene samples were grown by a physical method in a MBE chamber (DCA), as described in ref. e-gun, and the pressure was in the lower 10−7 mbar range.Graphene was grown in a multi chamber ultrahigh vacuum system (UHV) with a base pressure in the lower 10μ-Raman spectroscopy using Renishaw In-viaFlex spectrometer and green laser light (514 nm) with a spot diameter of about 500 nm. Transport measurements were performed with a 4-tip scanning tunnelling microscope  with a high resolution scanning electron microscope (SEM) used as a tip-navigation tool.Quality of graphene was evaluated by hν = 100 eV with linearly polarized light for Fermi surface mapping and at hν = 30 eV with circularly polarized light for the energy scans around the K point of graphene. The circular polarization in these experiments was selected in order to avoid the “dark corridor” problem along the Γ − K direction for graphene−11 mbar.Photoemission experiments were performed at the nano-ARPES microscope of the ANTARES beamline of the SOLEIL synchrotron, equipped with two Fresnel zone plates  for focalization of the synchrotron radiation beam and an order selection aperture (OSA) to eliminate higher diffraction orders. Samples are mounted on a nano-positioning stage placed at the coincident focus of the Scienta R4000 analyser and the FZP focal point, which is responsible for the sample rastering during the image acquisition. The spatial resolution is determined by the FZP resolution and the mechanical stability of the sample stage. Experiments were performed at photon energies p(2 × 2) surface cells, each containing eight dimers on one side and passivated with H atoms on the other side. Further calculation details can be found elsewherePseudopotential density functional theory (DFT) total energies and atomic and electronic structures were calculated by Quantum EspressoHow to cite this article: Dabrowski, J. et al. Understanding the growth mechanism of graphene on Ge/Si(001) surfaces. Sci. Rep.6, 31639; doi: 10.1038/srep31639 (2016).


Many potential applications of graphene require its precise and controllable doping with charge carriers. Being a two-dimensional material graphene is extremely sensitive to surface adsorbates, so its electronic properties can be effectively modified by deposition of different atoms and molecules. In this paper, we review two mechanisms of graphene doping by surface adsorbates, namely electronic and electrochemical doping. Although, electronic doping has been extensively studied and discussed in the literature, much less attention has been paid to electrochemical doping. This mechanism can, however, explain the doping of graphene by adsorbates for which no charge transfer is expected within the electronic doping model. In addition, electrochemical doping is in the origin of the hysteresis effects often observed in graphene-based field effect transistors when operating in the atmospheric environment. Graphene is a monolayer of carbon atoms arranged in a honeycomb lattice . With thIn this paper, we present an overview of two mechanisms of graphene doping by surface adsorbates, namely, electronic and electrochemical doping and consider appropriate atomic and molecular dopants.+/SiO2 substrate [g, the type of carriers and their concentration in graphene can be tuned. The sign of the induced carriers is opposite to the sign of the applied gate voltage. A positive Vg induces electrons while a negative Vg induces holes. It was shown that for graphene on Si+/SiO2 the concentration of charge carriers induced by this method can be as high as 1013 cm−2 [Charge carriers, either electrons or holes, can be induced in graphene by the application of an electric field or by chemical doping. The electric field effect doping is usually performed in graphene-based field effect transistors (FET), in which charge carriers are induced by changing the electric potential between graphene and a gate, which can be, for example, a Siubstrate ,13. By vs, and nitrogen, Ns, substitutional atoms and leads to p- and n-type conductivity, respectively [s-doped graphene behaves like an n-type doped semiconductor but exhibits a low charge carrier mobility [Chemical doping involves interactions of graphene with other chemical species . There aectively –16. HoweThe surface transfer doping is non-destructive and occurs due to the charge transfer between graphene and surface adsorbates. Two mechanisms of charge transfer doping can be distinguished, electronic and electrochemical doping. The electronic doping is a consequence of the direct charge transfer between graphene and an adsorbate. This requires a difference in electronic chemical potentials at an interface, which is determined by the relative positions of the graphene Fermi level and the highest occupied (HOMO) and lowest unoccupied (LUMO) molecular orbitals of an adsorbate. If the LUMO of the adsorbate lies lower in energy than the Fermi level of graphene, G + W for p-doping and ΔG − W for n-doping, where ΔG is the free energy for the molecular reaction and W is the work function of graphene. The work function of graphene is expected to be similar to that of graphite, about 4.5 eV [G can be estimated from the Nernst equation the barrier heights are unknown and it must be assumed that they are sufficiently low for the reaction to occur [The electrochemical doping of graphene occurs when certain surface adsorbates participate in electrochemical redox reactions in which graphene plays the role of an electrode. Such reactions occur spontaneously if the total Gibbs free energy is negative and the diffusion and reaction barriers are sufficiently low for the reaction proceed at room temperature. The total Gibbs free energy change is given by Δt 4.5 eV –18. Althto occur .Eredox to the graphene Fermi level. The Eredox is the equivalent of the Fermi level of an electrolyte solution. It measures the energy required to add or remove an electron in redox couples and is usually referenced to the standard hydrogen electrode (SHE) [G and Eredox measure the same property and are related by ΔG = −n·F·Eredox, where n and F are the number of electrons and the Faraday constant, respectively. Furthermore, while the Eredox level lies higher (lower) than the Fermi level of graphene (EF), electrons (holes) induced by a reaction will flow to graphene until equilibrium is reached, Eredox = EF, making graphene n- (p-)type doped.Whether a reaction will result in p- or n-type doping of graphene depends on the relative position of the electrochemical redox potential de (SHE) . Thus, ΔIn contrast to electronic doping, which occurs instantaneously, electrochemical doping is a time-dependent process, which is affected by the rate of the reaction and diffusion rates of participating species. Therefore, in the case the reaction or diffusion rates are slower than the rate of change of gate voltage electrochemical doping can lead to hysteresis effects which are often observed in graphene-based FET devices –24.Vg with the increase of the potassium concentration is a clear indication of n-type doping. It was also observed that the mobility of the charge carriers decreases with the increase of the doping concentration. This is attributed to the additional scattering caused by the ionized potassium atoms. In agreement with the experimental results, DFT calculations have shown that K atoms act as electron donors [1 electronic level of potassium, The ability to dope graphene either n- or p-type has been demonstrated for various atomic and molecular adsorbates. Electropositive elements that easily donate their outer shell electrons are expected to be n-type dopants. Indeed, density functional theory (DFT) calculations predicted group I–III metals to be efficient electron donors for graphene –28. Elecn donors –28. ElecThe doping properties were also explored for different transition metal clusters  deposited on graphene by molecular beam epitaxy (MBE) . The Ti 4-TCNQ), shown in Whereas one expects that electropositive adsorbates will dope graphene n-type, electronegative species should lead to p-type doping. By using angle-resolved photoemission spectroscopy Gierz et al.  demonstr4-TCNQ molecule was considered to be a good candidate for p-type doping of graphene. The effect of the F4-TCNQ molecule on the electronic properties of epitaxial graphene was studied by synchrotron-based high resolution photoemission spectroscopy (PES) [4-TCNQ  . The inc [4-TCNQ  suggestsmolecule .The p-type doping of graphene can also be achieved by depositing other organic molecules such as pyrenetetrasulfonic acid (TPA) or tetracyanoethylene (TCNE) –38, show6H5CH3), It was observed that the exposure of graphene to a humid atmosphere causes p-type doping whereas exposure to toluene  and acidic (pH 1) conditions, respectively. Therefore, the reaction is inhibited in basic conditions and promoted in acidic conditions since the concentration of OH− is low.The value of Δ−4.82 eV . Under t2 substrate in the presence of O2 and H2O was confirmed experimentally by Raman spectroscopy measurements [2 and H2O leads to a stronger doping than the exposure to O2 or H2O alone. The hysteresis effects commonly observed in graphene FETs doped under atmospheric conditions [The electrochemical p-type doping of graphene on a SiOurements . It was nditions –24 were Similarly, the n-type doping of graphene caused by toluene can be explained by the electrochemical mechanism. In this case it is difficult to determine which particular reaction takes place. One possibility is the electrochemical oxidation of toluene to benzyl alcoholThis reaction is spontaneous as the change of the total Gibbs free energy is −0.5 eV. The released electron can be trapped by graphene and make it n-type doped.Vg at different stages of the toluene-doping experiment. Firstly, the sample was annealed in vacuum and the resistance peak at positive Vg, Vg showing that toluene acts as n-type dopant on graphene, R(Vg) measurement. The position of the resistance peak changes with the direction of the Vg sweep. The hysteresis vanished when the toluene is pumped out, but the doping effect remains, +/SiO2 substrates [2 interface. Indeed, the presence of water layers on the surface of SiO2 films grown on Si has been confirmed by Kelvin probe microscopy, X-ray spectroscopy and FTIR measurements [2. It has been found that for graphene FETs operating under ambient conditions the hysteresis is suppressed when Si+/SiO2 substrate was covered with a thin hydrophobic layer [2 interface. The binding energy of OH− to the SiO2 network was estimated to be about 0.3 eV indicating that OH− can easily diffuse through SiO2 at room temperature [− at the graphene/SiO2 interface and thus the rate of the reaction is influenced by the gate voltage. A positive Vg results in the reduction of the OH− concentration at the graphene interface promoting the further dissociation of H2O molecules, which consequently increases the concentration of holes in graphene, as it was observed experimentally [Hysteresis effects are often observed in graphene FETs based on Sibstrates –24. Suchurements –43. Wateic layer . Anotheric layer , supportperature . Therefomentally .3 and enhanced by NO2 [Some of these redox reactions were first discussed for diamond and carbon nanotubes ,45 and wd by NO2 . Similard by NO2 –50. It w4-TCNQ, have been proved to be very efficient p-type dopants. This type of doping occurs instantaneously and should not cause hysteresis effects. However, the ionized dopants become additional charge scatterers leading to a reduction in the charge carrier mobility.In this article we have revisited the surface dopants of graphene and the charge transfer mechanisms. The deposition of atoms or molecules is an effective method to control the type and concentration of charge carriers in graphene. Surface charge transfer can be mediated by two mechanisms, electronic and electrochemical doping. The electronic doping occurs by direct exchange of electrons between graphene and the adsorbates, either atoms or molecules. Electropositive elements that easily donate their outer shell electrons, such as potassium, are expected to be n-type dopants while adsorbates with a strong electron affinity, such as FThe electrochemical doping of graphene occurs as a result of redox reactions that can take place near the graphene surface. Unlike electronic doping it does not reduce the charge carriers mobility but it may require appreciable time to occur since the reaction and diffusion barriers have to be overcome. Moreover, the electrochemical doping model can explain the hysteresis effects usually observed in graphene based field effect transistors when operating in atmospheric environment.


The discovery of graphene and its unique properties has inspired researchers to try to invent other two-dimensional (2D) materials. After considerable research effort, a distinct “beyond graphene” domain has been established, comprising the library of non-graphene 2D materials. It is significant that some 2D non-graphene materials possess solid advantages over their predecessor, such as having a direct band gap, and therefore are highly promising for a number of applications. These applications are not limited to nano- and opto-electronics, but have a strong potential in biosensing technologies, as one example. However, since most of the 2D non-graphene materials have been newly discovered, most of the research efforts are concentrated on material synthesis and the investigation of the properties of the material. Applications of 2D non-graphene materials are still at the embryonic stage, and the integration of 2D non-graphene materials into devices is scarcely reported. However, in recent years, numerous reports have blossomed about 2D material-based biosensors, evidencing the growing potential of 2D non-graphene materials for biosensing applications. This review highlights the recent progress in research on the potential of using 2D non-graphene materials and similar oxide nanostructures for different types of biosensors . A wide range of biological targets, such as glucose, dopamine, cortisol, DNA, IgG, bisphenol, ascorbic acid, cytochrome and estradiol, has been reported to be successfully detected by biosensors with transducers made of 2D non-graphene materials. The developments in material science are the driving force of technological progress. In addition, it may be strongly argued that the creation of new materials of different dimensionality and functionality is the primary prerequisite for any likely significant breakthroughs to be made. The invention of graphene has unambiguously demonstrated that the properties of two-dimensional (2D) materials can be different and in many ways far superior to those of the bulk counterpart.2/g) 3−/4−. Under optimum conditions, the amperometric signals decrease linearly with the target DNA concentrations ranging from 0.1 pM–1 nM. The detection limit was observed as low as 20 fM with an excellent selectivity, acceptable stability and reproducibility.Huang et al.  reportedThe authors concluded that the good analytical performance is attributed to the synergistic effect of acetylene black and the unique microstructure of CuS nanosheets. This can open up new opportunities for sensitive and selective detection of specific sequence DNA and provide a promising platform for biosensor design for other biomolecular detection .3N4) is a new semiconductor material with p-conjugated graphitic planes formed by the sp2 hybridization of carbon and nitrogen. The prominent feature of this semiconductor is that it demonstrates the electrochemiluminescence (ECL). Probably the first report on the synthesis of g-C3N4 was by Zhang et al. [3N4 nanosheets prepared by a “green” liquid exfoliation route from bulk g-C3N4 in water were investigated. It was found that 2D g-C3N4 demonstrated enhanced intrinsic photoabsorption and photoresponse compared to the bulk g-C3N4. High stability, good biocompatibility, superior physiochemical properties and large surface area have triggered interest in 2D g-C3N4 nanosheets as a material for ECL biosensors. Indeed, it was recently reported for biosensors based on 2D g-C3N4 for the detection of DNA [Graphite-like carbon nitride  is a novel semiconductor material with wide band gap (Eg = 5.2 eV), which makes it almost an insulator. h-BN is usually present in the form of flakes or sheets and has become widely used recently in the fields of electronics, catalysis and sensing. 2D nanosheets of BN consist of alternating boron and nitrogen atoms, representing an extra smooth surface. It has to be noticed that until now, only several applications of h-BN as a biosensor were reported, while most of them are still in the embryonic stage; while even the nanosheets allotropes—nanotubes of BN—were reported as promising nanotransducers for functionalization of biosensitive layers [Hexagonal boron nitride , which is a significant compound involved in many chemical and biological processes. The electrochemical sensor, based on nanocomposites consisting of BN nanosheets and Au NPs, was reported before [2O2 as a byproduct, like glucose or cholesterol [BN was reported for the successful detection of hydrogen peroxide  with green fluorescence, was reported. The sensor was proven to be an efficient platform for cell imaging, due to the nanocomposite’s strong fluorescent intensity, stability, water solubility, ry, etc. .Another type of nanocomposite was made based on h-BN nanosheets and Au nanoparticles as a label and applied for an immunosensor for the detection of interleukin-6 by the fluorescence and electrochemistry approaches. The sensor demonstrated excellent performance, which the authors attributed to the high surface area and morphology for antibodies’ immobilization .2, MoO3, WO3 and ZnO [Another type of inorganic non-graphene 2D material is metal oxides, which have recently stimulated plenty of interest as immobilizing matrixes for biosensor development. Nanostructured oxides of metals, such as zinc, iron, cerium, tin, zirconium, titanium and magnesium, have been found to exhibit interesting nanomorphological, functional biocompatible, non-toxic and catalytic properties . Among t and ZnO ,86, have2 nanosheet as the energy acceptor was proposed by Yuan et al. [2 surface. This resulted in the energy transfer from the fluorophore to MnO2 and the respective quenching of the fluorescence. Exposure of the aptamers with OTA molecules changes the conformation of the aptamers, which reduces the exposure of the nucleobases. As a consequence, the physisorption of the aptamers on MnO2 surface is weakened. Thus, the emission of the fluorophore is expected to be recovered, which enables the recognition and quantification of the target [For the first time, a homogeneous FRET sensing protocol using a MnOn et al. . The bioe target .et al. [in vivo sensing of ascorbic acid (AA) in rat brain. Single-layer MnO2 nanosheets were used for suppressing the fluorescence of 7-hydroxycoumarin. The mechanism for the fluorescence suppression is attributed to a combination of an inner filter effect (IFE) and a static quenching effect (SQE). The combination of IFE and SQE leads to an exponential decay in fluorescence intensity of 7-hydroxycoumarin with increasing concentration of MnO2 nanosheets in solution. Such a property allows optimization of the concentration of MnO2 nanosheets in such a way that the addition of reductive analyte  will to the greatest extent restore the MnO2 nanosheet-suppressed fluorescence of 7-hydroxycoumarin through the redox reaction between AA and MnO2 nanosheets. Compared to the turn-on fluorescent method through first decreasing the fluorescence to the lowest level by adding concentrated MnO2 nanosheets, the method demonstrated here possesses a higher sensitivity, lower limit of detection and wider linear range. Upon the use of ascorbate oxidase to achieve the selectivity for AA, the turn-on fluorescence method demonstrated can be used for in vivo sensing of AA in a simple, but reliable way [Different from those reported for the traditional two-dimensional nanosheets, a sensing mechanism was reported by Zhai et al.  for in vable way .et al. reported a facile surfactant-templated synthetic strategy for water-dispersible nanoplatelets of MnO2 and then established it as a new biosensing platform for probing and recognizing biomolecular interactions in a homogeneous solution. The sensing strategy is based on the attachment of the ssDNA on MnO2, with the DNA strand interacting non-covalently with nano-MnO2 by the van der Waals force between nucleotide bases and the basal plane of MnO2. This platform possesses three excellent features: (i) MnO2 with an excellent dispersibility in water can be synthesized in great force at room temperature and used as a quencher without further processing; (ii) the MnO2-based biosensor is low cost and can finish the biomolecular assay within a few minutes; (iii) the biosensing strategy can be applied to other types of molecular probes by simply changing the sequences of the ssDNA to a specific target [He c target .et al. reported a design of FET, using the 2D molybdenum trioxide (MoO3) material. As a protein model, bovine serum albumin was used. α-MoO3 nano-flakes, with the majority of nano-flake thicknesses being equal to or less than 2.8 nm, were stacked in a nanostructured film, forming the conduction channel. The FET demonstrated impressive response time (≤10 s). The authors explain this due to the high permittivity of the 2D α-MoO3 nano-flakes. It has been concluded that the 2D α-MoO3 system offers a competitive solution for future biosensing applications [Balendhran ications .i.e., bulk or nanostructures. Thus, Vabbina et al. reported label-free, highly sensitive and selective electrochemical immunosensors based on 2D ZnO nano-flakes (ZnO-NFs) which were synthesized on Au-coated substrates using a simple one-step sonochemical approach. Selective detection of cortisol using cyclic voltammetry (CV) is achieved by immobilizing anti-cortisol antibody (Anti-Cab) on the ZnO nanostructures (NSs). 2D ZnO-NFs provide unique sensing advantages over bulk materials. 2D-NSs with a large area in the polarized (0001) plane and a high surface charge density could promote higher Anti-Cab loading and, thus, better sensing performance. Beside a large surface area, ZnO-NSs also exhibit higher chemical stability, high catalytic activity and biocompatibility. The measured sensing parameters are in the physiological range with a sensitivity of 7.74 mA/M with the lowest detection limit of 1 pM, which is 100-times better than the conventional enzyme-linked immunosorbent immunoassay (ELISA). ZnO-NS-based cortisol immunosensors were tested on human saliva samples, and the performance were validated with a conventional (ELISA) method, which exhibits a remarkable correlation. The developed sensors can be integrated with a microfluidic system and a miniaturized potentiostat for point-of-care cortisol detection, and such a developed protocol can be used in personalized health monitoring/diagnostics [Zinc oxide is a semiconducting material with plenty of advanced properties, such as high catalytic efficiency, biocompatibility, chemical stability in physiological environments and low toxicity. It possesses a high isoelectric point (IEP) of about 9.5, which makes it a rather prospective candidate for biosensing applications . 2D ZnO gnostics .p-type conductivity  has been used in many electronic applications and sensors. Such a metal oxide can have many well-defined nanostructures with different dimensionalities, possessing unique electronic and optical properties. For instance, due to the size confinement effect, the 2D CuO has an enhanced optical band gap (gE = 2.15 eV) [et al. [et al. [−1·cm−2, a fast response time of ∼2 s, a relatively wide linear dynamic range of 0.1–4.0 mM, a low detection limit of 50 nM and good anti-interference ability.Copper oxide being a narrow gap semiconductor with 2.15 eV) . Recentl2.15 eV) ,95. Most2.15 eV) ,97. In p2.15 eV) ,97 repor [et al.  designedvs. no band gap in graphene) or bulk crystals (the highest surface-to-volume ratio in 2D). Using the 2D non-graphene materials, the biosensors can be designed for different types, mainly electrochemical  or optical (by quenching the fluorescence or retaining it via aptamers use).It is revealed that the family of 2D non-graphene inorganic materials is undeniably forthcoming for the application as a transducer material in biosensors. This is indirectly proven by the growing research interest, reflected in a rising number of papers during the last few years. The scientific reports clearly demonstrate the certain advantages of 2D non-graphene materials over graphene . It is shown that many different biological agents can be successfully immobilized on the surface of 2D non-graphene materials, providing sensing ability for a wide range of biological targets, as is demonstrated in 2, WS2, VS2, CoS, CuS). Specifically, MoS2 is the most reported non-graphene 2D material in the biosensing area. This may be explained by its several advantages, including comparatively simple synthesis procedure and possibility of direct (CVD) growth. Another important advantage of MoS2 for biosensors is that it has polarized planes, which favors the van der Waals interaction and promotes the biosensitive layers’ immobilization, for both enzyme-based and non-enzyme electrochemical biosensors. The realization of the FET-based biosensing device with MoS2 as a channel results in obtaining a highly sensitive, single-molecule detection compatible sensor. Eventually, the performance/sensitivity of the MoS2-based FET biosensor was reported to be 74-fold better than that one of the graphene-based one. In the optical type of biosensors, MoS2 has high fluorescence quenching ability, good detection limits and fast response time. Via functionalization of MoS2 by aptamers, the optical biosensor demonstrates high sensitivity with a simple design and low cost. Finally, nanocomposites containing MoS2 together with graphene and/or metal nanoparticles were reported to be efficient transducers for the electrochemical type of biosensors of a wide range of analytes.Chalcogenides are the dominant materials among the non-graphene 2D, reported in biosensors’ design  of about 9.5. All the above makes 2D ZnO therefore extremely promising for biosensing applications. This, however, is still compromised by its practical unattainability, which will be an exciting materials researcher’s task for the next few years. Finally, we would like to conclude that the present tendency of growing the 2D materials library will result in the appearance of new candidates, which will definitely join the biosensing area soon.


The ideality factor of the graphene/ZnO nanowire Schottky diode is ~1.7, and the Schottky barrier height is ~0.28 eV without external Vg. The Schottky barrier height is sensitive to Vg due to the variation of Fermi level of graphene. The barrier height increases quickly with sweeping Vg towards the negative value, while decreases slowly towards the positive Vg. Our results are helpful to understand the fundamental mechanism of the electric transport in graphene-semiconductor Schottky diode.Graphene-semiconductor interface is important for the applications in electronic and optoelectronic devices. Here we report the modulation of the electric transport properties of graphene/ZnO nanowire Schottky diode by gate voltage (V The ideality factor of the graphene/ZnO NW Schottky diode is ~1.7, and the Schottky barrier height is ~0.28 eV. Further gate controlling tests demonstrate that the Schottky barrier height is sensitive to the gate voltage, which mainly stems from the high sensitivity of Fermi level of graphene to the gate voltage. The barrier height increases quickly by applying negative gate voltage, while decreases slowly with increase of the positive gate voltage. Our results are helpful to understand the fundamental mechanism of the electric transport in graphene-semiconductor Schottky diode and may be useful for its application in nanoelectronics.via the processes as presented in 2 layer  and G peak (~1589 cm−1) can be clearly observed, while the defect related peak is quite weak. The Raman intensity ratio between the 2D mode and the G mode is ~3, indicating the nature of monolayer graphenesd) of the monolayer graphene as a function of the back gate voltage (Vg) as applying the source-drain voltage (Vsd) of 0.2 V between the electrodes B1-B2. The Isd of graphene can be changed by Vg, suggesting that the Fermi energy level of graphene can be effectively tuned by Vg. The current-voltage (I-V) curve of the graphene/ZnO NW hetero-junction is plotted in The inset in I) passing through the Schottky barrier is determined by the thermionic emission of electrons, which can be written asISAT is the saturation current, q is the elemental charge, η is the ideality factor R is the series resistance, k is the Boltzmann constant, T is the absolute temperature, ϕSB is the Schottky barrier height, A is the interface area of the Schottky barrier, and A* is the Richardson constant A ~ 1 μm2 and the experimental temperature T ~ 300 K, according to η ~ 1.7. The barrier height without gate voltage can be deduced to be ϕSB ~ 0.28 eV. Since the work function for intrinsic graphene is about 4.6 eV and the electron affinity of ZnO NW is about 4.7 eV, the analysis is reasonable according to the experimental results.The formation of Schottky barrier at the interface between graphene and ZnO NW is due to the difference of work function between the two materials. The barrier height can be described as sd-Vsd curves under different Vg were measured. The Isd-Vsd curves between electrodes B1 and A1 of the graphene/ZnO NW hetero-structure under different Vg are presented in g sweeping from −80 V to 0 V, the Isd at the same positive Vsd increases quickly, and then increase slowly with Vg further increasing from 0 V to 80 V. The Isd-Vsd curves at the negative Vsd side exhibit no apparent variation with Vg. Using sd-Vsd curve under different Vg. The variation of Schottky barrier height as a function of Vg was plotted in g from 0 to −80 V, while decreases slowly as varying Vg from 0 to 80 V. This gate voltage modulation of the Schottky barrier height in graphene/ZnO NW hetero-junction is attributed to the linear dispersion relationship of the energy band in graphene, which results in the high sensitivity of Fermi level of graphene to Vg.In order to further investigate the behavior of the Schottky barrier of the graphene/ZnO NW diode, the source-drain Ig. The electron transfer from ZnO to graphene results in the energy band bending near the ZnO NW surface and the formation of a depletion region. The Schottky barrier height is g from 0 to −80 V, the ϕSB of graphene continuously increases, leading to the notable increases of ϕSB .


Pain in arthritis may be experienced in regions outside the affected joint, and hyperalgesia may even be widespread. The spread of pain is usually attributed to mechanisms in the central nervous system. We investigated whether rats with antigen-induced arthritis (AIA) exhibit peripheral changes in skin innervation remote from the inflamed joint.After immunization, unilateral AIA in the knee joint was induced in rats. Intraepidermal nerve fibre density was determined by immunohistochemical staining for protein gene product 9.5 (PGP 9.5) and for nerve fibres expressing calcitonin gene–related peptide (CGRP), substance P (SP), transient receptor potential vanilloid 1 , β-tubulin, and growth-associated protein 43  in paw pad skin and back skin. Cluster of differentiation 11b (CD11b)-positive macrophages and CD3-positive T cells were quantified in skin, and macrophages were quantified in the lumbar dorsal root ganglia. In addition, pain-related behaviour was assessed.Intraepidermal nerve fibre density (PGP 9.5) and the numbers of fibres expressing CGRP, SP, TRPV1, or β-tubulin did not show a significant change in the acute (3 days) or chronic phase (21 days) of AIA compared with control rats that were only immunized. However, paw skin and back skin revealed a significantly higher number of nerve fibres expressing GAP-43 at both the acute and chronic stages of AIA. The skin of arthritic rats in these regions did not contain a greater density of CD11b and CD3 immune cells than the skin of control rats. Enhanced expression of GAP-43 in nerve fibres of the skin was not related to hyperalgesia in the joint, but it accompanied persistent secondary cutaneous hyperalgesia in the skin remote from the inflamed joint.Although the innervation of the skin remote from the joint did not show significant abnormalities of the other nerve fibre markers, the rapid and persistent increase of GAP-43 expression is conspicuous. The data suggest that immune-mediated arthritis is associated with changes in skin innervation remote from the inflamed joint, although the skin is not inflamed, which may contribute to symptoms in nonarticular tissue remote from the affected joint. Chronic joint diseases such as rheumatoid arthritis and osteoarthritis are usually associated with chronic pain in the affected joints. Careful clinical examination has shown, however, that pain is often not restricted to the affected joints. Patients with localized osteoarthritis and rheumatoid arthritis often exhibit allodynia (a reduction of pain thresholds in the innocuous intensity range) and hyperalgesia (increased pain perception on noxious stimulation) in the entire limb , 2. ThesHowever, pain states are not entirely characterized by augmented functional activity in the peripheral and central nociceptive systems. An intriguing finding is the change of the innervation density of diseased joints. Inflamed synovial tissue derived from experimental arthritis and from patients with rheumatoid arthritis showed a significant reduction of nerve fibre density. Some authors reported a reduction of peptidergic sensory afferents –16, wherChanges of innervation density are likely to be caused by local factors in the joint. However, to our knowledge, whether changes of the innervation density in joint diseases are restricted to the affected joint, or whether such changes also occur remote to the afflicted joint, has never been systematically tested. We therefore addressed this question in the present study using the AIA model in the rat, an animal model of monoarticular inflammation usually induced in the knee joint, which reproduces major aspects of clinical rheumatoid arthritis. Rats with AIA show a highly reproducible pathology with well-defined acute and chronic states. Pronounced inflammation of the affected knee joint with local swelling and hypersensitivity, as well as hyperalgesia in areas remote from the inflammatory process , have been described , 20. SpeThe study on AIA was performed on 25 female Lewis rats . All experiments were carried out in accordance with the European Communities Council Directive (86/609/EEC) for the care and use of laboratory animals and were approved by the Thuringian State Office of Food Safety and Consumer Protection, Department of Consumer Protection, Veterinary, Pharmaceuticals (registration number 02-013/08).n = 25) were immunized by two subcutaneous injections of 500 μg of methylated bovine serum albumin  prepared in saline, emulsified with 500 μl of complete Freund’s adjuvant (Sigma) supplemented with 2 mg/ml Mycobacterium tuberculosis strain H37Ra . Fourteen days after the second injection, monoarticular arthritis was induced by a further injection of mBSA  into the left knee joint cavity (n = 20). The five immunized-only rats served as controls.Arthritis was induced as previously described . In brien = 10, acute AIA) or at 21 days  after induction of AIA, the rats were anesthetized by intraperitoneal injection of sodium thiopentone  and killed by transcardial perfusion with heparin-enriched, phosphate-buffered saline (PBS) and 4 % phosphate-buffered formalin.Either at 3 days  as described previously . The difUsing an incapacitance tester , weight-bearing of the inflamed hind limb was assessed as a functional measure of pain-related guarding behaviour. The animals were placed with their hind paws on scales inside a plastic cage, and the weight force on the two scales was measured three consecutive times while the animals sat calmly in the cage after acclimation. From these averaged values, the relative resting weight (expressed as a percentage) of the inflamed hind limb was evaluated [weight of inflamed hind limb × 100 %/(weight of inflamed hind limb + weight of noninflamed hind limb)], as described previously .Primary hyperalgesia at the inflamed knee was assessed with a dynamometer  . At the As previously described , 4, secoAfter the animals were killed, hind paw pad skin , lumbar paraspinal back skin , and lumbar dorsal root ganglia (DRGs) from spinal levels L1–L5  were dissected separately. The specimens were then processed and immunostained as described previously . BrieflyIENFs immunoreactive to PGP 9.5, CGRP, SP, β-tubulin, and GAP-43 were counted in a blinded manner along the length of the tissue section (approximately 3–8 mm) according to the guidelines developed by the European Federation of Neurological Societies (EFNS) . Thus, oCD11b and CD3 immunoreactivity in the dermis and ED1 immunoreactivity in the DRGs were quantified by computerized image analysis. Images were captured using an Olympus DP70 camera mounted on an Olympus BX50 microscope and analysed using Olympus analySIS software. After applying the colour separation for red to blank out the counterstaining, positive immunostaining was highlighted by setting the grey-level detection limit to a fixed threshold, and the area of highlighted immunoreactivity obtained as a percentage of the field scanned. The threshold was kept constant for all specimens analysed that were immunostained with the same marker. For the skin  and DRGs  five and three microscopic fields (magnification × 40) were analysed, respectively, and the mean values were used in statistical analysis.U test (two-tailed). Behavioural changes were examined with the Wilcoxon matched-pairs signed-rank test (two-tailed) and intra- and interobserver correlations as well as IENF correlations with Spearman’s correlation coefficient. Significance was assumed at p < 0.05.Descriptive statistics were created and statistical tests were performed using Prism version 5.03 for Windows software . Group differences for the immunostaining data were assessed using the nonparametric Mann–Whitney p = 0.0059) for the acute AIA vs. baseline values . This pain-related guarding behaviour subsided gradually by day 21 (chronic state)  Fig. .p = 0.0058), demonstrating primary hyperalgesia at the inflamed knee. By day 21, the mechanical thresholds had increased again to values close to baseline . This indicates a trend towards secondary hyperalgesia remote from the inflammation , some extending almost to the stratum corneum  and chronic (p = 0.0007) AIA as compared with controls  with the increase of GAP-43 being significant in acute AIA (p = 0.0159), but not in chronic AIA (p = 0.0952), compared with controls .Intra- and interobserver agreements for the counts of IENFs were highly significant, as verified by correlations  and CD3-immunoreactive (T cells) cells appeared to be of similar density within the three groups examined Fig.  and b. Tp = 0.0007) in acute AIA animals compared with controls on the ipsilateral side .ED1-immunoreactive cells (macrophages) were detected around sensory neuronal perikarya of lumbar DRGs Fig.  – c. Theide Fig.  and on tWe next examined potential correlations between ED1 in the lumbar DRGs and GAP-43–positive IENFs in the paw pad and between ED1 and pain-related behaviour. Figure R = −0.7930, p < 0.0001)   Fig.  and with06) Fig. . Control06) Fig. . This unIn the present study, we investigated whether localized arthritis in the knee joint causes neuronal changes in the skin remote from the joint. Neither overall IENF density nor the numbers of fibres expressing CGRP, SP, TRPV1, or β-tubulin showed a significant change in the acute or chronic phase of AIA compared with control rats. In contrast, the paw and back skin revealed a significantly higher number of nerve fibres expressing GAP-43 in both the acute and chronic stages of AIA. Importantly, the skin of arthritic rats in these regions did not contain a higher density of CD11b and CD3 immune cells than these regions in control rats. However, the lumbar DRGs of both the inflamed and noninflamed sides did exhibit a pronounced invasion of macrophages in the acute stage of inflammation.GAP-43 has received considerable interest in basic as well as clinical neuroscience research because significant changes of GAP-43–positive nerve fibres were identified in human disease states. A reduction of IENF density is considered a hallmark of classical small-fibre peripheral neuropathy. In patients with neuropathy in the course of type 2 diabetes, IENF density was significantly lower than in controls , 31, corGAP-43 is a membrane protein associated with axonal development, axonal regeneration, remodelling, and neuronal plasticity , 37. It Whether alterations of GAP-43 are per se  associated with sensory changes such as pain or sensory abnormalities is currently unknown. In clinical studies, researchers often use changes in the expression of GAP-43 as an indicator of neuronal changes, mostly as an indicator of regeneration or regenerative potential. However, researchers in most studies have not reported whether patients had symptoms such as pain or sensory abnormalities. Interestingly, investigators in one study reported that patients with diabetic neuropathy and pain had higher GAP-43/PGP 9.5 ratios than patients with diabetic neuropathy without pain . HoweverThe present data show some aspects related to this problem. First, in the present experiments, the density of GAP-43–positive IENFs per se did not correlate to hyperalgesia, because at day 3 of AIA the numbers of GAP-43–positive IENFs were scattered from the normal range to enhanced levels but the drop of pain thresholds was pronounced in all animals. Furthermore, at day 21, weight-bearing and primary hyperalgesia at the knee were clearly reversed; yet, all rats exhibited an increase of GAP-43–positive IENFs in the ipsilateral paw at this time point. This suggests that the reversal from a significant pain state in the deep tissue may be associated with an increase of GAP-43–positive IENFs (at least in some areas) see Fig. . HoweverIt is important to note that the behavioural changes in experimental arthritis models correspond to observations in humans. Rats with unilateral arthritis in the knee joint also exhibit local mechanical hyperalgesia at the contralateral healthy knee joint, and they show enhanced sensitivity to mechanical and thermal stimuli at the hind paws , 4. WideWhether humans with rheumatoid arthritis show enhanced GAP-43 expression has not been explored, to our knowledge. However, patients with rheumatoid arthritis do not have significantly altered IENF densities compared with healthy subjects . This daAlthough the sensory consequences of altered GAP-43 expression are unclear, the consensus based on many studies is that neuropathy or local inflammatory changes may be associated with changes in GAP-43 expression and that an upregulation of GAP-43 indicates a regeneration of nerve fibres, whereas a reduction of GAP-43 is associated with a loss of regenerating nerve fibres. The novel finding of the present study is that the GAP-43 expression in nerve fibres was increased in skin remote from an inflamed joint. The present findings are the first, to our knowledge, to indicate that IENFs remote from the joint show changes in arthritis.The question regarding mechanisms which might be involved in the upregulation of GAP-43 in nerve fibres following arthritis in the skin remote from the joint is intriguing. Our data indicate that this process started within the first 3 days of AIA and persisted in some skin regions up to 21 days. In the AIA model, lumbar DRG neurons do not express activating transcription factor 3, a marker for neuronal damage, at the acute or chronic stage . Thus, tIn the present study, we observed, as in two previous studies, a bilateral invasion of macrophages into the lumbar DRGs of both the inflamed and noninflamed sides , 52. ThiWhether the invasion of macrophages into the DRGs is linked to enhanced GAP-43 expression is unknown. The data shown in Fig. On the basis of the present study, we report the novel observation that changes of innervation in arthritis models occur not only in the inflamed joint but also in healthy skin remote from the joint. Because GAP-43 is thought to indicate processes of structural neuronal plasticity, the present findings suggest that arthritis is associated with neuronal changes in skin innervation remote from the inflamed joint. Although the precise role of enhanced GAP-43 expression in mechanisms of pain is not known, the data show that localized immune-mediated arthritis may cause more widespread neuronal alterations in the peripheral nervous system. Such neuronal changes may contribute to long-lasting secondary hyperalgesia in the skin remote from the inflamed joint. Our findings should stimulate research into the regulation and functional importance of GAP-43 and regenerating nerve fibres in arthritis and chronic, widespread pain.


Graphene deposited on various substrates has attracted the attention of the scientific and technical communities for use in a wide range of applications. Graphene on substrates is commonly produced by two types of methods, namely, methods that require a transfer step and transfer-free methods. Compared with methods that require a transfer step, transfer-free methods have a simpler procedure and a lower cost. Thus, transfer-free methods have considerable potential to meet the industrial and commercial demands of production methods. However, some limitations of the current transfer-free methods must be overcome, such as the high temperatures encountered during production, the relatively long manufacturing times, incompatibilities for both rigid and flexible substrates, and an inability to extend the process to other two-dimensional (2-D) atomic crystals. In this work, a room-temperature rubbing method is developed for the rapid transfer-free production of defect-free polycrystalline graphene on rigid and flexible substrates. Starting with inexpensive commercially obtained graphite powder, mono- and few-layer graphene can be fabricated directly on various substrates, with an average production time of less than one minute (from raw graphite to graphene on the substrate). Importantly, this method can be extended to other 2-D atomic crystals. Graphene has attracted intense scientific and technical interest during the past decade, as its extraordinary properties make this material suitable for a wide range of applications, including electronics, photonics, energy, and mechanics12335678989101112131415891011121314158910111213141541718192) and flexible  substrates, and (4) the methods are difficult to extend to other two-dimensional (2-D) atomic crystals.In recent years, transfer-free methods based on different synthesis have been explored, including chemical vapour deposition (CVD) growth with metal catalysts, such as Cu, Ni, Ga, etc.212223242527282930313233343521373839402122232425262728293031323334353637383940Herein, a room temperature rubbing method is developed for the rapid transfer-free production of defect-free polycrystalline graphene on various types of substrates (including both rigid and flexible substrates). Starting with low-cost commercially available graphite powders, mono- and few-layer graphene were directly fabricated with an average production time of less than one minute (from raw graphite to graphene on substrates). Importantly, this rubbing method can be extended to other 2-D atomic crystals. Thus, this process has great potential to meet a number of industrial and commercial demands: it is more likely to produce polycrystalline films than the expensive single-crystalline films242518222719171257114428352) and flexible  substrates instead of only being suitable for flexible substrates; (2) both mono and few-layer graphene can be produced instead of just few-layer graphene; (3) the average time from raw graphite powders to final graphene films is reduced from approximately 10 minutes to less than one minute; (4) the procedures are more environmentally friendly because none of the auxiliary materials are discarded; and (5) importantly, the advantages of the previous rubbing method are maintained, including room temperature and low-cost production, together with the capability of being extended to other 2-D atomic crystals.This rubbing method is an important improvement over our previous methods46All of the auxiliary materials, including the poly (PET) films, the polished silicon (Si) wafers, and, most importantly, the reusable surfaces that are built on the polishing sandpapers, are reusable. The surface morphologies of the auxiliary materials are given in The reusability of the auxiliary materials means that we only needed to complete the sandpaper-rubbing see  and doubAccording to our experiments, these production cycles could continue for more than 20 rounds (commonly 22 to 34 rounds). If we count the rounds as 20, then the total time is 10 + 9 × 20 = 190 min, and the total sample number is (10 to 20) × 20 = 200 to 400. Therefore,1 sample only takes 0.475 to 0.95 min, in other words, within one minute.2) as rigid substrates, whereas PET, polyethylene (PE) and polyurethane (PU) were employed as flexible substrates. All of the chosen substrates have smooth surfaces (surface roughness within 1 nm). The results of graphene on different substrates are presented in In this work, we chose a Si wafer and quartz glass -type defects577425051The elemental purity of the graphene is another important feature to characterize the quality of the produced graphene. In this work, energy dispersive spectroscopy (EDS) was used to detect the different elements present in our graphene sample. The result  reveals (1) How many samples can a reusable surface produce? (2) If possible, then how can the reusable surface be restored for further production?Cyclic operability is one of the main advantages of the proposed method. There are two important issues: th sample on, relatively large differences in the macro-performance (a considerably higher resistivity and higher transparency) and significant changes in the micro-morphology  and the micro-morphology . For example, for 40 s of soft-contact-rubbing, the number is 400/40 = 10, whereas the number of samples increases to 400/20 = 20 for 20 s.P), friction speed (V), and friction time (t). t was selected as the core parameter to be adjusted for the following reasons:(1) Under industrial conditions, t is easier to adjust and control than P and V. (2) Relatively higher P and V are favourable to improve the production efficiency; however, P·V has a direct relationship with the power requirements of the equipment, and such power is limited. Thus, only a relatively small adjustment range is possible for P and V. Consequently, t is the best parameter to describe the controllability and cyclic operability of the method.During each rubbing step, there are three important parameters that impact the controllability: friction pressure : the initial rubbing uses PET as the smoothing material and is followed by smoothing with a polished Si wafer. The impacts of smooth-rubbing-time and smoothing materials (PET or Si) on the number of deposited graphene layers are compared in Control over the number of graphene layers that are deposited is the other important parameter. The layer numbers were obtained from thickness results of AFM tests . These results indicate that the rigid humidity sensor can be used at a relatively high temperature, whereas the PET humidity sensor has the advantage of high flexibility during operation.Polycrystalline grain boundaries can have a strong influence on the properties of graphene films, and also allow the preparation of materials with novel properties7425051ency see . After pency see . The reaThe influence of the graphene sample size on its properties is crucial for industrial and commercial purposes. Notably, the results in 2), tungsten disulfide (WS2), etc.In recent years, 2-D atomic crystals have been explored extensively, and examples of such crystals include those from hexagonal boron nitride (h-BN)1561565455562 and WS2) produced on different substrates by the rubbing method contained within. SEM images of the raw materials are given in 2 substrates are given in 2 atomic crystals, which form a film with individual crystals on a scale of several micrometres. 2 and monolayer h-BN, which were transferred from SiO2 to TEM grids. 2 atomic crystals on a SiO2 substrate.The results showing 2-D atomic crystals on SiOA1g) peaks in the Raman spectra exhibit clear red shifts: approximately 2 cm−1 for few-layer MoS2 and approximately 5 cm−1 for monolayer MoS2. In addition, blue shifts of −1 and approximately 2 cm−1 for few-layer and monolayer MoS2, respectively. Such results were consistent with results from the literature on Raman spectra of bulk MoS2 and MoS2 atomic crystals15455562 atomic crystal flakes. 2 atomic crystal on Si with thicknesses of approximately 1.06 nm.The results of 2-D atomic crystals on Si are given in 2 atomic crystals on PET. 2 on PET that presents atomic crystals on a scale of several micrometres, similar to the case of MoS2.The results of 2-D atomic crystals on flexible PET substrates are given in 2 and WS2 atomic crystals on Si, and just like graphene, most of these atomic crystals are monolayer and bilayer. The similarities between 2 atomic crystals/SiO2 sample. While the similarities of 2 atomic crystals on PET.The controllability and cyclic operation of the method as applied towards 2-D atomic crystals are shown in The rubbing procedures involve the wear processing of the sandpaper surface, e.g., during the double-smoothing-rubbing step, the surface of the sandpaper is worn by PET and the polished Si wafer, while the soft-contact-rubbing step results in the sandpaper’s surface being worn by different substrates. Thus, classical friction and wear theories can be applied to explain the mechanisms behind the rubbing procedures.According to the adhesive wear mechanismDuring the early stages, the rates of wear (d’) on sandpaper’s surface is proportional to pV. Because a certain pV value is already chosen, d’ is approximately constant at the beginning of the soft-contact-rubbing step. Thus, the amount of wear on the sandpaper’s surface increases with rubbing time (t), as does the amount of graphene transferred to the substrates.ad) can be considered proportional to the friction coefficient, as described in Equation According to Archard’s viewThus, at a later stage of the soft-contact-rubbing, the rate of wear (d’) on the sandpaper’s surface is decreased, because graphene that was already transferred to the substrates would decrease the friction coefficient between the sandpaper and the substratesTherefore, a polished Si wafer is chosen because it exhibits a similar surface roughness to PET and has a much higher hardness. According to Rabinowicz’ view on wearThree additional processing conditions  were carried out. Condition 1replaces the initial smoothing material (PET) in the double-smoothing-rubbing step with a polished Si wafer. Condition 2 replaces the second smoothing material (Si) in the double-smoothing-rubbing step with PET. Condition 3 changes the sequence of the double-smoothing-rubbing step .For Condition 1, the smooth region did not increase significantly as the rubbing time increased compare  even thoFor Condition 2, as described in Similar results were obtained under Condition 3 as under Condition 2  at room temperature by a rubbing method. This highly efficient and low-cost method used inexpensive commercial graphite as a raw material. Mono- and few-layer graphene films were fabricated directly on various substrates with an average production time of less than one minute. The size of the deposited graphene flakes was several to tens of micrometres. The graphene was characterized as having a high elemental purity, with defects consisting primarily of boundary (or edge) types. The rubbing method displayed good controllability of the graphene-covered area and of the number of graphene layers on the substrates. The graphene films produced by this method performed well in applications, such as humidity sensing, transparent heating, and strain sensing, thus demonstrating the potential industrial and commercial value of the technique. Furthermore, this rubbing method can be extended to other 2-D atomic crystals.2) glasses  and PET films were used as substrates  and WS2  powders are needed as raw materials for other 2-D atomic crystals.Poly (PET) films  were needed as initial smoothing material (light gray piece in All of the rubbing steps shared the following characteristics: (1) The rubbing action can be described as “revolution without self-rotation”. The rubbing diameter was 0.5 cm, and the rubbing rate was 200 rpm. (2) A commercial rubber  was needed to apply the appropriate pressures. It was affixed to the rubbing equipment, as shown in The commercial raw graphene powder was rubbed between two pieces of polishing sandpaper  by applyAfter this rubbing step, the polishing sandpaper surface would be covered by graphite sheets or other 2-D sheets  with a commercial rubber, the target substrate was rubbed with the reusable surface by applying a soft pressure (0.5 MPa for rigid substrates and 0.1 MPa for flexible substrates) for 20 to 40 s. This rubbing process fabricated the final samples .As shown in As shown in Initially, a proper amount of graphite or other 2-D raw powder was added and then rubbed between the two PET smoothing materials  by applying a soft pressure (0.1 MPa) for 100 s.Then, we rubbed the reusable surface on the PET smooth material by applying a soft pressure (0.1MPa) for 100s. The PET smoothing material can be reused for the next restorative rubbing step. From our experiments, the PET smoothing materials can be reused 20 times.Finally, we affixed the polished Si wafer (after a one-minute ultrasonic cleaning and drying) to the commercial rubber, and we rubbed the reusable surface on the Si by applying a soft pressure (0.2 MPa) for 200 s. The Si smoothing material could be reused for the next restorative-rubbing step, following the cleaning step described above. From our experiments, the Si smoothing materials can be reused more than 100 times.After the above three rubbing steps, the reusable surface was ready for another cycle.2 substrates) were characterized by TEM (JEM-2100F) after transferred onto TEM grid, and the elemental purity of the graphene was detected by EDS. The sizes and distribution of 2-D atomic crystals on SiO2 substrates were observed by optical microscopy (XSP-35). The morphology of graphene and other 2-D atomic crystals on PET substrates and the morphology changes of polishing sandpapers’ surfaces were characterized by SEM (Sirion 200 and Nova NanoSEM 450). Sheet resistance and optical transparency measurements of flexible transparent conductive films (graphene/SiO2 and graphene/PET samples) were taken by Four-probe tester (RTS-8) and UV-Vis Spectra photometry (Lambda 35), respectively.Raman spectra of graphene and other 2-D atomic crystals on Si substrates were characterized by 514 nm Raman spectrometry (INVIA), and the 10 μm × 10 μm area Raman mapping images were obtained from Raman laser whose spot size is 1 μm × 1 μm was moved by 1 μm. The thicknesses of graphene and other 2-D atomic crystals on Si substrates were tested by AFM . The edges of monolayer graphene and other 2-D atomic crystals (peeled from SiO2 and graphene/PET in a pre-defined pattern. The humidity sensing behaviours were tested in a constant temperature and humidity incubator (SPX-160B) at a constant temperature of 300 K while the relative humidity was changed from 30% to 90%.A 30 s soft-contact-rubbing was chosen for humidity sensing. The sensors were fabricated by printing silver pads (3 cm × 0.25 cm) onto 3 cm × 3 cm sized graphene/SiO2 and graphene/PET in a pre-defined pattern. A polycarbonate (PC) film was used as an insulating protective layer for security. The alternating current (AC) working voltage was provided by an adjustable regulated AC power supply. The heating temperature was measured using an infrared thermometer.A 40 s soft-contact-rubbing was chosen for transparent heating. The heaters were fabricated by printing silver pads (6 cm × 0.25 cm) onto 6 cm × 6 cm graphene/SiOFor strain sensing, a 25 s soft-contact-rubbing was chosen. The sensors were fabricated by printing silver pads (12 cm × 0.25 cm) onto 12 cm × 12 cm sized graphene/PU in a pre-defined pattern. The strain gauge factors were calculated by testing the resistivity changes under strain-on/off conditions. The strain was performed and measured by a strain equipment made by ourselves. In this study, “strain off” means 0% strain (without elongation), and “strain on” means approximately 3% strain (with elongation as approximately 0.36 mm).How to cite this article: Jiang, S. et al. One-Minute Room-Temperature Transfer-Free Production of Mono- and Few-Layer Polycrystalline Graphene on Various Substrates. Sci. Rep. 6, 19313; doi: 10.1038/srep19313 (2016).


In most applications based on chemical vapor deposition (CVD) graphene, the transfer from the growth to the target substrate is a critical step for the final device performance. Manual procedures are time consuming and depend on handling skills, whereas existing automatic roll-to-roll methods work well for flexible substrates but tend to induce mechanical damage in rigid ones. A new system that automatically transfers CVD graphene to an arbitrary target substrate has been developed. The process is based on the all-fluidic manipulation of the graphene to avoid mechanical damage, strain and contamination, and on the combination of capillary action and electrostatic repulsion between the graphene and its container to ensure a centered sample on top of the target substrate. The improved carrier mobility and yield of the automatically transferred graphene, as compared to that manually transferred, is demonstrated by the optical and electrical characterization of field-effect transistors fabricated on both materials. In particular, 70% higher mobility values, with a 30% decrease in the unintentional doping and a 10% strain reduction are achieved. The system has been developed for lab-scale transfer and proved to be scalable for industrial applications. Graphene has been shown to be an excellent material for opticalA manual method for graphene transfer is widely used578911121315161819202122252/Si wafers they tend to cause undesired mechanical defects on graphene films, which considerably degrade their electrical propertiesDespite of these drawbacks, the only existing automatic methods are based on roll-to-roll transfer processes27In this work we demonstrate a new system that automatically transfers the graphene layer to an arbitrary target substrate, avoiding the need of an operator and the waiting times of a typical manual transfer process. Furthermore, we demonstrate the improved carrier mobility and yield of the automatically transferred graphene, as compared to that manually transferred, as shown by the optical and electrical characterization of graphene field-effect transistors (GFETs) fabricated on both kind of materials. The system reported has been developed for lab-scale transfer, but it is fully scalable and, thus, suitable for industrial applications.The automatic transfer system is described schematically in The PTFE holding structure has been specifically designed to assist the automatic process . The choThe control electronics consist of an Arduino UNO microcontroller that is used for connecting, timing and reading the other system modules. A serial connection to a PC is used for programming and logging the process.Regarding the fluidics, two peristaltic pumps are used for introducing and removing liquid from the etch beaker. Chemical-resistant tubing connect the three containers of the liquid system: the etch beaker, and the DIW and the residues tanks. For the liquid flow, three states are available: idle, liquid introduction and liquid removal. By means of two check valves, the liquid can only flow from the DIW tank to the etch beaker and from the etch beaker to the residues tank, corresponding with the liquid introduction and liquid removal states in the peristaltic pumps system, respectively. The on/off pump conditions are controlled by means of relay switches.A contactless, ultrasound-based distance sensor reads directly the liquid level inside the etch beaker, providing the feedback signal for controlling the fluidics and determining the filling state. This sensor does not disturb the liquid surface, and is reliable even when the liquid composition is changing, as opposed to infrared sensors. The distance sensor is mounted on a micropositioner that allows fine tuning the initial distance before running the automatic process. Minimum and maximum liquid levels are defined in order to keep the graphene constantly floating in the liquid inside the container tube without touching the target substrate underneath till the very last step of the transfer process. This is a key characteristic of the automatic transfer system, as compared to the manual transfer methods, where the sample is changed abruptly to different liquid solutions using an intermediate holding substrate. Thus, the all-fluidic manipulation prevents the graphene from any strain, damage, or contamination induced by the intermediate holding substrate.The heating plate along with the temperature sensor form the temperature control unit. A proportional-integral-derivative controller is used for setting the temperature, being optimized for the working temperature range of the system (RT - 60 °C).Finally, a fan positioned on top of the container tubes is activated during the last step of the transfer process to help drying the samples.μm thickness), has been used as protective polymer. Finally, the distance sensor on top of the beaker is checked and readjusted if needed, and the tubing end is introduced at the bottom of the beaker. After this point, the system operation is fully automated with no need of operator intervention.Before starting the automatic system operation, the target substrate is loaded onto the substrate tray. For some cases, such as chemically-reactive target substrate, the substrate tray can be loaded onto the system just before the end of the process. This way, the target substrate will only contact pure DIW. For flexible substrates, such as thin PET films, a rigid holder should be used to fix the substrate to the substrate tray. For preparing the transfer process, the whole PTFE structure is introduced inside the etch beaker, with the alignment base and container tubes in place as shown in 2 membranes on 2 × 2 cm2 substrates, 400 ml of DIW are needed for the rinse, though a system design optimization could reduce this amount.In After the dilution loop, the critical part of the transfer process starts, filling the etch beaker with DIW up to the maximum liquid level , and theOnce the automatic transfer process ends, the target substrate is unloaded from the system. In In addition to the technological advantages inherent to an automated process , an automatic transfer process is expected to provide better quality graphene in terms of cleanliness (i.e. lower amount of etchant residues) and structural integrity, since the manipulation of the graphene is done in a smoother way with no intermediate holding substrate involved. The usage of an intermediate substrate in the manual transfer may cause undesired effects, as a quick capture movement is needed to perform several steps. For example, the membrane is forced an unwanted tensile strain when raised from the liquid, as the membrane is partially floating while the liquid is perturbed. Other undesired effect may be wrinkle formation around etchant residues, that may promote the adhesion of trapped ions on the graphene surface. An explanatory schematic of both mechanisms can be found in et al.μm-thick Cu foil. The chosen target substrates are 2 × 2 cm2 pieces diced from a 300 nm-thick SiO2/Si In order to demonstrate the improvement of the properties in automatically-transferred graphene, the performance of GFETs fabricated on manually and automatically transferred samples was compared. Thus, two groups of samples from the same graphene material were processed with the same conditions (see Methods), except for the graphene transfer method: the first group was transferred manually following a method derived from Sun ack-gate  inset.μ)   and the μ)  . The lat2V−1s−1). As discussed above, the gradual dilution procedure used in the automatic transfer process is likely to remove more efficiently the etchant from the graphene surface in contact with the liquid, thus reducing the unintentional doping concentration. Additionally, the all-fluidic manipulation of the graphene membrane in the automatic transfer process avoids the mechanical stress induced during the successive DIW rinse steps and the final transfer step to the target substrate of the manual method. Nonetheless, for both types of samples, the standard deviation in the mobility is similar, indicating that the dispersion may be related with the intrinsic properties of the commercial CVD-graphene used in all cases.In the case of the mobility, a clear improvement is observed for the automatically transferred samples, with a 70% increase in the mobility mean value as compared to the manually transferred samples. This is consistent with the decrease of 30% in the unintended doping for the automatically transferred samples, indicating less adsorbed impurities than in the manual method and, thus, less scattering centers reducing the mobility of the carriers in the graphene film. These results confirm that the automatic method developed degrades less the electrical properties of the material. The values reported for the manual transfer are in good agreement with literature values for the same target substrate and graphene source2V−1s−1) to eight-fold for the most demanding threshold value (2000 cm2V−1s−1). A similar behavior is found for the unintentional doping level, with a seven-fold increase in the yield for the automatically transferred devices compared with the manually transferred ones for doping concentrations as low as 1012 cm−2.It is also important to point out that the automatic transfer method provides a much larger yield. The yields of the manual and automatic transfer processes, expressed as the fraction of devices with higher mobility or lower doping values than certain thresholds, are shown in et al.Raman spectroscopy mapping has been performed in the channel area of several GFETs fabricated on both automatically and manually transferred graphene samples in order to evaluate the amount of strain and doping present in each kind of samples and correlate them with the results obtained from the electrical characterization of the devices. The analysis is based on the correlation of the frequencies of the Raman modes G , the automatic system also provides higher quality graphene. GFETs fabricated on the automatically transferred graphene present higher mobility values, lower unintentional doping and strain, and better yields as compared to those fabricated on manually transferred material. The system, initially developed as a lab-scale tool, is fully scalable for industrial applications, allowing the large-scale transfer of graphene films with superior quality to arbitrary substrates.2O2:H2SO4 1:3 vol.) during 5 minutes and rinsed afterwards thoroughly in DIW. N2 drying is performed after each DIW rinsing step. This ensures the cleanliness of the PTFE pieces and reduces their hydrophobicity for the following steps. Next, the container tubes are immersed in an aqueous solution of polystyrene sodium sulfonate (PSS) (2% in mass) for 30 seconds, rinsed in DIW, and dried with N2 afterwards. After this, another 30-second immersion in an aqueous solution of polydiallyldimethylammonium chloride (PDDA) (2% in mass), DIW rinse and N2 drying are performed. A last PSS treatment as the previous one is performed. Therefore, the final coating is composed of a layered PSS/PDDA/PSS structure.The PTFE tubes enclosing the graphene membrane during the automatic transfer process (container tubes) were surface-treated with SAMsμm-thick Cu foilhas been used as starting material for GFET fabrication. Oxygen plasma has been used for insulatingthe transistors and Ti/Au (10/200 nm) contacts were deposited by e-beam evaporation. Deviceswith gate lengths between 10 and 20 μm and widths ranging from 100 to500 μm have been fabricated. The Si n++ doped substrate, coated with 300 nm of thermally-grown SiO2, has acted as the back-gate. For the characterization, the samples were introduced inside a Janis-CCR10 probe station, which was used for degassing the last residues in graphene at 500 K for 3 hours under vacuum conditions (10−5 mbar). After letting the probe station cool down and without breaking vacuum, the current-voltage characteristics for a large number of devices in each sample were measured at room temperature. Back-gate voltage values between −40 and 65 V and a drain-source voltage of 0.2 V were used.Commercially-available monolayer CVD graphene grown on 25 μm2 in size, scanned with steps of 0.33 μm.A WITec Alpha 300AR Raman confocal microscope has been used for the Raman spectroscopy mapping of the channel area of the GFETs. Raman spectra were obtained in backscattering geometry using a ×50 objective lens  in ambient conditions. A 532 nm wavelength laser set to a power of 3 mW was used as excitation source. Typical mapped areas were 110 × 8 How to cite this article: Boscá, A. et al. Automatic graphene transfer system for improved material quality and efficiency. Sci. Rep.6, 21676; doi: 10.1038/srep21676 (2016).