The humidity sensing characteristics of different sensing materials are important properties in order to monitor different products or events in a wide range of industrial sectors, research and development laboratories as well as daily life. The primary aim of this study is to compare the sensing characteristics, including impedance or resistance, capacitance, hysteresis, recovery and response times, and stability with respect to relative humidity, frequency, and temperature, of different materials. Various materials, including ceramics, semiconductors, and polymers, used for sensing relative humidity have been reviewed. Correlations of the different electrical characteristics of different doped sensor materials as the most unique feature of a material have been noted. The electrical properties of different sensor materials are found to change significantly with the morphological changes, doping concentration of different materials and film thickness of the substrate. Various applications and scopes are pointed out in the review article. We extensively reviewed almost all main kinds of relative humidity sensors and how their electrical characteristics vary with different doping concentrations, film thickness and basic sensing materials. Based on statistical tests, the zinc oxide-based sensing material is best for humidity sensor design since it shows extremely low hysteresis loss, minimum response and recovery times and excellent stability. The regulation of humidity is important for human comfort, storage of various goods, industrial process control, high-tech instruments, and plenty of advanced sectors . In induIn 1450, Nicolas Cryfts invented a hygrometer which is the first humidity measurement instrument on record. This hygrometer used wool to determine the changes of humidity in air ,3. Over Humidity is evaluated by different functions such as vapor pressure, saturation vapor pressure, dew/frost point temperature, and relative humidity . Vapor pRelative humidity  is a ratio of the vapor pressure (VP) of moist air to its saturation vapor pressure (SVP) at a given temperature, which is expressed by 3), defined as the amount of water vapor contained in a unit volume of dry air, i.e., mixing ratio mr  and the ratio with respect to saturation is defined as saturation deficit psd (mbar).Humidity can also be expressed in other ways like absolute humidity , food processing and storage, medicine, ecology, agriculture, mineral processing, fuel quality control, and aerospace. Finally the concluding remarks are provided in Section 5.2.For many sophisticated applications, there has been a keen attempt to reduce the size of the sensors using the most advanced technologies collectively called miniaturization. Miniaturized humidity sensors mainly depend on five different transduction principles: hygrometry, capacity, resistance, gravimetry and optical properties.2.1.For many decades hygrometric sensors have been using in old devices that are classified into four basic types—mechanical, dry bulb-wet bulb, chilled mirror, LiCl dew point—for the measurement of the humidity of air, which is converted into mechanical energy –10. ThesAdvantages:Low cost;No power supply is required;It provides a primary rather than a secondary measurement of dew point;High accuracy;High repeatability;Traceable to N.I.S.T. or other national standards labs;Wide dew point range;Wide temperature range;Low hysteresis;No drift.Disadvantages:More complex, may be more expensive;Narrow flow rate range;Periodic cleaning may be required;High uncertainty (±2% to 5%).Generally, hygrometric sensors have two different materials with different thermal expansions coefficients, such as a polysilicon membrane and a hygroscopic material such as polyimide. The polyimide as a hygroscopic coating expands due to the absorption of water. Due to the unequal expansion that occurs between the polyimide and polysilicon, a change in the bending structure of the membranes is observed. The change in bending is converted into an electrical signal via a piezoresistor. The hygrometric sensor has some advantages as well as some disadvantages which are listed below:2.2.R(hr) represents the resistance at relative humidity (RH) at a specific humid condition h, Rr0 is the resistance at zero humidity, and a and b are the constants.The most common resistive sensors are based on electrical resistance. The electrical resistance of a material is the opposition to the passage of an electric current through that conducting material. Since the humidity can change the electrical resistance or impedance of a material noticeably, resistive humidity sensors have been developed using this principle. Generally the all resistive sensors have four layers: substrate, interdigital electrode, humidity sensitive film and protective layer which are shown in RS) can be measured using an expression given in Rh refers to the resistance at certain humidity and R0 represents the original resistance of the sensors at air of 3% RH.The sensing response (4BF4-PEG600 thin film resistive type humidity sensor is based on cellulose acetate (CA), ammonium tetrafluoroborate (NH4BF4) and polyethylene glycol (PEG600) . L. L106]. et al., had shown that 0.45 mol % of Li-doping in a γ-Fe2O3-based humidity senor is critical, since it showed maximum linearity . M. M2O3 se3.3.2.+ addition in a LaCo0.3Fe0.7O3 thick film sensor, the resistance decreases effectively as the frequency increases at a low RH range and the difference in resistance value between two working frequencies becomes smaller at a high RH range . T. T+ addi3.3.3.i.e., in the range of 10−11–10−12 F). The capacitance value increases with increased Li doping concentration in iron oxide film over the full RH range at 1 kHz frequency and at high RH (90%), the capacitance value increased by about one order in magnitude  in the low frequency range  and in the high frequency region , the capacitance becomes almost independent of RH , the value of capacitance increases with the increasing value of RH  and in the high frequency region (10–100 kHz), the capacitance becomes almost independent of RH, is illustrated in Due to the addition of K3.3.4.+ ion may improve the response time of an iron-based humidity sensor. The response times of MgFe2O4 (in the 53%–98% RH range) and Mg0.9Sn0.1Fe2O4 (in the 0%–53% RH range) sensors, which are 5 min and 3 min, respectively, is shown in + doping in an iron-based humidity sensor, the response and recovery times in a humidity range of 33%–95% RH are 100 s and 120 s, respectively . I. I+-ion ty range .3.3.5.+-ion-doped iron oxide perovskite type nanocrystals show narrow hysteresis loops during cyclic humidity operation. The hysteresis of a 2 wt % K2CO3-doped sample between humidification and desiccation processes over the whole humidity range with 1 V, 100 Hz and at room temperature is shown in 1−xKxCo0.3Fe0.7O3−δ perovskite humidity sensor is observed . O. O125]. 2 nanoparticles, polypyrrole and AgNO3 based humidity sensor, impedance varies significantly with RH with addition of different doping concentration. The impedance decreases with increases of doping concentration in all relative humidity range. The main advantages of the conducting polypyrrole polymers (PPy) are relatively good environmental stability and most importantly its surface charges can easily be modified by different dopants by easy synthesis method . P. P128]. s method . It has y sensor . The basng, 2007 . It has ng, 2007  gives fang, 2007  sensor.5 to 7.0 × 104 Ω with an increase in RH from 11% to 95% with greater linearity and good sensitivity (0.0370), but copolymer film has poor linearity and less sensitivity (0.0305) ..170].3.9.2) has being selected for advanced humidity sensors due to its unique properties, such as high thermally and excellent chemically stability, high toughness, high strength, and good catalytic activities, which make it more attractive compared to other materials in wide range of applications. Very little work has been reported on ZrO2-based humidity sensors so far. Wang et al. [2 thick film humidity sensor, which was made of ZrO2 nanoparticles with an average grain size 20 nm, was used to deposit it as a thick film on a silicon substrate. Y3+-doped and Mg2+-doped zirconia thick film humidity sensors have also been investigated by Su et al. [Many kinds of materials like ceramics, organic polymers, semiconductors, and solid electrolytes have been studied for humidity analysis. However, recently zirconia -doped ZrO2 humidity sensors [Like the other materials, the impedance decreases as the frequency increases and the impedance became independent of the humidity with increasing frequency. It has been found that the best linearity of the impedance–RH characteristic appears at frequencies between 100 Hz and 1 kHz for ZrO sensors , however sensors , as depi3.9.3.2-based sensors change strongly with doping agent. For example, the response time of a Y3+-doped ZrO2 humidity sensor is 30 s and for a Mg2+-doped sensor it is 5 s; and the recovery time for both the Y3+ and Mg2+-doped ZrO2 sensor is 5 s [2 humidity sensor are 130 s and 60 s, respectively [2+-doped ZrO2 sensor has the smallest response and recovery time, and can be considered as the best compared to the pure ZrO2 or Y3+-doped ZrO2 sensors.The response and recovery times of ZrOr is 5 s . The resectively . Therefo3.9.4.2 sensor is about 8% RH [3+- and Mg2+-doped ZrO2 sensors, respectively [2-based ceramic sensors, for less hysteresis value of a Y3+-doped sensor can be selected as the most suitable material for humidity measuring devices.The humidity hysteresis of the pure ZrOut 8% RH , while oectively . Therefo3.10.et al. [3. The changes in electrical characteristics  with the variation of frequency, temperature and relative humidity of sodium-based sensor materials are explained in the following subsections.Oxide ceramic materials have mostly been used in humidity sensor applications. Sodium oxide-based sensors also are being tried in humidity sensing devices due to its attractive thermal, physical and chemical stability like that of other ceramic materials. However, the main drawback of normal ceramics-based humidity sensors is their insufficient sensitivity over a wide humidity range, as well as lack of reversibility and drift in base resistance with time due to chemisorption of water molecules. Therefore, to overcome this problem many researchers are trying to use sodium-based materials to promote the sensitivity. Among them Na- and K-montmorillonite  sensors et al.  develope3.10.1.et al. [Su et al.  observed3.10.2.3-based humidity sensors [3-based humidity sensor has a better linearity than the pure Na-montmorillonite one.In both pure Na-montmorillonite-  and NaTa sensors , the imp3-based sensor the best linearity is observed at 100 Hz  is an important biomarker which has a significant role in the human body. Ammonia is present in all body fluids, mainly as ammonium ion (NH4+) but also in the form of NH3. A high concentration of NH3 is toxic to the human body. During the last decades, considerable effort has been put into detecting gaseous exhaled breath ammonia (eNH3) as a surrogate for blood or urine detection. Schmidt et al. [Advanced telehomecare (THC) systems use humidity sensors to monitor serious changes in chronic conditions as well as other health risks including floods, fires and gas leaks. A team led by Haick at the Israel Institute of Technology (Technion) made a humidity sensor breathing tests to detect lung cancer which contains multiple gold nanoparticle-based chemiresistors that can detect chemicals through changes in electrical resistance. The approach for breath-volatile organic compound (VOC) collection and preconcentration by applying needle traps was developed and optimized by Filipiak et al. . Here hut et al.  developeBreath monitoring is essential during certain imaging and surgical procedures where the patient needs to be sedated or anesthetized . Humiditet al. [et al. [To study the progression of a diagnosed illness or to evaluate the health of a person it is very important to monitor the breath. It is not at all wise to use electronic breathing sensors when patients are in a magnetic resonance imaging (MRI) system, or during any oncological treatment, which may create hazards for the patient . A simplet al.  and an a [et al. .et al. [The most familiar and common form of communication is voice communication. Due to some hereditary reasons or acquired impediments or due to other reasons like an accident, there are many people who suffer from speech/hearing difficulties. A humidity sensor-based language recognition system that focuses on the moisture included in devoiced breaths as a method for communication support in persons with speaking difficulties was developed by Morisawa et al. .4.2.Humidity controlled incubators are one of the essential pieces of equipment in tissue engineering applications. Normally 50%–60% RH is maintained for growing animal cells in the incubator.4.3.The shelf life of fruits and vegetables may change with humidity. Excessive water loss from fruits during storage or transportation often reduces its shelf-life. Depending on the type of fruits and the storage methods, the environment can be infected with different bacteria . Therefo4.4.et al. [Analysis of the root distribution of plant and its water absorbing ability is an important subject of considerable interest in ecology and agriculture. It gives a better understanding of the behaviour of different crops under sub-optimal environments, so that the improvement of the quality of modelling of root water uptake in different hydrological environment is possible . The effet al.  and it w4.5.The most widely used areas of humidity and moisture sensors are in structural health monitoring (SHM) –197 appl4.6.To reduce erosion in mountain areas humidity sensors is used by different regulated methods such as check dams, often made of concrete or wood-logs, which decrease the water speed during storm events, allowing sediment to settle .4.7.The study of moisture content in soil has also been a subject of considerable interest in mineral processing plants. Manual gravimetric drying moisture determination methods are still currently employed by most mineral processing plants, but they fails to provide timely and accurate information . An effi4.8.et al. [in situ wireless sensing system for a proton exchange membrane fuel cell (PEMFC) application.In many ways, biomass is a new source of power. Biomass is the energy which is contained inside plants and animals. The variation of moisture contents affects its energy-producing ability. The moisture content in biomass fuel is therefore an important parameter, which often fluctuates . By usinet al. ,189 deve4.9.in situ measurements in space, most spectacularly of the near-surface atmospheric water content on Mars [Recently, humidity sensors have been used to perform  on Mars –203. The4.10.et al. [The Conseil Européen pour la Recherche Nucléaire (European Council for Nuclear Research or CERN)  is the wet al. . This wo4.11.Humidity controlled dark rooms are used for many electronics materials or electronic devices during manufacturing. Power circuit boards (PCBs) are manufactured in controlled humidity dark rooms. Nowadays for low power consumptions in most of the industrial application, radio frequency identification (RFID) tags  based fl5.i.e., >3% for hysteresis, >19 s for response and recovery times), very good  and excellent . Since stability is the change in resistance or capacitance curves with respect to humidity over a long time, according to observation, it is directly graded as good, very good and excellent according to our flatness for a particular humidity sensor material.Humidity sensors for measuring relative humidity based on different types of materials, including carbon, vanadium, iron, silicon, polymer, tin, titanium, zinc, zirconia, and sodium have been reviewed extensively. The electrical properties of humidity sensors such as resistance and capacitance, the effects of temperature, frequency, and relative humidity, sensitivity, response time, hysteresis and stability have been compared in detail for various materials. For different sensor materials, the electrical properties are changed significantly with the doping concentration of different materials, film thickness of the substrate and the resulting morphological changes. The basic principle of six different types of humidity sensors and their typical advantages as well as disadvantages are discussed in the miniaturization section. Based on our overall review, the general electrical responses such as resistance, capacitance, hysteresis, response and recovery times, stability and their best characteristic features for different materials with respect to relative humidity, frequency and temperature are listed in Although response and hysteresis loss are a little less in polymeric sensors compared to zinc oxide-based humidity sensors, the recovery time is significantly larger in polymeric sensors than that of zinc oxide-based sensor materials. Based on our review, it has been found that the zinc oxide-based sensor material is the best for humidity sensor design due to its extremely low hysteresis loss, minimum response and recovery times, and excellent stability. Humidity sensors have a tremendous range of applications in different important fields such as biomedicine, SHM, food processing and storage, medicine, ecology, agriculture, mineral processing, fuel quality control, aerospace, the electronics industry and high-energy physics applications. The humidity conditions, including temperature and % RH, are also very important for different manufacturing or processing sectors as well as their specific areas. Therefore, the development of more integrated humidity sensors for multi-field applications would be highly fascinating topic for the new generation of humidity sensing devices. The review has provided examples that represent the diversity of applications and the demand for RH or moisture sensors in industry today. Therefore, it can be expected that different advanced humidity sensors made of hybrid or doped materials using the latest nanotechnology will be able to monitor a wide range of application sectors.


Confocal Raman spectroscopy has emerged as a major, versatile workhorse for the non-invasive characterization of graphene. Although it is successfully used to determine the number of layers, the quality of edges, and the effects of strain, doping and disorder, the nature of the experimentally observed broadening of the most prominent Raman 2D line has remained unclear. Here we show that the observed 2D line width contains valuable information on strain variations in graphene on length scales far below the laser spot size, that is, on the nanometre-scale. This finding is highly relevant as it has been shown recently that such nanometre-scaled strain variations limit the carrier mobility in high-quality graphene devices. Consequently, the 2D line width is a good and easily accessible quantity for classifying the crystalline quality, nanometre-scale flatness as well as local electronic properties of graphene, all important for future scientific and industrial applications. Raman spectroscopy has become an invaluable tool for graphene characterisation, yet the nature of the broadening of the Raman 2D line remains unclear. Here, Stampfer et al. show that the Raman 2D line width is a measure of nanometre-scale strain variations in graphene on insulating substrates. Graphene combines several highly interesting material properties in a unique way, promising unprecedented material functionality. This makes graphene increasingly attractive for fundamental research as well as industrial applications2456810731415In this article, we show that the experimentally observed Raman 2D line width is a measure of nanometre-scale strain variations in graphene on insulating substrates, that is, it contains valuable information on local  flatness, lattice deformations and crystal quality of graphene. Our findings solve the long-standing question of the nature of the observed broadening of the Raman 2D line and also link this quantity to the electronic transport properties of graphene, making it a valuable quantity for classifying the quality of graphene. To prove that the experimentally observed 2D line width depends on sub-spot-size strain variations and lattice deformations, we employ the following strategy.B-field dependent. In agreement with existing theory19202123B-field of ∼8 T, the G line becomes almost independent of electronic properties such as charge carrier doping, screening, or electronic broadening.We start by showing that by combining Raman spectroscopy with magnetic fields, electronic broadening contributions for the Raman G line width can be strongly suppressed. Since in perpendicular magnetic fields the electronic states in graphene condense into Landau levels (LLs), the interaction between electronic excitations and lattice vibrations becomes We observe that, under these conditions, the G line width nevertheless exhibits strong variations across graphene flakes. In particular, we show that the G line width is significantly increased in regions where the graphene flake features bubbles and folds, that is, in correspondence with increased structural deformations.B-fieldFinally, we show that at 8 T, there is a (nearly) linear dependence between the G line width and the 2D line width, implying that there is a common source of line broadening. According to the previous points, the broadening must be related to structural lattice deformations. This finding is further supported by a detailed analysis of the relation between the area of the 2D peak and its line width. By analysing the relation between the G and 2D line width, we find that nanometre-scale strain variations constitute a dominant contribution to the observed line broadenings. Importantly, the 2D line has been shown to only very weakly depend on the 2 and hBN as illustrated in 2 usually feature lower carrier mobilities of around 103–104 cm2/(Vs), indicating a detrimental influence of SiO2 on the electronic properties of graphene. In this regard, our structure gives us the invaluable capability of probing a single graphene sheet exposed to two different substrates  sheet is partly encapsulated in hexagonal boron nitride (hBN) and partly sandwiched between SiO2 areas can be clearly distinguished in the map (see highlighted regions I and II). While analysing the Raman spectra of every point on the map, it is evident that the G lines recorded in the hBN-encapsulated area are broader than in the SiO2 supported area  is used to suppress Landau damping of the G mode, leaving ΓG unaffected from electronic contributions. The coloured data points stem from Raman maps (B=0 T) of chemical vapour deposition (CVD)-grown graphene flakes that were transferred onto SiO2 by a wet chemistry-based transfer. These graphene sheets contain doping values of nel>3 × 1012 cm−2, which corresponds to Fermi energies EF>200 meV  or to the presence of defects (increasing the electron-defect scattering)38402D and the integrated area of the 2D peak (area2D) we can exclude such a hypothesis. In 2D versus the region-normalized area2D for both B=8 T and B=0 T, highlighting the very weak B-field dependence of Γ2D. More importantly, we observe that the area of the 2D peak does not depend on Γ2D, contrary to what is expected in presence of a variation of the electronic broadening γ  delivered through a single-mode optical fibre, where the spot size is limited by diffraction. Using a long working distance focusing lens with a numerical aperture of 0.80, we obtained a spot size of ∼400–500 nm. For the low-temperature Raman measurements, we employ a commercially available confocal Raman setup that allows us to perform spatially resolved experiments at a temperature of 4.2 K and magnetic fields of up to 9 T. We use an excitation laser wavelength of 532 nm with a spot diameter on the sample of ∼500 nm. For detection, we use a single-mode optical fibre and a charge-coupled spectrometer with a grating of 1,200 lines mmet al.−1 wide Lorentzianset al.2D as a function of area2D. For a laser energy of 2.33 eV, the electron–phonon contribution for the electronic broadening is 81.9 meV, which leads to Γ2D=12.1 cm−1. However, when we choose γ to be twice this value, to account for additional electron–electron interaction2D equal to 17.9 cm−1. These values can be understood as a theoretical expectation of the 2D line width for a perfect graphene lattice disregarding any broadening from averaging effects over different strain values within the laser spot.For the computation of the double-resonant Raman cross-section we employ an approach based on Fermi's golden rule generalized to the fourth perturbative order as described in detail in Reference Venezuela How to cite this article: Neumann, C. et al. Raman spectroscopy as probe of nanometre-scale strain variations in graphene. Nat. Commun. 6:8429 doi: 10.1038/ncomms9429 (2015).Supplementary Figures 1-3


The molecular machinery responsible for cytosolic accumulation of misfolded TDP-43 in amyotrophic lateral sclerosis (ALS) remains elusive. Here we identified a cullin-2 (CUL2) RING complex as a novel ubiquitin ligase for fragmented forms of TDP-43. The von Hippel Lindau protein (VHL), a substrate binding component of the complex, preferentially recognized misfolded TDP-43 at Glu246 in RNA-recognition motif 2. Recombinant full-length TDP-43 was structurally fragile and readily cleaved, suggesting that misfolded TDP-43 is cleared by VHL/CUL2 in a step-wise manner via fragmentation. Surprisingly, excess VHL stabilized and led to inclusion formation of TDP-43, as well as mutant SOD1, at the juxtanuclear protein quality control center. Moreover, TDP-43 knockdown elevated VHL expression in cultured cells, implying an aberrant interaction between VHL and mislocalized TDP-43 in ALS. Finally, cytoplasmic inclusions especially in oligodendrocytes in ALS spinal cords were immunoreactive to both phosphorylated TDP-43 and VHL. Thus, our results suggest that an imbalance in VHL and CUL2 may underlie oligodendrocyte dysfunction in ALS, and highlight CUL2 E3 ligase emerges as a novel therapeutic potential for ALS. Amyotrophic lateral sclerosis (ALS) is a life-threatening neurodegenerative disorder that is characterized by progressive muscle atrophy and weakness. The majority of ALS cases are caused by abnormal conformation of ALS-linked proteins and by defective RNA handling, both of which induce various downstream cascades, including excitotoxicity, endoplasmic reticulum stress, mitochondrial dysfunction, glial dysfunction, proteasome impairment, unexpected secretion, and neuroinflammation12467The exact mechanism of RNA mishandling in TDP-43 proteinopathy is unclear, but a loss of nuclear TDP-43 due to cytosolic mislocalization is considered a likely mechanism. Indeed, aggregate-prone TDP-43 proteins are readily mislocalized in the cytosol and sequester nuclear TDP-4391045VHL is an anti-oncogenic gene that was originally identified as a cause of von Hippel Lindau disease, a dominantly inherited tumorigenic disorder in multiple organs. The VHL protein is a substrate binding protein in the CUL2 E3 complex and has been implicated in diverse pathways, including ubiquitination of hypoxia inducing factors (HIF)151819Here, we found that the von Hippel Lindau (VHL)/cullin-2 (CUL2) E3 complex recognized and ubiquitinated misfolded TDP-43 and promoted clearance of fragmented forms of TDP-43. in vitro ubiquitination and a reversible covalent immunoprecipitation assayBecause of the potential failure to seize labile protein complexes in specific conditions such as ubiquitination, we integrated Based on the finding that VHL and CUL2 colocalize with cytosolic inclusions of TDP-43, we investigated whether VHL/CUL2 recognizes misfolded forms of TDP-43 more than normally folded TDP-43. We previously reported that the conformation of TDP-43 is tightly regulated by two cysteine residues in RRM1, the substitution of which (C173S and/or C175S) leads to formation of aggregate-prone TDP-43 that shares diverse features of pathogenic inclusions in ALSWe next attempted to identify the site in TDP-43 molecules that is recognized by VHL. First, a pull-down study using domain deletion mutants revealed that RRM2, but not RRM1 or the C-terminus, was required for the interaction with VHL . Interesin vivo ubiquitination assay showed no clear trend toward increased ubiquitination of FL TDP-43 by CUL2  rather than in motor neurons by immunohistochemistry. Why TDP-43 inclusions in motor neurons and oligodendrocytes showed different colocalization patterns for VHL remains elusive, the abundant expression and the more active roles of VHL/CUL2 in oligodendrocytes for their maturation from OPCs could underlie the interaction between VHL and misfolded TDP-43 than in motor neurons1819In conclusion, we identified VHL/CUL2 proteins as a novel E3 ligase for misfolded forms of TDP-43. The substrate-binding protein, VHL, plays bidirectional roles by promoting ubiquitin-mediated degradation of fragmented TDP-43 at proteasomes with CUL2, and by stabilizing TDP-43 at JUNQ when upregulated. The deregulation of this pathway would cause oligodendrocyte failure in ALS. Further investigation of regulation of the VHL/CUL2 balance may suggest therapeutic strategies for ALS.in vitro ubiquitination2, 2 mM DTT, 0.5 μg ubiquitin aldehyde , 5 μg ubiquitin, 30 μg S100 lysate 0.5 mM ATP, 10 mM creatine phosphate, and 0.5 μg of creatine phosphokinase in a total volume of 20 μl) was incubated for 30 min at 37 °C. The disulfide-dependent cross-linker, Dithiobis[succinimidyl propionate] (DSP), was then added to the mixture at a concentration of 40 mM and was allowed to cross-link for another 30 min. Disulfide formation was terminated with excess l-cysteine . TDP-43-FLAG proteins were pulled down with anti-FLAG affinity beads , and disulfide-mediated binding proteins were released by mild reducing conditions (50 mM DTT at 37 °C for 30 min). The eluates were separated on an acrylamide gel and visualized with a fluorescent dye . Positive bands, which disappeared in the absence of ATP or TDP-43-FLAG, were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as previously describedTDP-43-interacting proteins during the ubiquitin-proteasome reaction (UPR) were investigated with a combination of the reversible covalent-linked immunoprecipitation (ReCLIP) assayRabbit polyclonal anti-DYKDDDD antibody and mouse anti-GFP antibody were purchased from Cell Signaling Technology  and Nacalai Tesque , respectively. Rabbit polyclonal anti-Cullin2 (CUL2) and anti-TDP-43 antibodies were purchased from Abcam  and Proteintech , respectively. Rat monoclonal anti-HA antibody was obtained from Roche . Mouse monoclonal antibodies against actin (C4), elongin B (D-5), and elongin C (56) were obtained from Santa Cruz Biotechnology . Mouse monoclonal anti-FLAG antibody (M2) was obtained from Sigma. Rabbit anti-ubiquitin, Lys48-specific antibody was obtained from EMD Millipore . Mouse anti-Hsc70/Hsp70 antibody was purchased from StressGen . Mouse anti-HIF1α antibody was obtained from Abcam. Goat anti-mouse IgG Alexa fluor488 and fluor405 were purchased from Invitrogen , and goat anti-rabbit IgG CF568 was from BIOTIM . Mouse monoclonal anti-human VHL was generated as previously describedMammalian expression plasmids for TDP-43 tagged with FLAG (pcDNA3-TDP-43-FLAG) or EGFP (pEGFP-N3-TDP-43) were constructed using a conventional PCR technique as described previouslyThe mutant C-terminal fragment 35 kDa  tagged with FLAG (pcDNA3-CTF35-FLAG) and CUL2 tagged with Myc (pCMV-Myc-CUL2) were constructed using conventional PCR and subcloned into pcDNA3-FLAG using the BamHI/XhoI sites or pCMV-Myc using the BamHI/KpnI sites. Human VHL tagged with HA (pHM6-HA-VHL) was constructed using conventional PCR and subcloned into pHM6-HA using the HindIII/EcoRI sites. The primer pairs are described in 2 and 100% humidity. HeLa cells and HEK293A cells (Invitrogen) were maintained in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum and penicillin/streptomycin . LipofectamineTM 2000 (Invitrogen) and FuGene HD Transfection Reagent  were used for transfection of the plasmid according to the manufacturer’s protocols. For the RNA knockdown experiment, short interference RNA pairs were transduced into cells using Lipofectamine2000 and Magnetofection system . Incubation time for the overexpression and siRNA knockdown transfection before the harvest, cells were 48 h and 96 h, respectively, unless mentioned.All cultured cells were maintained at 37 °C in 5% COAt 48 h after the transfection, cultured cells were lysed in co-immunoprecipitation buffer  or RIPA buffer , containing protease inhibitor cocktail (Roche) and PR-619 , a de-ubiquitination inhibitor for ubiquitination assay. In several experiments, RIPA-insoluble fractions were solubilized in 8 M urea. Ten percent of each cell lysate was analyzed as the total cell lysate, and the remaining 90% was incubated with anti-FLAG M2 affinity gel (Sigma) or beads coated with anti-HA antibody  at 4 °C overnight. The immunoprecipitates were eluted into sodium dodecyl sulfate (SDS) sample buffer. Samples were reacted in one-third volume of 4× SDS buffer, denatured at 95 °C for 5 min, separated on a 5–20% or 15% polyacrylamide gel (Wako), and transferred onto a PVDF membrane (Millipore) for Western blotting. Proteins were detected using an enhanced chemiluminescence system , and the densitometric analysis of protein was performed using Image J softwareAt 48 h after the transfection, cultured cells were fixed in 4% paraformaldehyde in PBS, pH 7.2, for 20 min at room temperature and permeabilized with 0.1% Triton X-100 containing 5% normal goat serum as blocking agent. Cells were reacted with primary antibody (4 °C overnight) and subsequently with fluorescence-tagged secondary antibody  for 1 h at room temperature. Cells were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescent images were obtained using a confocal laser microscope FV1000-D IX81  or fluorescence microscope .Information about the antibodies used is described in Protein half-life was estimated 48 h after transfection by chronological chase analysis at 5, 10, and 24 h after CHX treatment (100 μg) of HEK293A cells to inhibit protein synthesis. The cells were directly lysed in the SDS sampling buffer containing 100 mM DTT and were denatured at 70 °C for 20 min. The remaining amount of protein as estimated by densitometry was expressed as the percentage of the residual protein level compared with that at the starting point (0 h). To determine the effect of CUL2 on transgene expression, cells were transfected with siRNAs for CUL2 , incubated for a further 48 h, and subcultured before transfection with TDP-43 plasmids for the chase study.The total RNA samples were purified from the transfected cells using a commercially available kit (Invitrogen) and were converted to cDNA with reverse transcriptase . The levels of mRNAs for VHL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the synthesized cDNAs were analyzed with the SYBR quantitative PCR kit (Toyobo) and real-time PCR Detection Systems (BIO-RAD). GAPDH was used as an internal standard, and the relative expression levels of mRNAs were calculated by the ΔCT method according to the manufacturer’s protocol using the included software (BIO-RAD). The primer sequences are described in 6 cells/dish. At 48 h after the transfection, all lysates were analyzed for proteasome activity, which was determined at 360 nm excitation and 480 nm emission using a multi-plate reader .Chymotrypsin-like activity was measured as proteasome activity using a commercially available kit . HEK293A cells were seeded in 6-cm culture dishes at a density of 1 × 10All experiments were approved by and performed under the guidelines of the Kyoto University ethics committee. Informed consent was obtained from all individuals or their guardians before the autopsy analysis. Postmortem lumbar spinal cords from five patients with a definite diagnosis of sporadic ALS and five neurological disease controls other than ALS  were analyzed. At autopsy, spinal cord blocks from the lumbar level were paraffinized after fixation in 10% buffered formalin. Sections with 6 μm-thickness were deparaffinized and antigen retrieved by autoclaving (20 min at 120 °C), using Histofine deparaffinizing antigen retrieval buffer, pH 6 . Sections were then incubated overnight with primary antibodies in PBS containing 3% bovine serum albumin at 4 °C. The bound antibodies were visualized by the peroxidase polymer-based method using a Histofine Simple Stain MAX-PO kit (Nichirei) with diaminobenzidine as the chromogen. Double immunofluorescence was performed with the similar protocols, except fluorescence-tagged secondary antibodies were used, as mentioned above.Primary cultures of embryonic rat oligodendrocytes and their precursor cells (OPCs) were prepared as previously descriedet al. 2010). The spinal cord was homogenized in a tissue lysis buffer (20 mM HEPES–KOH (pH 7.4), 120 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Triton X-100, and protease inhibitor cocktail) and then incubated for 1 h at 4 °C, and the supernatant was collected for Western blotting. The protein amount was adjusted according to Bradford assay . All study protocols were performed respecting the dignity of animal lives and were approved by the animal experimental committee of the Shiga University of Medical Science (Document# 2009-12-2).Transgenic mice harboring human G93A SOD1  were housed and bred as previously reported . The difference between the two groups was assessed by Stuent’s t test. How to cite this article: Uchida, T. et al. CUL2-mediated clearance of misfolded TDP-43 is paradoxically affected by VHL in oligodendrocytes in ALS. Sci. Rep.6, 19118; doi: 10.1038/srep19118 (2016).


Graphene devices for radio frequency (RF) applications are of great interest due to their excellent carrier mobility and saturation velocity. However, the insufficient current saturation in graphene field effect transistors (FETs) is a barrier preventing enhancements of the maximum oscillation frequency and voltage gain, both of which should be improved for RF transistors. Achieving a high output resistance is therefore a crucial step for graphene to be utilized in RF applications. In the present study, we report high output resistances and voltage gains in graphene-on-silicon (GoS) FETs. This is achieved by utilizing bare silicon as a supporting substrate without an insulating layer under the graphene. The GoSFETs exhibit a maximum output resistance of 2.5 MΩ∙μm, maximum intrinsic voltage gain of 28 dB, and maximum voltage gain of 9 dB. This method opens a new route to overcome the limitations of conventional graphene-on-insulator (GoI) FETs and subsequently brings graphene electronics closer to practical usage. The enormous interest in graphene for electronic device applications originates from its outstanding charge transport properties and atomic-scale thickness1214fT) with the help of gate length scalingfT values as high as 400 GHz were reported from state-of-the-art graphene RF FETs789101112131415fT, the maximum oscillation frequency (fmax) and intrinsic voltage gain (AV0) are more important factors for RF devicesfmax values as high as 105 GHz were reportedmax and AV0 values of conventional GoIFETs mainly originate from the absence of current saturation in the output characteristics due to the absence of a band gap and the Klein tunneling effectIntensive research activities examining graphene RF FETs thus far have mainly focused on achieving higher cut-off frequencies  and thereby degraded RF performance. Thus, by suppressing the generation of the other charged carrier in the channel near the drain, the graphene device is able to obtain a higher ro.It is therefore highly desirable to obtain robust current saturation in the output characteristics from graphene FETs, which would result in a higher n0)22One of the methods that has been attempted to suppress the generation of the other charged carriers is the strengthening of the gate electrostatic control. In other words, the influence of the drain voltage is weakened with respect to the graphene channel near the drain side. Thus, previous attempts have focused on increasing the gate coupling by increasing the gate capacitance1920h-BN) has been adopted as a bottom gate dielectric to minimize n022h-BN exhibits lower charged impurities and provide higher optical phonon energy than a conventional SiO2 substrate19n0, which was proportional to the charged impurities, disorder, and thermal excitation, was found to significantly affect the drain conductance , indicating that the silicon substrate successfully acted as an insulator under the reverse bias condition. In contrast to the reverse bias condition at VDS < 0 , which results in the doping of the graphene and degradation of the graphene devices2 were reported to be around 2 ~ 5 × 1011 cm−2, while those on the silicon surface were around 0.3 ~ 2.8 × 1010 cm−2 312 than graphene/Si interfaces.The cumulative results of the maximum ro is inversely proportional to RC for the GoSFETs, while ro exists irrespective of RC for the GoIFET. The lower contact resistance in the GoSFETs compared to that of the GoIFETs is attributed to the presence of additional charge injection into the graphene from the Si bottom substrate, in addition to the metal pad. Both Pd and Si atoms are able to inter-diffuse through the defects in the graphene, such as carbon atom vacancies, thereby form a conductive Pd silicide (ro and μ can be observed in 2 (50 meV)34The suppression capabilities that prevent the generation of the other polarity carrier in the channel near the drain are directly affected by the electron potential energy in the Si, which is influenced by the metal/graphene voltage drop. Therefore, it is expected that the maximum silicide  in the Sro was proportional to the maximum AV0, and that a maximum AV0 as high as 28 dB was achieved from the GoSFETs, which is comparable to that of Si-based RF FETs. In order to verify the performance of the voltage amplifier, a common-source voltage amplifier was implemented with a P-GoSFET, as shown in V as high as 9 dB was obtained at a load resistance (RD) of 50 kΩ  on a heavily doped p-type silicon wafer (resistivity of ~0.01–0.05 Ω∙cm). For the GoSFETs, the thermal oxide (50 nm) in the active region on lightly doped n- or p-type silicon (resistivity of ~1–5 Ω∙cm) was patterned using a buffered oxide etchant, and the graphene was transferred immediately after the removal of the photoresist and the native oxide. The subsequent processes were identical for both the GoSFETs and GoIFETs. After patterning the channel graphene, the source and drain were defined using a Pd (5 nm)/Au (50 nm) lift-off process. Next, 20 nm of Al2O3 was deposited by atomic layer deposition (ALD) process on the graphene surface. Prior to the ALD process, 0.5 nm aluminum layer was evaporated on the graphene surface as a nucleation layer. The ultra-thin aluminum nucleation layer is soon oxidized during the ALD process. The 150 nm Al gate metal was formed using a thermal evaporator and the lift-off process. Finally, the device went through a thermal budget of 300 °C for 12 hrs.The graphene used in this work was synthesized using the inductive coupling plasma chemical vapor deposition method on a thin copper film, and it was transferred using the typical wet transfer method with PMMA. For the conventional GoIFETs, the graphene was transferred onto thermally grown SiORG = 1 MΩ, RD = 10, 30, 50 kΩ) and capacitor (CG = 1 μF) set on a breadboard, as well as a DC supplier and function generator, were connected. The transistors were staged in a probe station and connected to the circuit with a biaxial cable. The input signal was sinusoidal with an amplitude of 20 mV and a frequency of 1 kHz.All of the devices were measured at room temperature in ambient air with a B1500 parameter analyzer. In order to implement a common source voltage amplifier with a graphene–on–silicon transistor, the external resistances .


A set of simple stopping criteria is presented, which improve the efficiency of iterative root finding by terminating the iterations immediately when no further improvement of the roots is possible. The criteria use only the function evaluations already needed by the root finding procedure to which they are applied. The improved efficiency is achieved by formulating the stopping criteria in terms of fractional significant digits. Test results show that the new stopping criteria reduce the iteration work load by about one-third compared with the most efficient stopping criteria currently available. This is achieved without compromising the accuracy of the extracted roots. The drawback of such stopping criteria is that they shift the responsibility for providing accurate results from the program developer to the user. Also, functions exist for which such stopping criteria fail  those that rely on the user to specify a tolerance within which the roots are needed and 2) those that seek to terminate the iterations automatically when an iterate has been reached whose accuracy cannot be improved. Both categories are widely used  stopping criteria already exist. They are reviewed in §3. The new stopping criteria are then presented in §§4, 5 and 6. Finally, the new and the old criteria are compared in §7 in terms of efficiency and accuracy.3.3.1zi is declared a root ifA(zi) and B(zi) are both equal to p(zi) but with p(zi) calculated in different ways: A(zi)=p(zi) with p(zi) evaluated as usual by Horner's method. B(zi) must be written as B(zi)=D(zi)−C(zi), where D(z)=zp′(z) and C(z)=zp′(z)−p(z). Both polynomials D(z) and C(z) must be reduced analytically to their simplest form and evaluated separately by Horner's method before B(zi)=D(zi)−C(zi) is calculated. Analytical reduction results inA(zi) and B(zi) approach zero, |A(zi)−B(zi)| will initially be smaller than either |A(zi)| or |B(zi)|, thus preventing the stopping criterion from being satisfied. But, as |A(zi)| and |B(zi)| grow smaller, round-off errors will dominate |A(zi)−B(zi)| before they dominate |A(zi)| and |B(zi)|, thus providing an opportunity for the stopping criterion to be satisfied when |A(zi)−B(zi)| has lost all its significant digits.Igarashi  providesp(zi), p′ (zi) and p′′(zi) are needed by the iteration procedure itself, and D(zi) and C(zi) are needed by the stopping criterion. Thus, Igarashi's stopping criterion adds two-thirds to the work load per iteration. The criterion is included in the comparisons reported in §7.If a third order iteration procedure, such as Laguerre's method (e.g. Orchard ), is use3.2f(z): iterate zi is declared a root ifA(zi) and B(zi) are both equal to f(zi) but with f(zi) calculated in different ways: A(zi)=f(zi) with f(zi) in its standard analytical formulation. B(zi) must be written as B(zi)=D(zi)−C(zi), where D(z)=zf ′(z) and C(z)=zf ′(z)−f(z). Both functions D(z) and C(z) must be reduced analytically to their simplest form before B(zi)=D(zi)−C(zi) is calculated. W must be equal to either 1.0 or 0.5 if f(z) is algebraic, and W must be equal to 0.01 when f(z) is transcendental. It is not clear what value W should have if f(z) has both algebraic and transcendental terms. Igarashi gives only an abbreviated explanation of how this works. Presumably, the basic explanation is similar to the one proposed above for polynomials. Igarashi also gives only an abbreviated explanation for the choice of W. W=1.0 appears to be based on the assumption that the evaluations of algebraic functions and polynomials incur similar round-off errors, so the same stopping criterion can be used for both. But Igarashi also suggests that W=0.5 can be used instead to relax the stopping criterion to allow for errors in experimental data and/or the conversion of experimental data from decimal to floating-point binary. Finally, Igarashi chooses W=0.01 to further relax the stopping criterion for transcendental functions in order to account for the truncation errors incurred by the intrinsic transcendental functions used to evaluate A(zi), D(zi) and C(zi).Igarashi  providesf(zi), f′ (zi) and f′′(zi) are needed by the iteration procedure itself, and D(zi) and C(zi) are needed by the stopping criterion. Thus, Igarashi's stopping criterion adds two-thirds to the work load per iteration. The criterion is included in the comparisons reported in §7.If a third order iteration procedure, such as Ostrowski's method (e.g. Orchard ), is use3.3zi=xi+jyi is accepted as a root of the complex polynomialc0|<ϵg0(1+ϵ)n5 and |d0|<ϵh0(1+ϵ)n5. c0, d0, g0 and h0 derive from the recurrences:ϵ=2t− is the machine epsilon, i.e. t is the bit length of the floating-point significand, e.g. t=53 for IEEE double-precision. A rigorous derivation of this stopping criterion is given by Grant & Hitchins . The calculation is demonstrated below.sm is the length of the floating-point significand used, e.g. sm=53 bits and sm=113 bits, respectively, for IEEE double-precision and quad-precision. qm is the order of the iteration procedure to which the stopping criterion is applied, e.g. qm=3 for Laguerre's method and Ostrowski's method.Also in stopping criterion #1, a,b. These examples use iterates z1, z2 and z3 from sm=24 bits, as in IEEE single-precision. Also shown are the corresponding iteration increments e2=|z2−z1| and e3=|z3−z2|. A trailing ‘b’ designates a binary number.Stopping criterion #1 is explained with reference to table 2s2 in table 2a is the number of MLBs of z1 and z2 following iteration #2. s2 is equal to the number of leading zero bits of e2 (i.e. 6) plus a fraction produced by (i) removing the radix point and the leading zeros from e2 to produce the bit-string 101 1111 0001 1110 000b, (ii) taking two's complement of the bit-string to get 010 0000 1110 0001 111b, and (iii) placing a radix point in front of the complement to produce the binary fraction 0.010 0000 1110 0001 111b = 0.256893. This fraction is added to the number of leading zeros of e2 to get s2=6+0.256893=6.256893, as shown in table 2a. s2=6.256893 states that z1 and z2 have 6.256893 leading bits in common. The not function in table 2a is used as shorthand for the above procedure for finding the fractional part of s2. A simple numerical procedure for calculating s is outlined in Nikolajsen . The criteria are applied in the following order of descending efficiency: #3, #1, #2 and #4. The order of application affects the percentage usage of the criteria because two criteria are often satisfied simultaneously.ei>ei−1 is replaced by ei≥ei−1 because ei>ei−1 fails to activate when ei approaches zero monotonically and then remains equal to zero. For complex root finding, Ward's criterion is applied to the real and imaginary iteration streams separately because Ward was found in one instance to be triggered prematurely when applied directly to the complex iteration stream.Ward's stopping criterion zi+1is a root if zi+1=zi. Without this, G&H does not get invoked when the start value by chance hits a root.G&H's stopping criterion is supplemented by the following criterion: 7.2a shows the total number of determinant evaluations needed to find all the eigenvalues of dense, random, non-symmetrical matrices of orders 5 through 250 using JLN's and Ward's stopping criteria. The same random matrices are used for both. The matrices are reduced to upper Hessenberg form to allow the eigenvalues to be extracted by Laguerre's method, as outlined in Nikolajsen 2. All the roots are double roots and the single root Laguerre formula is used to slow the convergence rate to linear for half the roots in order force stopping #2 and #4 into action. The results are shown in In the previous polynomial examples, convergence is so rapid that stopping criteria #2 and #4 have hardly been used. Their use is demonstrated here by calculating the roots of polynomial type 2-type polynomials. The 19.2% usage of stopping criterion #4 is still surprisingly low. The raw data suggest the following explanation. The lower accuracy of double roots accelerates the loss of accuracy of the deflated polynomials. As a result, the double roots still to be extracted gradually lose accuracy and split apart to become single roots, whose convergence rate is much greater. As a result, the faster stopping criteria, #1, #2 and #3, gradually take over from #4. This is also reflected in figure 5a by the progressive levelling off of both the Ward and the JLN lines as the number of function evaluations per root drops due to the greater speed of convergence of the emerging single roots.The percentage usage of JLN stopping criteria #1, #2, #3 and #4 changes from 74.3%, 0.5%, 25.1% and 0%, respectively, for b and 4b shows that this remains at 82. The reason appears to be that, once the double roots have split, any further deterioration in their accuracy is greatly reduced because they have become single roots that can be extracted with twice the number of MLBs. Of course, the first roots extracted will still be double roots, so the maximum accuracy cannot exceed half the single root accuracy, as also indicated by comparing figures 5b and 4b.A corresponding reduction might be expected in the maximum polynomial degree at which all the roots can be matched unambiguously to their exact counterparts. But comparison of figures 5b again confirms that both are equally accurate. The exception is the blip in the Ward graph at polynomial degree 52. The raw data suggest that this is due to a small variation in the linear rate at which MLBs are gained when the first of a pair of double roots is extracted by the single root Laguerre formula. In this particular case, a very slow constant gain of 1.8 bits per iteration is interrupted by a premature loss of 0.6 bits, which triggers Ward's stopping criterion prematurely, reducing the number of correct decimal digits of the 21st root from 12.5 to 11.7 . As this small error propagates through the subsequent deflations, it gradually reduces the accuracy of the subsequent roots to the point where the number of correct decimal digits of the poorest root drops from 8.8 to 6.1, as indicated in figure 5b. This could be avoided by putting a lower limit on the loss needed to trigger Ward, but that would reintroduce the problem which was solved in §7.1 by replacing ei>ei−1 by ei≥ei−1.The coincidence of the JLN and Ward graphs in figure 5si+2≤si+1 by si+2≤si+1−1 in stopping criterion #4. But that would lead to triggering difficulties in many other cases. The conclusion is that neither JLN stopping criterion #4 nor Ward is fail-safe at extremely slow convergence rates that can occur when the single root Laguerre formula is applied to a double root. But this is unlikely to happen in practice, when a sustained linear rate of convergence would be used to trigger a switch to the double-root Laguerre formula.JLN stopping criterion #4 avoids this failure only by chance, i.e. because tiny differences between the iterates (caused by faster termination of previous roots) happen to produce a Laguerre iteration sequence that does not include a tiny premature loss of digits. This potential problem could be avoided by replacing p2(z). In other words, the advantage of JLN over Ward, in terms of efficiency, is reduced from 37.1% for p2(z)-type polynomials to 4.76/42.70=11.1% for [p2(z)]2-type polynomials. The main reason is the more frequent need for JLN stopping criterion #4, whose efficiency is no better than Ward's. The reduced advantage of JLN is also evident from the closer proximity between Ward and JLN in figure 5a compared with figure 4a.The total number of function evaluations is 39 288 with Ward and 34 905 with JLN. The total number of roots found is 8+12+16+⋯+84=920. Thus, the average number of function evaluations per root is 39 288/920=42.70 with Ward and 34 905/920=37.94 with JLN. Thus, JLN reduces the number of function evaluations per eigenvalue by an average of 4.76, compared with 7.32 for Polynomials with several other distributions of known roots were also tested, as were polynomials with random roots, for a total of approximately 739 polynomials of different degrees and levels of ill-conditioning with a total of approximately 60 227 roots. All the tests confirm the findings of this and the previous sections. Results are shown in the electronic supplementary material.7.4W=0.01 for transcendental functions and W=0.5 or W=1.0 for algebraic functions.)Igarashi's, Ward's and JLN's stopping criteria are implemented in a general nonlinear root finder based on Ostrowski's square root iteration formula in the complex domain (e.g. ). (‘Igarf1 is so simple that its exact roots are known. f2(z)=0 is one of two purely transcendental frequency equations for a beam with three equal-length spans on four pinned supports [f3(z)=0 is the frequency equation for lateral vibration of a beam with flexible, pinned supports at each end [Results are presented for the following sample functions:supports . f3(z)=0each end .W=0.01 for purely transcendental functions like f1 and f2 but does not make it clear which W-value should be used for functions like f3, which has both algebraic and transcendental terms. A preliminary value of W=0.01 is used for f3 because it is the least stringent and therefore the most likely to trigger Igarashi. This is an attempt to avoid Igarashi's failures to stop when a root has been found, as experienced earlier for both matrices and polynomials.Igarashi prescribes the use of f1, f2 and f3. The average number of function evaluations per root are shown in Two hundred and fifty roots are extracted for both f1, f2 and f3. Inspection of the raw data shows that this is because (1) neither Iga, Ward nor JLN terminate the iterations until convergence has been completed and (2) in all cases, the first converged iterate and all subsequent iterates are exactly equal. No attempt was made to explain (2): the workings of the root solvers are beyond the scope of this paper. The important conclusion here is that all three sets of stopping criteria provide equal accuracy. Igarashi's previous failures to stop are not encountered with functions f1, f2 and f3.In terms of accuracy, Iga, Ward and JLN provide the exact same values for all 250 roots of both p1(z) is the polynomial tested in §7.3.1. The subtraction of z5/2 is used as a simple means of making f4 non-polynomial without introducing discontinuities in the f4-derivatives needed by Ostrowski. f4 has n roots, one for each of the n intersections between p1(z) and z5/2.Purely algebraic, non-polynomial functions are not common in practice but, for completeness, the roots of the following such functions are found:n=8,12,16,…80, with W=1.0 used for Igarashi. f4 turns out to be quite a challenge for Ostrowski's method, which fails to find the nth root for n=28, 44, 60 and 68, regardless of which set of stopping criteria is used. The four peaks in figure 7a are due to the additional iterations carried out in the fruitless search for the final root. The three topmost data points of each peak are the peak values for Iga, Ward and JLN, in the order shown for n=68.The results are shown in b gives the relative accuracy of the stopping criteria in terms of log10|( f4)|max, i.e. the logarithm of the largest absolute f4-value at the roots. The exact f4-values at the roots are of course zero. Any log10|( f4)|max-value less than −34 is set to −34 .The exact roots are unknown, so figure 7b, the accuracy of all three sets of stopping criteria is virtually the same except for (i) the tiny dip in the Iga line at n=28, (ii) the huge dips in the Iga line at n=72 and 80, and (iii) the huge dip in the Ward line at n=80. These dips do not signal any momentary improvement in accuracy. Rather, they occur for the following reasons:n=28, 32 roots for n=72, and 35 roots for n=80,(1) when Igarashi is applied, Ostrowski is only able to find 21 roots for n=80, and(2) when Ward is applied, Ostrowski is only able to find 35 roots for n roots are found, log10|( f4)|max becomes unrepresentatively small. This is because the roots are found roughly in ascending order of modulus, and the size of each log10|( f4)|-value is roughly proportional to the modulus of the corresponding root, so when only the small-modulus roots are found, the large log10|( f4)|-values are missing.(3) when so few of the As shown in figure 7These problems appear to be related to relatively weak global convergence properties of Ostrowski's method. They are therefore beyond the scope of this paper and will not be pursued. It is also not clear why these problems are not encountered when JLN's stopping criteria are applied.n=28, 60, 64 and 72, respectively. With W=0.5 instead of 1.0, the total number of failures grows to 32. As before, the iterations are stopped automatically by the root finder, so the failures do not affect the apparent accuracy of Iga. But, as before, these failures are deemed to be unacceptable, thus disqualifying Igarashi's stopping criterion in its current form.Inspection of the raw data shows that Iga fails to stop, when a root has been found, on a total of 25 occasions: once, twice, three times, 10 times and 11 times for The average number of function evaluations per root is 12.391 with Ward and 9.070 with JLN for all the root extractions related to 8.(1) Grant & Hitchins tended to trigger prematurely, leading to multiple failures. Igarashi occasionally failed to trigger, leading to multiple redundant iterations and uncertainty as to whether a root had been found. Both were therefore rejected in their current formulations.(2) Ward and JLN were the only criteria that did not fail. Both provided the maximum possible accuracy of the results but JLN did so more efficiently, reducing the number of function evaluations by one-third without any deterioration in the accuracy of the results.(3) Ward's criterion is by far the simplest and most easily implemented. So when simplicity is more important than efficiency, Ward's criterion is preferable. JLN's criteria are preferable in all other cases.A new set of stopping criteria for iterative root finding and eigenvalue extraction has been presented, which terminates the iterations immediately when no further improvement of the results is possible. The new criteria, called JLN, have been tested numerically against the existing stopping criteria of Igarashi ,5, GrantThe range of testing reported here is of necessity limited and must be regarded as preliminary. But it is considered to be sufficiently convincing to warrant further consideration of the JLN stopping criteria when high efficiency is desired.A numerical implementation of the JLN stopping criteria is available from the author on request.


These heterostructures offers excellent current-carrying abilities whilst offering the prospect of a fast, low cost and easy methodology for device applications. The use of ND monolayers is also a compatible technology for the support of large area graphene films. The nature of the C-H bonds between graphene and H-terminated NDs strongly influences the electronic character of the heterostructure, creating effective charge redistribution within the system. Field effect transistors (FETs) have been fabricated based on this novel herterostructure to demonstrate device characteristics and the potential of this approach.Graphene on hydrogen terminated monolayer nanodiamond heterostructures provides a new way to improve carrier transport characteristics of the graphene, offering up to 60% improvement when compared with similar graphene on SiO The detailed material properties of graphene on ND surfaces with and without hydrogen termination treatments have been investigated using Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy. It has been shown that the hydrogen termination treatment not only removed surface contamination from as-deposited monolayer NDs, but also provided a suitable linkage between ND and the graphene layer to form a conductive path, as is demonstrated by impedance spectroscopy (IS) measurements. Also, here the carrier mobility of graphene on hydrogen terminated ND (GrHND) has been compared to graphene on hydrogen terminated SCD (GrHSCD). In addition, to Hall mobility measurements, top-gate graphene transistors with two different gate lengths of 200 nm and 500 nm have been fabricated using a focused ion beam (FIB) tool; and thus, this study has demonstrated a new approach for commercial graphene transistor fabrication.In this paper, a cost effective and mass producible method to fabricate monolayer ND films that are capable of tuning the properties of graphene for the fabrication of Field-Effect Transistors (FETs) is demonstrated. Compared to similar graphene transferred onto SiO2/Si substrate (300 nm SiO2) is shown in 2/Si substrate with an ND-water solution, then an ultra-sonic bath treatment to attach the NDs, followed by drying. Thermal hydrogenation was then performed . The surface coverage and roughness of the ND layers were determined by atomic force microscopy (AFM), as shown in etc20−1 is characteristic of the C = O stretching band involved in carboxylic acid groups and anhydride functionalities−1 region is attributed to the OH of adsorbed water. Various peaks relating to other ND surface groups  are observed in the 1000–1500 cm−1 range−1 have been reduced. The free and adsorbed -OH peaks have disappeared in the region of 3000–3600 cm−1, while the peak at 2923 cm−1 has become much stronger, being assigned to the C-H stretching of the hydrogenated ND surface coupled with the CHX bands at 1461 cm−1 (CH2) and 1377 cm−1 (CH3)A schematic view of the GrHND heterostructures supported on a SiOubstrate 0 nm SiO22g phonon at the Brillouin zone centre and the D peak is due to the breathing modes of an sp2 phase and defects for those activations3/sp2 bonded phase can be expected for graphene on NDs and H-NDs. The dangling bonds from ND and H-ND surfaces evidenced in the FTIR results lead to the observed shifts in the Raman peaks as compared to unsupported graphene layers392, here, the G peak positions for the graphene-ND heterostructures have an upshift from 1581 cm−1 to 1586.2 cm−1 and 1585.7 cm−1 for untreated ND and H-ND substrates respectively41−1 for ND (2698 cm−1) and H-ND (2690 cm−1) substrates, respectively, due to hole doping−1 2D peak of graphene on SiO2. The 2D peak widths for both heterostructures are within 25–28 cm−1 range, suggesting the graphene is monolayer in nature2D/IG can be used to provide additional information2D/IG2D/IG for both heterostructures are 1.06 and 1.17 respectively, proving electron transfer between the interface and thus high levels of doping of the graphene has occurred; for low doping the 2D peak is expected to be 3–5 times stronger than the G peak3/sp2 ratio3 signal, leading to the heterostructure here being formed from sp3/sp2 carbon mixtures. Both FTIR and Raman results show a clear modification occurs to the graphene lattice with H-termination treatment of the NDs. The charge transfer between graphene and NDs introduces disorder and high doping to graphene, hence tuning the graphene electronic properties.Raman spectroscopy was next used to study the graphene-ND hetrostructure to investigate the chemical nature of the carbon materials present332 and sp3 atoms present2 hybridized C atoms in ND (graphite structure) and graphene443 hybridized C atoms due to the formation of C-H and the C–C bonds in the ND film4446472 contribution since NDs are partially covered by a thin layer of graphite3 hybridization of the deposited carbon by saturation with hydrogen atoms, whilst thermal annealing  leads to graphitization and an increase of the sp2 phase463 phase contributing to the D peak143 bonds are likely to affect the translational symmetry of C = C sp2 bonds in graphene, which is similar to the effect of hydrogenating graphene3 coverage was determined as θ/(1 + θ) where θ is defined as the intensity area of sp3 and sp2 peaks3 coverage was found to be ~19%, while for graphene transferred on H-ND films the sp3 coverage increased to ~37%. As a result, the hydrogen terminated ND film has also indirectly hydrogenated the monolayer graphene to an H-coverage of approximately 37%−19% = 18%. , where A0 is the pre-exponential factor; KB is the Boltzmann constant and Ea is the activation energy representing the energy barrier for the thermal activation process55The electrical characteristics of the heterostructures were examined using AC impedance spectroscopy (IS) measurements. Two Au/Ti (300 nm/10 nm) contacts were deposited on the top-side corners of square samples. IS was carried out in vacuum at temperatures ranging from room temperature to 500 C as shown in 5150532/Si is 132 cm2/Vs  segments, and amorphous carbon at the grain boundaries13 cm−2 to a highest point of 1.3 × 1013 cm−2 at 400 K, then dropped slightly to 1.2 × 1013 cm−2 at 450 K. The carrier density of GrHSCD shows a linear increase with temperature from 6.4 × 1012 to 8.5 × 1012 cm−2 at room temperature to 450 K.The sheet resistance and carrier densities for both heterostructures have been plotted against temperature as shown in 2O and CO2 to produce H3O+, HCO3− and H+ layers shown in Although GrHSCD has lower a carrier density which enables an increase in the mobility2O3 dielectric layer with 10 nm thickness was grown by atomic layer deposition (ALD Ultratech Fuji F200). Detailed fabrication information is given in the fabrication method section. et al. measured the Dirac point voltage of their graphene FETs formed on DLCet al. suggested that the aluminium nanoparticles present in their work tuned the Dirac point, which then induced n-type dopingds/dVg) × (L/WCiVds), where L is the channel length (5 μm), W is the channel width (500 nm) and Ci is the areal capacitance per unit area between the channel and gate (8 × 107 F/cm2)2/Vs, which is similar to the measured Hall mobility.Top gate FET devices were fabricated on GrHND heterostructures with different gate channel sizes of 200 nm and 500 nm, respectively. A focussed-ion-beam tool, giving a potential feature size smaller than 10 nm, was used to define the source, drain contacts, gate electrodes. FIB was also used for local material deposition for contact formation using tungsten carbide (WC). A top-gate Al3/sp2 carbon present has changed. From IS measurements performed at various temperatures, it is indicated that the hydrogen links both ND and the graphene layer to create a charge transfer process which induces a conductive interface layer between graphene and ND. The mobility of GrHND increases some 60% compared with similar graphene on SiO2/Si and is comparable with observations GrHSCD, which is not viable for commercial applications. Moreover, over the whole temperature range, GrHND demonstrated stable carrier mobility compared to GrHSCD which is vital for commercial applications. Whilst the mobility of the CVD-derived graphene layers here was modest, it can be expected that the advantage of the use of a supported ND layer, over SCD, DLC or SiO2/Si would be maintained for higher quality CVD-grown graphene layers as they emerge as commercial materials. FETs fabricated on this novel type of herterostructure GrHND demostrated comparable characteristics to those FETs on graphene-SCD, whilst offering the prospect of low cost large area production of graphene electronics.A systematic study of the properties of graphene-ND heterostructures has been carried out and the operation of an FET on GrHND structures demonstrated. Due to the prospects for low cost, easy deposition and an associated high thermal conductivity, monolayer ND is a promising candidate as a support material for graphene. It appears a better solution than other novel materials such as SCD or UNCD in case of the need for high current-carrying device capacity and mass reproducibility. The properties of graphene on NDs with and without H-termination have been investigated using FTIR, Raman spectroscopy, and XPS. The C-H bond present on H-terminated NDs, strongly influences the heterostructure properties that result from depositing graphene on the NDs. The presence of H-termination leads to the appearance of a so-called D peak, and to a shift in the G and 2D peaks, suggesting the ratio of sp2/Si substrates with 300 nm thickness of SiO2. Nanodiamonds were supplied prepared in solution from ultra-dispersed detonation nanodiamonds . Deposition involved immersing the substrate material in the ND solution (0.05 g/L of NDs) followed by sonication (10 min). Hydrogen termination for NDs was performed using a Seki AsteX AX6550 reactor with the following parameters for 5 hours: microwave power, 600 W; H2 200 sccm; pressure 10 Torr; temperature 500 C. CVD grown monolayer graphene on a copper foil (Graphene Supermarket) was coated with PMMA. The PMMA-coated graphene was treated with Iron nitrate solution to etch away Cu foil. The floating PMMA coated graphene was removed and cleaned in DI water to transfer onto H-terminated NDs samples. The PMMA was removed by acetone.As shown in The surface morphology of the various films measured over 10 μm × 10 μm and 1 μm × 1 μm regions was measured using scanning atomic force microscopy  in tapping mode for the H-terminated NDs. The chemical composition and bonding states of the films were characterized using Fourier transfer infrared spectrometry  in transmission mode and X-ray photoelectron spectroscopy  with the Al K α line as the exciting source. A micro Raman spectroscopy system (Renishaw Invia) operating at 514.5 nm was used to study the film microstructure, where the laser output power used was 1 mW. Impedance spectroscopy was applied in the range of 0.1 Hz to 10 MHz in a vacuum at elevated temperature from room temperature to 500 C using an Autolab electrochemical system. Hall Effect measurements  were performed to determine graphene carrier mobilities using the “van der Pauw” method, where by four Ohmic metal contacts (10 nm Ti-300 nm Au) were placed on the topside of the graphene layers at room temperature.2) and material-specific preferential milling can be performed by introducing reactive gases in the vicinity of the electron or ion probe. The electron column delivers the imaging abilities of the SEM which is less destructive than FIB imaging. SEM imaging of the graphene sample before milling identified clean area suitable for FET fabrication. Keep one clean graphene area 5 μm × 10 μm then other surrounding area around 100 μm × 110 μm were milled by using a 30 kV Ga+ beam with a current of 2 nA to remove the graphene and nanodiamond layers for FET definition. Before depositing S/D/G contact patterns, an insulator material (SiO2) was deposited on graphene surrounding area (50 nm thickness). Three contacts patterns 40 μm × 40 μm were coated by Tungsten Carbides with thickness of 200 nm using a 30 kV/2 nA beam. A final gentle “polish” with Ga+ ions (30 kV/ 5 pA) was used to clean the graphene area edge and remove side damage. Finally connect contact pattern with graphene area edge by WC using a 30 kV/50 pA beam. After the deposition of 10 nm Al2O3 layer by Atomic layer deposition (ALD), the Al2O3 layer on big gate pattern was removed then one narrow gate channel was built on top. In this study, the gate sizes are 500 nm × 5 μm and 200 nm × 5 μm.Graphene field-effect transistors were fabricated using a Carl Zeiss Focused Ion Beam Microscope and an Atomic Layer Deposition system. A dual-beam instrument (Carl Zeiss XB1540 cross-beam Focussed-Ion-beam microscope) was used to pattern samples without using masks, giving a feature size as small as 10 nm. The instrument combines a FIB and a SEM column in the same chamber and is fitted with a gas-injection system to allow local material depositions .


In this article, we have theoretically investigated the performance of graphene-hexagonal Boron Nitride (hBN) multilayer structure  to demonstrate all angle negative refraction along with superior transmission. hBN, one of the latest natural hyperbolic materials, can be a very strong contender to form a hyper crystal with graphene due to its excellence as a graphene-compatible substrate. Although bare hBN can exhibit negative refraction, the transmission is generally low due to its high reflectivity. Whereas due to graphene’s 2D nature and metallic characteristics in the frequency range where hBN behaves as a type-I hyperbolic material, we have found graphene-hBN hyper-crystals to exhibit all angle negative refraction with superior transmission. Interestingly, superior transmission from the whole structure can be fully controlled by the tunability of graphene without hampering the negative refraction originated mainly from hBN. We have also presented an effective medium description of the hyper crystal in the low-k limit and validated the proposed theory analytically and with full wave simulations. Along with the current extensive research on hybridization of graphene plasmon polaritons with (hyperbolic) hBN phonon polaritons, this work might have some substantial impact on this field of research and can be very useful in applications such as hyper-lensing. Negative refraction2346623434111314151314156Graphene, widely used in both photonics and plasmonics in a broad frequency range because of its unique and tunable optical properties, has been recently used in constructing HMMs10171820212223242526In this article, we have considered the graphene-hBN multilayer structure for constructing a hyper crystal. Graphene shows both better mobility and high relaxation time, if hBN is the substrate material20211314152830σ of graphene depends on frequency (ω), chemical potential (μc), relaxation time (τ) and temperature (T). It can be determined using the Kubo formalisms34The optical conductivity The tangential permittivity of graphene is expressed as,ε0 is the free space permittivity and tg represents the thickness of monolayer graphene. As graphene is a two dimensional material, the normal electric field cannot excite any current in the graphene sheet. So the normal component of the permittivity is given by εg,⊥ = 137εg,⊥ can be approximated as the permittivity (~2) of graphite. In this article, the graphene that we have considered has only a few layers . The relaxation time of graphene indicates its purity, and it can vary from fs (femto-second) to ps (pico-second) range. We have considered the relaxation time as τ = 200 fs in all calculations. This is a reasonable value as considered in literature for different graphene based devices1017181516Here ε∞ and γ indicate the high-frequency dielectric permittivity and the damping constant, respectively. These data have been taken fromεuu becomes negative.where u =  represents the transversal  and u = z represents longitudinal  axes. LO and TO respectively stand for longitudinal and transversal optical phonon frequencies. Hyper-crystals can be constructed by a stack of17−1. In hBN, two Reststrahlen bands are non-overlapping1315LO = 825 cm−1, ωTO = 760 cm−1) εxx = εyy > 0 and εzz < 0 and in the upper Reststrahlen band (URB),  εxx = εyy < 0 and εzz > 0 and thus leading to the Type I and II hyperbolic response, respectively.For a general one dimensional anisotropic crystal or hyper-crystal, each layer has the permittivity tensor of the form,The basic Bloch equation for a p-polarized wave in case of an anisotropic photonic crystal has been derived as,, can be given by,Here dk0stands for the wave-number in free-space.where, zd ≪ 1 is the sufficient condition for effective medium theory. But for hyper-crystal, the situation is slightly differentk waves (k > k0). So, effective medium theory cannot properly describe the physical properties in hyper crystal, such as the band gap, originating from the high-k waves in either or both layers. This failure of effective medium theory in hyper crystal0) and for moderate high-k waves (up to k ~ 20 k0), effective medium theory should still work, where layer thicknesses (d1 and d2) and wavelength (λ) play a critical role. Effective permittivity tensor for a hyper-crystal can thus be derived from Now, for isotropic case, where both layers have isotropic permittivity , k occurs only in the lower Reststrahlen band (LRB) where hBN shows type I hyperbolic behavior. In the upper Reststrahlen band (URB), hBN shows type II hyperbolic behavior and propagating waves  (k < K0) are not allowed to propagate in either hBN or GhHC. The perpendicular permittivity of graphene is constant because it does not depend on the chemical potential of graphene. As a result, the effective perpendicular permittivity of GhHC is independent of the chemical potential of graphene. On the other hand, the effective tangential permittivity of GhHC is a strong function of the chemical potential of graphene. With higher chemical potential, the effective tangential permittivity of GhHC reduces from bare hBN to a value closer to the permittivity of ambient medium (air). TO = 760 cm−1). But the lower value of the imaginary part of the perpendicular permittivity at the upper edge of the LRB (ωLO = 825 cm−1) plays an important role in absorption as discussed later on.z,hc) can be given by,From To demonstrate whether effective medium theory works, and to what extent if it does, here we do not put any arbitrary tensor values in ωc = 1600 cm−1 and ωc = 800 cm−1 respectively for graphene’s chemical potential, μc = 0.5 eV. Other values of chemical potential of graphene can also be chosen for GhHC. Here, we have chosen μc = 0.5 eV arbitrarily for illustration purpose. In ωc = 1600 cm−1 is also shown. The shaded regions indicate the regions where no propagating wave is allowed. The red symbols represent the exact calculation (ωc = 800 cm−1 (in the LRB) and ωc = 1600 cm−1 (in the URB), hBN behaves as a type-I and type-II hyperbolic material, respectively. From 170).culation  and the culation . At ωc =Any (meta) material which possess hyperbolic dispersion , negative refraction especially the all-angle negative refraction will be available from such (meta) materials23789783rs,hc) and wave-vector  as a function of incidence angle at wavenumber 810 cm−1 for different values of chemical potential of graphene. rs,hc) as a function of wavenumber and incidence angle for μc = 0.25 eV. The black dashed lines indicate the angle of refraction equal to zero (Transition between positive and negative refraction). Only in the LRB (760 cm−1 ≤ ω ≤ 825 cm−1), the effective perpendicular permittivity of GhHC becomes negative .4445ene. cf. . But in ene. cf. . But in ene. cf. , effectiene. cf. . As a reene. cf. ; εxeff bAlong with transmission, we want to clearly mention about the absorption properties of GhHC. Although the resonance of absorption of both the hBN and GhHC is at the beginning of the lower Reststrahlen band (LRB) cf. , the max994445y) of a p-polarized wave, incident from air to hBN and GhHC  using EMT. z,hc)/Im is a well-known and accepted value for characterizing materials which possess negative refraction9TM,hc) for TM polarization and the figure of merit (FOM) respectively calculated using the effective medium approximation as a function of wavenumber for different potential values of chemical of graphene. Although for higher chemical potential values, the FOM decreases. But higher transmission is achieved for higher chemical potential values of graphene which mainly occurs due to the reduction of reflection at the ambient medium (air)-GhHC interface.The FOM = Re. We have found that an all-angle negative refraction is achievable in such a hyper crystal with much higher transmission compared to bare hBN. Transmission and negative refraction properties can be controlled by tuning the chemical potential of graphene. Here negative refraction originates from the optical properties of hBN and transmission from the structure can be fully controlled by graphene without hampering negative refraction. Such properties are quite remarkable, and experimental demonstration of such phenomena should be possible with current experimental setup. We have also presented an effective medium description of the hyper crystal in the low-k limit, which might be useful in theoretical calculations and it has been validated by numerical calculations. We believe that, with the current ongoing extensive investigation on the hybridization of graphene plasmon polaritons with (hyperbolic) hBN phonon polaritons2223242526How to cite this article: Sayem, A. A. et al. Negative Refraction with Superior Transmission in Graphene-Hexagonal Boron Nitride (hBN) Multilayer Hyper Crystal. Sci. Rep. 6, 25442; doi: 10.1038/srep25442 (2016).


The development of chemical strategies to render graphene viable for incorporation into devices is a great challenge. A promising approach is the production of stable graphene dispersions from the exfoliation of graphite in water and organic solvents. The challenges involve the production of a large quantity of graphene sheets with tailored distribution in thickness, size, and shape. In this review, we present some of the recent efforts towards the controlled production of graphene in dispersions. We also describe some of the chemical protocols that have provided insight into the vast organic chemistry of the single atomic plane of graphite. Controlled chemical reactions applied to graphene are expected to significantly improve the design of hierarchical, functional platforms, driving the inclusion of graphene into advanced functional materials forward. Various methodologies for the production of graphene and chemically modified graphene have been described during the last years . Among t2 lattice is partially disrupted into layers containing sp2–sp3 carbon atoms. The most drastic example is probably graphene oxide (GO), where the extreme oxidation of the carbon lattice causes a detriment in the electrical and mechanical properties of the sheets [To exfoliate graphite by wet chemistry techniques, it is necessary to decrease the π–π staking interactions between the graphene layers. In achieving this, the spe sheets . As consTo date, the exfoliation of graphite in dispersions has been obtained by a number of mechanochemical methods. Each one of these strategies presents advantages and disadvantages depending on the type of application. In this contribution, we summarize some of the chemical procedures used to obtain graphene dispersions for various applications. Special emphasis is placed on the liquid-phase exfoliation of graphite by ultrasonic techniques, since this is a straightforward process for the production of clean graphene layers ready for organic functionalization. Importantly, the exfoliation of graphite by ultrasonication is a very versatile technique that has already demonstrated its effectiveness in dispersing graphene in solvents with different physical and chemical properties. This flexibility allows the incorporation of additives such as surfactants , antioxiAs previously mentioned, a number of chemical strategies can be followed for the production of graphene in dispersions. Until now, the most widely used methodology has involved the production of GO . In thisAs an alternative, charged species can be used to induce the exfoliation of graphite. For example, the addition of electrons from alkali metal intercalation compounds resulted in the spontaneous exfoliation of graphite in water or polar solvents. This procedure yields stable solutions of negatively charged graphene sheets .As charged, intercalating species, ionic liquids are considered a green alternative. The negligible vapour pressure, thermal stability, wide electrochemical potential window, good conductivity, recyclability, and the high dielectric constant of ionic liquids induce the exfoliation of graphite by weakening the π–π stacking interactions. Indeed, the use of ionic liquids is considered a highly versatile and industrially scalable method for the preparation of graphene nanomaterials .Some neutral molecules are also able to exfoliate graphite by producing polarized π-systems . For exaThe methodologies used for the exfoliation of graphite described thus far might pose a challenge to the further modification of graphene layers by covalent organic reactions. The presence of additional chemical species could yield unexpected products. Thus, different approaches are followed for the production of clean graphene sheets. For example, graphene can be produced by supercritical solvent exfoliation of graphite. In this procedure, solvents reach or exceed their critical point, presenting outstanding wetting properties, low interfacial tension, low viscosity, and high diffusion coefficients. Under these conditions, graphite is exfoliated as high-quality graphene .A versatile and simple strategy to exfoliate graphite in liquid phase is based on the use of ultrasonic wave treatment. Ultrasound techniques have important applications in a wide range of materials synthesis strategies . The phy−2). This relatively high surface energy results in solvents with high boiling points and high surface tensions, for example, N,N-dimethylformamide (DMF) and N-methyl-1,2-pyrrolidone (NMP), which are well-known as good dispersing solvents for carbon nanostructures such as fullerenes and carbon nanotubes (CNTs) [Ultrasonic processes are successful at exfoliating graphite only if the net energetic cost of the process is very small. The enthalpy of mixing depends on the affinity between graphene layers and solvent molecules. Then, in order to obtain high yields of exfoliated graphite, the surface energy of the solvent must compete with the surface energy of graphite (≈70–80 mJ∙ms (CNTs) –25. UsinAs an alternative, it is possible to prepare graphene dispersions in chloroform, acetone, or isopropanol. These are all volatile solvents that have generally lower surface tensions, and are therefore more easily evaporated. Unfortunately, in order to achieve graphene layers of reasonable concentrations in volatile solvents, it is necessary to increase the ultrasonication time, which increases the oxidative processes that cut the graphene sheets into smaller layers, resulting in a higher content of oxidized carbon atoms .The exfoliation of graphite in water is also possible by adding surfactants as stabilizing agents. For example, graphite and sodium cholate were ultrasonicated in water for long periods up to 400 h . This prN-(2-mercaptopropionyl)glycine (tiopronin) was reported [As previously mentioned, ultrasonication is a highly energetic process that produces strongly oxidizing chemical species, such as radicals and peroxides. These radicals typically oxidize carbon atoms in graphene close to the defects and at the edges. Under prolonged periods of ultrasonication, the oxidation can cut graphene sheets into small pieces. A well-known strategy to mitigate the damage induced by radicals in different biological processes is through the addition of antioxidant. Following this approach, the ultrasonication of graphite upon addition of reported . TiopronAn important potential application of exfoliated graphene is the tailored production of other carbon nanostructures such as fullerenes  and CNTsThe proposed mechanism for the formation of the MWNTs from graphene is as follows : (1) theFrom an organic chemistry perspective, graphene is a very exciting material as it can be functionalized on the two faces, at the edges and at the defects. Initially, graphene was expected to behave as a chemically inert surface, similar to graphite. However, it was experimentally and theoretically shown  that funThere are two types or graphene edges: zigzag and armchair. Zigzag edges are considered more reactive as compared to the armchair ones . DefectsThus, reactions occur at both faces of the graphene layer and different types of edges show different chemical reactivity. Additionally, defects such as vacancies, adatoms or substitutional atoms might act as active chemical sites on the graphene layer . These fThe covalent organic functionalization of graphene is necessary for several purposes. For example: (1) to increase the dispersibility in common organic solvents and water and (2) to combine the properties of graphene with the properties of other functional materials such as nanoparticles, polymers or chromophores –39. The Organic reactions might present different reactivity on the graphene surface when compared with other carbon nanostructures. The characterization of functionalized graphene layers typically requires the use of advanced analytic techniques. Graphene layers in dispersions are normally present at low concentrations and functional groups appear only sporadically. Thus, high-resolution techniques such as X-ray photoelectron spectroscopy (HR-XPS) and HR-TEM are very useful for the identification of functional groups.2 lattice of carbon nanostructures is the azomethine ylide [A common organic reaction used for the functionalization of carbon nanostructures such as fullerenes, nanotubes, nano onions, nano horns and currently graphene, is the use of dienophiles as reactive species. One of the most successfully applied dienophiles to form covalent bonds with the hexagonal spne ylide . This inThe 1,3-dipolar cycloaddition, performed on graphene by employing an aldehyde and an α-amino acid as precursors, attacks both graphene faces and edges. This reactivity was observed by introducing protonated terminal amino groups that selectively bind gold nanorods allowing the recognition of the reactive sites by low resolution TEM . The HR-In order to acquire more knowledge related to the organic chemistry of exfoliated graphene by ultrasonication, carboxylic groups where introduced on the graphene sheets by applying the 1,3-dipolar cycloaddition. This product was compared with fresh exfoliated graphene by the derivatization of both products with PAMAN dendrons using an amide-bond condensation reaction . Using AAs expected, for the 1,3-dipolar cycloaddition reaction, the TEM micrographs showed Au NPs completely dispersed on graphene faces and edges. In contrast, for the amide-bond condensation reaction, Au NPs were mainly found on the graphene edges. These results were corroborated by HR-XPS analysis of the products. A higher concentration of functional groups was found in graphene functionalized by using the 1,3-dipolar cycloaddition. With this, we demonstrated that graphene sheets could be selectively functionalized on the borders or in the entire lattice .These results corroborate the success of the production of graphene layers by ultrasonication techniques. Oxidized carbon atoms on graphene sheets are introduced mainly on the edges of the layers when compared with GO. Additionally, the presence of the adatoms is not revealed by HR-XPS or HR-TEM.4POM) anionic catalyst by electrostatic interactions, as illustrated in As described above, organic chemical reactions can be chosen to attack the complete graphene sheets or mostly the edges. Strategies such as this enable the integration of the outstanding properties of graphene in different applications. For example, graphene–polyoxometalate hybrids are considered important materials for the development of artificial photosynthesis. The properties of graphene enhance the electrocatalytic performance of the catalytic polyoxometalate and minimize the applied overpotential, increasing the long-term robustness of the device . Recentl2 surfaces [N,N,N-trimethylbenzenammonium, as illustrated in 4POM in the charged functional moieties by complementary electrostatic interactions were introduced. Comparing the insertion of a shorter moiety by the diazonium arylation reaction with the positively charged dendrons attached by the 1,3-dipolar cycloaddition described before, we confirmed the interplay of electrostatic forces and π-electron clouds on graphene. The proximity of the Ru4POM to the graphene surface in the arylation reaction leads to a tight immobilization.In order to introduce higher loadings of functional materials, stronger chemical reactions for graphene functionalization can be achieved by the attack of radical species. As reactive radical species, aryl diazonium salts are attractive and popular sources. These radicals covalently graft to carbon spsurfaces . Using t4POM molecules were identified on functionalized graphene surfaces by low voltage aberration-corrected TEM (AC-TEM) [4POM anions on functionalized graphene was performed. When comparing the Ru4POM anions accompanying short carbon moieties with those attached to dendrons with longer carbon chains, a higher degree of motion and frequency were observed for the latter, as shown in Finally, the optical properties of graphene for the high contrast imaging of the two previously described graphene–polyoxometalate nanohybrids can be exploited. For this, Ru(AC-TEM) . FollowiSubstantial efforts have been performed for the mass scale-up and low-cost production of graphene. Thus far, wet chemical strategies have been demonstrated as the most efficient for the inclusion of graphene in low performance applications. To improve the physical properties of chemically produced graphene, it is necessary to gain better control of the experimental techniques by increasing the knowledge of the physical and chemical properties of both graphite and graphene. Likewise, it is critical to acquire detailed comprehension of the organic chemistry of the two dimensional material. The tailored chemical design of graphene platforms is essential for the development of new exciting functional materials.