Dielectric-graphene-dielectric (DGD) structure has been widely used to construct optical devices at infrared region with features of small footprint and low-energy dissipation. The optical properties of graphene can be manipulated by changing its chemical potential by applying a biased voltage onto graphene. However, the excitation efficiency of surface wave on graphene by end-fire method is very low because of large wavevector mismatch between infrared light and surface wave. In this paper, a dielectric-semiconductor-dielectric (DSD) tapered waveguide with magnetic tunability for efficient excitation of surface waves on DGD at infrared region is proposed and analyzed. Efficient excitation of surface waves on DGD with various chemical potentials in graphene layer and incident frequencies can be attained by merely changing the external magnetic field applied onto the DSD tapered waveguide. The electromagnetic simulations verify the design of the proposed structure. More importantly, the constituent materials used in the proposed structure are available in nature. This work opens the door toward various applications in the field of using surface waves. Surface plasmon polaritons (SPPs) on metal surface have attracted a lot of attention from all over the world for its promising optical property, such as supporting subwavelength surface wave234810111214The wavelength and extension length of surface waves can be reduced to dozens of nanometer under infrared illumination by replacing metal with two-dimensional material, graphene. Therefore, the use of graphene provides us a path to make an ultra-compact device2021There are many methods for launching the surface wave on graphene, such as prism coupling excitationIn this work, a semiconductor tapered waveguide with magnetic tunability for efficient end-fire excitation of gaphene surface waves at infrared region for the demand of real application is proposed and analyzed. High coupling efficiency of excitation of graphene surface waves with different chemical potentials in graphene layer and different incident frequencies can be attained by merely changing the external magnetic field applied onto the tapered waveguide. Compared to previous investigations, this work focuses on adding another degree of freedom for manipulating the coupling efficiency between semiconductor surface waves and graphene surface waves by applying an external magnetic field.. x = 0) . The disWhen the external magnetic field is applied onto InSb, its dielectric constant becomes a dielectric tensor whose components depend on the magnitude of magnetic field. Therefore, the optical property of InSb can be manipulated by the applied magnetic field. This is the physical mechanism of magnetic tunability for the proposed structure. The simulated dielectric tensor of InSb under Voigt configuration is expressed ase, m* and N represent the electron charge, the effective mass and the electron density of free electron, respectively, and B is the magnitude of applied external magnetic field; and m* = 0.014 m0 (m0 is the electron mass in vacuo), where For modeling the monolayer graphene, the numerical formula for conductivity (from Kubo formula) and relative permittivity are described as below, respectively30313233e, 2 V−1s−1, which is calculated from where shown in  is almosshown in . The carThe physical mechanism of efficient excitation of graphene surface wave in the proposed semiconductor tapered waveguide is described as follows. First, the surface waves on the first section of DSD are effectively excited by a plane wave normally incident upon the end face of tapered waveguide (i.e. the end-fire method). . This effective excitation of surface wave is ascribed to a much smaller wavevector mismatch between incident light and surface wave. Second, the surface waves propagate in the tapered DSD with wavelength compression due to the gradual increase of their wavevectors. Since InSb is a lossy material (the loss is modeled by the collision frequency in the simulation), the decay of surface waves on the tapered DGD is inevitable. Finally, the graphene surface waves on DGD are efficiently excited at the interface between the fifth section of DSD and DGD because of their wavevectors close to each other. The magnetic tunability of the tapered DSD stems from the fact that the applied magnetic field can manipulate DSD’s dispersion curve.12 rad/s, which is excited directly by the end-fired method. In the simulation, the TM-polarized plane wave  is launched at the beginning of DGD structure (i.e. x = 0). However, the coupling efficiency is only 5.53%. (The method to determine coupling efficiency is listed in Methods).d). In d, as shown in insets (i)~(v) of d in front of graphene can effectively reduce the wavevector mismatch for exciting surface wave on graphene. Furthermore, since the thickness of InSb layer is larger than that of monolayer graphene, the extinction cross section of the DSD-graphene composite structure also increases. After the surface wave is excited at position of x = 0 (shown in cm−1) vs. momentum (k/k0). (See k0 is the wavevector in free space). The propagation lengths of surface waves (defined as the lengths after which the intensities decrease to 1/e) on DGD and DSD at incident frequency of 26.8 × 1012 rad/s are also calculated. Based on d = 300 nm  are 25.9 shown in , its wavd and magnitude of applied external magnetic field B) on the dispersion curves of DGD and DSD structures are also investigated. d and d = 80 nm intersect each other  as For improving the feasibility and dynamical reconfigurability of the proposed tapered waveguide, the effects of changing key parameters . The dispersion relation of DSD with an applied magnetic field under Voigt configuration is also derived in Methods. B. Insets (i), (ii), and (iii) of B = 0, 1, and 1.67 Tesla, respectively, at incident frequency of 28.6 × 1012 rad/s. They confirm that the surface waves can propagate on DSD structure and the wavelength decreases with the increase of B’s magnitude. Comparing B = 0 is suitable as a waveguide connector to efficiently excite the surface wave on graphene with its 12 rad/s. When B between 1 and 1.67 Tesla. B’s values equal to 0, 1.33 and 1.67 Tesla with incident frequency of B = 1.33 Tesla. This result is ascribed to that the dispersion curves of surface waves on DGD with B = 1.33 Tesla are very close to each other at this incident frequency . Here the DSD structure is the same as the fifth section of the proposed tapered waveguide  in 12 rad/s, respectively. They show that, except for 30 × 1012 rad/s, the surface waves with all other frequencies are efficiently excited on DGD. The surface waves of 30 × 1012 rad/s are only excited in the DSD region. All the results agree with the prediction of dispersion curves in 12 rad/s is very small (5.53%) due to momentum mismatch between surface wave on DGD and light line. However, when the tapered waveguide is adopted, the coupling efficiency at the same frequency increases to 68.7% (insets (ii) of B = 0). For 23 and 17.2 × 1012 rad/s with B = 1 and 1.67 Tesla, respectively, the coupling efficiencies of surface waves on DGD are also substantially improved (insets (iii) and (iv) of d of DSD structureFinally, the frequency tunability of surface wave on DGD that is excited by using the proposed tapered DSD waveguide is examined. The dispersion curves of surface wave on DGD with ases see . Here thIn this work, parylene thin film can be used as the substrate in our proposed design. Its relative permittivity is close to 3.0 at the operating frequenciesases see .In summary, the DSD tapered waveguide with magnetic tunability for efficient excitation of surface waves on DGD at infrared region is proposed and analyzed. Excitation of surface waves on DGD with various chemical potentials in graphene layer and incident frequencies can be attained by merely changing the values of external magnetic field applied onto the DSD tapered waveguide. The FEM electromagnetic simulations verify the design of the proposed structure. More importantly, the constituent materials used in the proposed structure are available in nature. Hence, our methodology can be applied in the field of use of surface waves, such as multi-functional material (device), real-time subwavelength imaging, and high-density optoelectronic components. It will provide us a path to overcome the major issue of energy dissipation of surface waves in conventional metallic material .All the simulated results shown in The dispersion relation of surface wave on each three-layered DSD structure without applying the external magnetic field is described as,where When the external magnetic field is applied onto the DSD structure, the dispersion relation is transformed into the following form:where The coupling efficiency of excitation of surface wave on DGD is defined as How to cite this article: Cheng, B. H. et al. Tunable tapered waveguide for efficient compression of light to graphene surface plasmons. Sci. Rep.6, 28799; doi: 10.1038/srep28799 (2016).


In this paper we detail a novel semi-automated method for the production of graphene by sonochemical exfoliation of graphite in the presence of ionic surfactants, e.g., sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB). The formation of individual graphene flakes was confirmed by Raman spectroscopy, while the interaction of graphene with surfactants was proven by NMR spectroscopy. The resulting graphene–surfactant composite material formed a stable suspension in water and some organic solvents, such as chloroform. Graphene thin films were then produced using Langmuir–Blodgett (LB) or electrostatic layer-by-layer (LbL) deposition techniques. The composition and morphology of the films produced was studied with SEM/EDX and AFM. The best results in terms of adhesion and surface coverage were achieved using LbL deposition of graphene(−)SDS alternated with polyethyleneimine (PEI). The optical study of graphene thin films deposited on different substrates was carried out using UV–vis absorption spectroscopy and spectroscopic ellipsometry. A particular focus was on studying graphene layers deposited on gold-coated glass using a method of total internal reflection ellipsometry (TIRE) which revealed the enhancement of the surface plasmon resonance in thin gold films by depositing graphene layers. The double-bonded structure of graphene is responsible for the electrical properties of the material as the movement of π-bonds between adjacent carbon atoms can be used to transmit an electrical current. Because of this electrical activity in particular, graphene is being examined as a base material in a number of different applications including sensor applications, for use in flexible electronics [Since its initial discovery and development by Novoselov et al.  graphenectronics .Graphene has reportedly been produced in a number of different ways. The method chosen for this research is sonochemical exfoliation in water in the presence of a surfactant, as first reported by Notley et al. . This meIn previous work  we manufIn this work a semi-automated technology of graphene production by sonochemical exfoliation of graphite in the presence of ionic surfactants is described in detail. The composite graphene-surfactant materials produced were characterised with NMR and Raman spectroscopy to confirm the formation of graphene. Thin films of graphene composites were deposited using the techniques of Langmuir-Blodgett (LB) and electrostatic layer-by-layer (LbL) deposition. Films composed of these new graphene composite materials were then characterised using SEM, AFM, and spectroscopic ellipsometry. The study of SPR in gold films coated with graphene using total internal reflection ellipsometry was carried out for the first time.−1 while the CTAB solution concentration was made up to 49.7 mg·mL−1. These solutions were prepared and then placed into a water bath heated to 50 °C to aid dissolution.Two different surfactants were used to synthesise graphene–surfactant complexes: sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB). Firstly the surfactant solutions were prepared by dissolving in water. The SDS solution was made to a concentration of 462.9 mg·mL−1 giving an addition rate of 16.2 mg·min−1 for SDS and 1.74 mg·min−1 for CTAB. The addition rate was crucial to maintain a surface tension of 41 mJ·m−2, which is both the optimum surface tension for graphene production and also the surface free energy of graphene. The graphite suspension (10–50%) was then placed into the reactor, and sonicated continuously for 120 min at a power of 50 W. A total of 3.15 mL of surfactant solution was added in each case over the course of the synthesis. The sonication step was carried out in a fume cabinet, as an aerosol containing potentially harmful graphene nanoparticles is produced at this stage. A schematic diagram of this apparatus is shown below  was re-suspended in 40 mL of deionised water. This was then centrifuged at 3500 rpm for 20 min. This process was repeated until no more graphene was contained in the supernatant. If a dry sample was required, then the graphene suspension was frozen and then placed into a freeze dryer (Labogene) for 3–4 days (depending on the sample size) and then subsequently into a vacuum oven at 70 °C overnight to remove all traces of water.−1·cm−1 [2O and placed into an NMR tube. Another tube containing the surfactant alone was also prepared. Both were scanned individually and then the spectra were compared to show shifts in peak position corresponding to interaction between the surfactant and the graphene. NMR experiments were performed on a Bruker 400 MHz spectrometer running topspin analysis software. This analysis works according to the nuclear Overhauser effect (NOE). The NOE, which is present in nuclear magnetic resonance (NMR) spectroscopy, can be used to determine the amount of contact between the surfactant and the graphene sheet. This is achieved by observing peak shifts that occur when two nuclei are within 5 Å of each other. The dried graphene sample was deposited onto a glass microscope slide and then scanned using a Renishaw In-Via Raman microscope at an excitation wavelength of 532 nm. Data were recorded between 100 and 3200 cm−1.Graphene–surfactant complexes were characterised by using a range of different techniques. The presence of graphene was determined by Raman microscopy. The concentration of graphene in solution was obtained by spectrophotometry. The absorbance of a graphene solution at a wavelength of 650 nm was determined and the Beer–Lambert law applied, using an extinction coefficient of 13.9 mg·mL−1·cm−1 . The int3+ and SO3−, respectively. Yet, the presence of alkyl chains and π-systems facilitated their solubility in chloroform, hence the use of the Langmuir–Blodgett (LB) technique (Nima 610 trough) was an obvious choice for the deposition of thin films. The standard LB procedure was applied [−1 solution of graphene(+)CTAB in chloroform was spread onto the surface of deionised water (Millipore). Surface pressure was then recorded using a Wilhelmy plate-based sensor. Because of the unknown ratio of graphene/surfactant within the complex the area per molecule (or repeated unit) was difficult to calculate, so the area was presented in actual units of cm2. Another method known as Langmuir–Schaefer (LS) deposition [Both composite materials obtained, e.g., graphene(+)CTAB and graphene(−)SDS, appeared to be soluble in water due to the presence of ionic groups, NMe applied : a 1 mg·position  was also−1 solutions of the above materials in deionised water. The mutlilayered films obtained were then characterised with scanning SEM combined with EDX (energy dispersing X-ray) elemental analysis (SEM NOVA) and AFM.Much better results (in terms of adhesion and surface coverage) were obtained by using a simple technique of electrostatic layer-by-layer (LbL) deposition, a well-established technique developed first for polyelectrolytes  and lateUV–vis spectra of graphene–surfactant samples are featureless showing almost constant absorbance over the spectral range of 350–800 nm, while the main absorption band of graphene lies in the UV region at about 280 nm. Therefore, optical properties of novel graphene–surfactant composites were studied via spectroscopic ellipsometry using a J. A. Woollam M2000 instrument operating in the spectral range of 370–1000 nm. The measurements were performed on graphene–surfactant films deposited on different substrates, i.e., glass, silicon, and gold-coated glass. Experimental parameters were found by fitting data, the procedure for which is explained in the Results and Discussion section below.−6 Torr using an Edwards 360 unit. Prior to LbL deposition, gold-coated slides were treated overnight in cystamine hydrochloride solution in order to make the gold surface positively charged. Then LbL deposition started with the layer of graphene(−)SDS (negatively charged) followed by deposition of graphene(+)CTAB (positively charged). This procedure was repeated four times, so that four graphene bilayers were deposited. Layer by layer deposition onto other substrates was performed in a similar manner.For the LbL deposition of alternating layers of graphene(−)SDS and graphene(+)CTAB on gold-coated glass slides, a gold film of approximately 25 nm thickness was thermally evaporated onto a 3 nm under layer of chromium (which was used to improve adhesion between the gold and the glass). Metal evaporation was carried out in a vacuum of 10The surface plasmon resonance (SPR) phenomenon in graphene films deposited on thin films of gold was studied in more detail using the method of total internal reflection ellipsometry (TIRE), which was developed in the last decade –14. TIREGraphene was synthesised using graphite suspensions of 10–50% using either SDS or CTAB as the surfactants. The concentration of the final graphene solution obtained from each synthesis was determined by spectrophotometry .1H NMR measurements of the graphene–surfactant complex, when compared to 1H NMR measurements of the surfactant alone, shows shifting of peaks representing hydrogens involved in the complexation interaction  is shifted downfield by complexation with the graphene. This means that the protons are deshielded by the presence of graphene. Since the carbon–sulfur bond is polar the electron density around the carbon atom is already lower than it would normally be in a carbon–carbon bond. Repulsion between the graphene and the sulfate group could cause lengthening of the carbon–sulfur bond. This could in turn lead to a lower electron density around the nuclei responsible for this peak .−1 (D) and 1620 cm−1 (G & D’). Additionally the peak labelled 2D is slightly broader, between 2650–2700 cm−1. This is indicative of graphene flakes with a high number of edge defects [The sample was also analysed using Raman spectroscopy  which, w defects .2-hybridised carbon materials, and is caused by stretching of the C–C bond. The reduction in intensity in the graphene spectrum compared with the graphite spectrum is caused by the reduced number of layers. The D and D’ bands are caused by disorder in the graphene flakes. The D’ band is present when there are surface defects, such as charging or other impurities adsorbed onto the surface. The D band is caused by edge defects such as a “zig-zag” or “chair” shape on the edge. Edge defects provide an enhancement to electrochemical systems by increasing the total capacitance of the electrode surface. Both the D and the D’ band are not present in pristine graphene with straight edges [The G band is present in all spht edges .2-hybridized systems and can be used to estimate the number of layers [The 2D band is also present in many spf layers . Howeverf layers .Typical surface pressure vs area diagrams of graphene(+)CTAB in - groups, positively charged graphene(+)CTAB was chosen; as this provided good adhesion between the substrate and monolayer.However, for LB deposition onto hydrophilic substrates such as glass or oxidised silicon, which are slightly negatively charged due the presence of OHAttempts at depositing thin films of graphene(+)CTAB using a traditional LB process, e.g., vertical dipping and withdrawing the substrate through the monolayer, were not successful since the transfer ratio was poor. The first withdrawal yielded about 60%. After that the layer was shown to peel off during subsequent dipping cycles. The overall transfer ratio by area (when substrate surface area was compared with graphene LB isotherm) was 10–20%. The most significant cause of this was poor adhesion of the first graphene layer to hydrophilic substrates. This could be improved in future work through the use of substrates with surface modifications that either enhance the surface charge or make the surface more hydrophobic. Much better results were obtained using the horizontal lifting method known as Langmuir–Shaefer (LS) deposition , in whicOrganised monolayer films obtained in this fashion were then characterised by using AFM (Nanoscope III) operating in tapping mode using Veeco cantilevers with silicon nitride tips having a radius of less than 10 nm. A typical AFM image of graphene(+)CTAB flakes deposited onto a piece of silicon wafer using LS method is shown in Sectional analysis of AFM image along the lines shown, allows for an estimation of the graphene(+)CTAB flake thickness at 2 nm, which is significantly higher than the reported thickness of pristine graphene of 0.355 nm . The obtIt is clear from these images that the surface coverage is not optimal. Additionally the graphene flakes were seen to change position and shape during scanning, suggesting poor adhesion to the silicon surface. This could potentially be overcome in future work by using surface-modified silicon wafers for sample deposition.The multi layered films obtained from layer-by-layer deposition method were characterised with scanning SEM combined with EDX (energy dispersing X-ray) elemental analysis (SEM NOVA) and AFM.Not all alternating combinations worked well, however. For example, the most promising combination of graphene(+)CTAB with graphene(−)SDS was not successful, while the alternation of graphene(+)CTAB with PAH (or with PEI) proved to be optimal. Deposition on glass or silicon samples was performed by electrostatic adsorption of PAH (or PEI) for 20–30 min followed by dipping into a solution of graphene(−)SDS for 10–15 min. This sequence was repeated several times with a typical incubation time of 10–15 min.Adhesion between graphene and substrate was greatly improved when using a branched polycation such as PEI. The AFM image of graphene(−)SDS deposited onto a layer of PEI in Sectional analysis performed on the sample shows the double and triple layers, and shows the thickness of a single flake at approximately 2.5 nm. This is a somewhat higher than that for LS films in The analysis of graphene films by spectroscopic ellipsometry has already been carried out by other groups, and so is fairly well described –21. A tyd) and dispersions, e.g., spectra of refractive index (n) and extinction coefficient (k) of graphene films can be found by fitting the above spectra to the model using the dedicated software by J. A. Woollam. In this particular case, the model of the reflecting system consists of the following three layers: (1) Si substrate; (2) the layer of native oxide (SiO2) which is typically present on the surface of Si; (3) the deposited layer of PAH/graphene(−)SDS. The ambient was air. Optical parameters for Si and SiO2 were taken from the J. A. Woollam database. The fitting was first performed for ellipsometric spectra of the Si/SiO2 substrate. For the set of data in 2 layer was performed first using the data for the bare Si substrate. The thickness of SiO2 layer obtained (d = 3.2 nm) was then fixed for consecutive fittings. The PAH/graphene(−)SDS film was considered as one layer in the following fittings which is justified by the fact that aliphatic chains of SDS interlock with PAH and form a mixed composite layer of PAH/graphene(−)SDS. Several possible models were tried for fitting PAH/graphene(−)SDS layers, and the best results were achieved using a Lorentz oscillator model from the J. A. Woollam data analysis software, which is given below as a dispersion function of a complex dielectric permittivity, ε(hν):The thickness values 2, Bk = 3.86 eV. The presence of the Lorentz peak in the IR region gives a featureless dispersion of k for graphene–surfactant composite films in the visible spectral range similar to that reported in [d value of the first bilayer appeared to be too high, the average thickness increment Δd = 3.63 ± 2 nm is reasonable and close to that observed with AFM.where εorted in –23. The orted in –23 at abd, n(λ) and k(λ) of the chromium/gold layer were found by fitting the data for uncoated samples, and then used as fixed parameters for subsequent fittings. The graphene layers were modeled through a Lorentz oscillator as before, the values for thickness obtained are plotted against the graphene layers deposited in Spectroscopic ellipsometry measurements were carried out on samples after each layer was deposited and similarly to the previous experiments, Ψ spectra did not change much while the Δ spectra exhibited downward shifts upon deposition of the layers. The ellipsometry data fitting was performed in a similar way as described above using a three layer model containing the substrate glass, the chromium/gold layer, and the graphene layer. The ambient was air. The parameters As mentioned above, the use of PEI as a binding layer alternating with graphene(−)SDS improving the deposition of graphene. The ellipsometry spectra recorded on samples of PEI/graphene(−)SDS deposited on gold-coated glass are shown in It is quite remarkable to observe spectral features around the plasmon-resonance frequency of the gold substrate, which are not related to graphene itself –23 but rThe samples were constructed by electrostatic LbL deposition of PEI and graphene(−)SDS on chromium/gold-coated glass slides, as described above. The results obtained are shown in Deposition of graphene layers made the SPR minimum even lower on the Ψ spectra while causing an additional phase shift on Δ spectra. The enhancement of the SPR peak due to the deposition of graphene layers is observed for the first time. It is interesting to note that graphene itself does not show plasmonic behavior in the visible spectral range as was proven by ellipsometry measurements shown before . At the A simple semi-automated technique for graphene production by aqueous sonochemical exfoliation of graphite in the presence of ionic surfactants, e.g., CTAB or SDS, was developed. Full automation could be potentially achieved by adding surface tension sensors to control the amount of surfactant being added to the reactor, thus maintaining a constant and optimum surface tension. The formation of individual graphene flakes and the interaction of alkyl chains of the surfactants with graphene were, respectively, confirmed with Raman spectroscopy and NMR measurements.The two different graphene-surfactant complexes produced (graphene(+)CTAB and graphene(−)SDS) appeared to be soluble in water and thus suitable for electrostatic LbL deposition. Both compounds were also found to be amphiphilic and soluble in chloroform, hence it was possible to form stable monolayers on the water surface. Thin films of the above graphene composites were deposited onto different solid substrates, i.e., silicon, glass and gold-coated glass, using either electrostatic LbL or LB (LS) deposition techniques. SEM and AFM study showed that LB (or LS) films of graphene(+)CTAB had poor surface coverage and adhesion to the substrate. Electrostatic LbL deposition of graphene by alternation of graphene with oppositely charged polyelectrolytes was much more promising in these aspects. Several combinations of materials were tried including the alternation of graphene(+)CTAB and graphene(−)SDS. The best results were achieved by alternation of graphene(−)SDS with PEI. AFM study allowed the estimation the thickness of an individual graphene–surfactant flakes of about 2.0–2.5 nm.The spectroscopic ellipsometry study of graphene thin films gave similar values for the thickness of the graphene–surfactant composite layer. While the dispersions of refractive index and extinction coefficient were modelled by a single Lorentzian peak lying in the IR region, the absorption peak of graphene at approximately 260 nm was outside the spectral range (370–1000 nm) of the ellipsometric instrument. Interesting results were obtained when studying the SPR effect in gold films coated with a few layers of graphene using both external and internal (TIRE) reflection ellipsometry. Even though graphene itself axhibits no spectral features associated with plasmon oscillations in the above spectral range, the deposition of graphene layers on gold progressively enhances the plasmon resonance in TIRE Ψ spectra and caused an extra phase shift in TIRE Δ spectra. This phenomenon can be explored in the future for enhancing the performance of SPR-based biosensors.


Here, we present QTLs associated with flowering time, maturity, plant height, and grain yield using high density single nucleotide polymorphic (SNP) markers in the same population. A mapping population of 158 RILs and the two parents were evaluated at five environments for flowering, maturity, plant height and grain yield under field conditions, at two greenhouse environments for flowering, and genotyped with a subset of 1809 SNPs out of the 90K SNP array and 2 functional markers (Ppd-D1 and Rht-D1). Using composite interval mapping on the combined phenotype data across all environments, we identified a total of 19 QTLs associated with flowering time in greenhouse (5), and field (6) conditions, maturity (5), grain yield (2) and plant height (1). We mapped these QTLs on 8 chromosomes and they individually explained between 6.3 and 37.8% of the phenotypic variation. Four of the 19 QTLs were associated with multiple traits, including a QTL on 2D associated with flowering, maturity and grain yield; two QTLs on 4A and 7A associated with flowering and maturity, and another QTL on 4D associated with maturity and plant height. However, only the QTLs on both 2D and 4D had major effects, and they mapped adjacent to well-known photoperiod response Ppd-D1 and height reducing Rht-D1 genes, respectively. The QTL on 2D reduced flowering and maturity time up to 5 days with a yield penalty of 436 kg ha-1, while the QTL on 4D reduced plant height by 13 cm, but increased maturity by 2 days. The high density SNPs allowed us to map eight moderate effect, two major effect, and nine minor effect QTLs that were not identified in our previous study using microsatellite and DArT markers. Results from this study provide additional information to wheat researchers developing early maturing and short stature spring wheat cultivars.We recently reported three earliness  Triticum aestivum L.) production increased from 627 million t in 2005 to 729 million t in 2014 (http://faostat3.fao.org). Canada is the seventh largest wheat-producing and the second largest wheat-exporting country. Average wheat yield in Canada has increased from 2.7 t ha-1 in 2005 to 3.1 t ha-1 in 2014, which is equivalent to an average yield increment of 35.7 kg ha-1 yr-1. Diseases and drought contribute to substantial reductions in overall wheat yields in Canada. Wheat breeders there aim to develop short, early maturing cultivars, with high grain yield and protein content, combined with resistance to major diseases, such as leaf, stem and yellow rusts caused by Puccinia sp., fusarium head blight caused by Fusarium graminearum and common bunt caused by both Tilletia tritici and T. laevis (http://www.pgdc.ca).Global hexaploid wheat , recombinant inbred lines (RILs), and near isogenic lines (NILs) [Wheat breeders, in addition to phenotypic selection, employ molecular markers in their breeding programs for different purposes, including parental selection, quality control analysis of advanced lines (cultivars) on genetic purity and identity, and for marker-assisted selection (MAS) . The uses (NILs) . In wheas (NILs) . Howevers (NILs) .QEps.dms-1B1 for both flowering and maturity, QEps.dms-1B2 for maturity and QEps.dms-5B1 for flowering time. One drawback of that study was low marker density (low genome coverage), which varied from 2 on chromosomes 4D and 6D to 57 on chromosome 2B, with an average of 23 markers per chromosome. DArT markers enable the simultaneous typing of several hundred polymorphic loci spread over the genome [vs. absent variation) of DArT markers is one of the major drawbacks, as homozygous dominant and heterozygous individuals cannot be easily identified. SSR markers are widely used by wheat researchers for different reasons, including wide availability, co-dominant inheritance, multiallelism, high polymorphism, uniform distribution, and high polymorphic information content [Taq DNA polymerases used for DNA synthesis add an extra base  at the end of the amplified fragments [Several mapping studies associated with grain yield and other agronomic traits have been conducted for many years –12. Recee genome –15, but  content , 17. SSRragments , which gragments . Hence, ragments . Furtherragments . Currentragments –24. A toragments . The conragments , which h6:7 at the University of Alberta using a single seed descent approach from a cross between two spring wheat cultivars, ‘Cutler’ and ‘AC Barrie’ [Vrn-A1a, recessive vrn-B1 and vrn-D1 vernalization alleles at Vrn1 loci, and the photoperiod insensitive allele Ppd-D1a. ‘AC Barrie’ possesses the same vernalization genes as the ‘Cutler’ with the photoperiod sensitive allele Ppd-D1b. ‘Cutler’ and ‘AC Barrie’ have the mutant Rht-D1b and wild type Rht-D1a alleles, respectively.The present study was based on a subset of 158 of the 177 RILs used in our previous study [ Barrie’ . ‘AC Bar Barrie’ . ‘Cutlerth May and 4th June), 2011 and 2012 at the University of Alberta South Campus Crop Research facility , Edmonton, Canada. Seeds from the F6:7 were initially used for phenotyping in 2007; subsequent phenotyping trials were conducted using seeds multiplied from bulk harvest of typical heads of the previous year. Each field trial was conducted in a randomized incomplete block design with two to three replications depending on seed availability. Each entry was planted in 1.35 x 2.0 m double rows in 2007 and 2008, and 1.35 x 1.8 m with six rows in 2011 and 2012; row spacing in all field trials was 22.5 cm. All field trials were conducted in rainfed conditions using standard agronomic and cultural practices recommended for the station. Each RIL was evaluated for number of days to flowering and maturity, plant height and grain yield. As described in our previous study [The 158 RILs and the two parents were evaluated five times for flowering time, maturity, plant height and grain yield under field conditions and twice for flowering time under greenhouse conditions [Genomic DNA was extracted from three weeks old seedlings using a modified Cetyl Trimethyl Ammonium Bromide (CTAB) method . DNA conPpd-D1  and Rht-D1  at the Agricultural Genomics and Proteomics lab, University of Alberta, Edmonton, Canada. PCR was performed in 96-well plates in a total reaction volume of 10 μL that consisted of 50 ng template DNA, 1× magnesium-free PCR buffer, 1.5 mM MgCl2, 0.50 μM of each of the forward and reverse primer, 0.20 mM of each dNTP, and 1 unit GoTaq® Flexi DNA polymerase. All PCR components were purchased from Promega, USA. PCR amplifications were performed using Gene-Amp PCR System 9600 (PE-Applied Biosystems) as follows: 3 min initial denaturation at 94°C, followed by 40 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec, and a final extension of 7 min at 72°C. PCR fragments were separated with QIAxcel Advanced  as described in the user’s manual using fast analysis kit .We also genotyped the RIL population and the parents with Ppd-D1 and Rht-D1) were constructed in two steps. First, ‘draft’ linkage maps were generated using the minimum spanning tree map (MSTMap) software [−10 and a maximum distance between markers of 15 cM. Second, the ‘draft’ maps were refined using the MapDisto version 1.7.5 software [Least square means, F statistics and heritability were obtained using PROC MIXED and PROC IML in SAS version 9.3 . We analysed each trial (environment) separately and then combined across all environments. Genotypes were considered fixed, while replications, blocks and years were considered random. For each trait, both test for normality and the frequency distribution were done using MiniTab v14. All SNPs that were monomorphic between the two parents and those with >20% missing data were excluded from linkage mapping. Linkage maps for the remaining SNPs and the two functional markers , which consisted of trait acronym, lab designation (dms = Dean Michael Spaner), and chromosome. In this study, QTLs that explained <10%, 10–20% and >20% of the total phenotypic variation (R2) were arbitrarily classified into minor, moderate and major effect QTLs, respectively. Genetic maps and QTL graphs were drawn using MapChart v2.1 [Composite interval mapping (CIM) was performed on the least square means of each trait using PLABQTL version 1.2  with theart v2.1 .-1 lower grain yield than ‘AC Barrie’. The average plant height among the 158 RILs varied from 66 to 104 cm, and required between 49 and 58 days for flowering and between 91 and 101 days for maturity. Mean grain yield of the RILs varied between 4.6 and 7.4 t ha-1  were used for QTL analyses, which is summarized in Among the 81,587 SNPs used for genotyping the RIL population, approximately 87% of the SNPs  were discarded for a number of reasons, including lack of polymorphism between the two parents, high (>20%) missing data, very high segregation distortion, and lack of linkage with other markers. The remaining 10,342 SNPs (12.7%) were incorporated in to the genetic linkage maps of the 21 chromosomes . HoweverQFlt.dms-2D), at 187 cM on 5A (QFlt.dms-5A.1), at 44 cM on 5B (QFlt.dms-5B), at 59 cM on 6B (QFlt.dms-6B.1) and at 5 cM on 7A (QFlt.dms-7A.1). The proportion of phenotypic variance explained by each flowering time QTL under greenhouse varied from 6.9% for QFlt.dms-5A.1 to 36.6% for QFlt.dms-2D. QFlt.dms-2D is the only major effect QTL for flowering time under greenhouse, flanked by the known photoperiod insensitive allele Ppd-D1a and SNP marker (wsnp_CAP11_c3842_1829821)  associated with the number of days to flowering and six QTLs  for degree days to flowering. Four of the QTLs for flowering time under field conditions  were common between the number of days and degree days. For maturity, we found four QTLs  for the number of days to maturity and five QTLs  for maturity in degree days, but only two QTLs (QMat.dms-2D and QMat.dms-7A.2) were common between the two datasets. To account for the difference in temperature across years, therefore, we only present details for the QTLs associated with flowering time and maturity in growing degree days.Composite interval mapping (CIM) was performed on the least square means estimated for individual environments and also combined across two environments for flowering time under greenhouse and five environments for flowering time, maturity, plant height and grain yield under field conditions. 1829821) . For the1829821) , Fig 3. 2 < 1.5%). The six QTLs associated with flowering time (in degree days) mapped at the proximal tip of 2DS (QFlt.dms-2D), at 94 cM on 3B (QFlt.dms-3B), at 41 cM on 4A (QFlt.dms-4A.1), at 253 cM on 5A (QFlt.dms-5A.2), at 118 cM on 6B (QFlt.dms-6B.2) and at 9 cM on 7A (QFlt.dms-7A.1). Each QTL individually explained between 6.3 and 25.4% of the phenotypic variance across the five environments , at 41 cM on 4A (QMat.dms-4A.1), at 37 cM on 4D (QMat.dms-4D.2) plus at both 13 and 42 cM on 7A (QMat.dms-7A.1 and QMat.dms-7A.2). Each maturity QTL explained between 9.1 and 16.2% of the phenotypic variance across all combined environments , flanked by a height reducing Rht-D1b gene and wsnp_CAP11_c356_280910. This QTL had a LOD score of 16.2 and accounted for 37.8% of the phenotypic variance for plant height across the combined data of the five environments. RILs that had the ‘Cutler’ alleles at the two flanking markers were on average 13.2 cm shorter than those RILs that possessed the ‘AC Barrie’ alleles. When individual environments were considered, QPht.dms-4D1 was consistently detected within the same confidence interval in all five individual environments (QYld.dms-2D) and at 34 cM on 5B (QYld.dms-5B), which explained 7.7 to 8.6% of the phenotypic variance for grain yield across five environments. RILs that had the ‘AC Barrie’ alleles at the two flanking markers of QYld.dms-2D and QYld.dms-5B produced on average 436.0 and 321.8 kg ha-1 more grain yield than those RILs that had the ‘Cutler’ alleles. When individual environments were considered, each QTL was detected only in a single environment , maturity (QMat.dms-2D) and grain yield (QYld.dms-2D). As in our previous study [per se QTL; hence, both QFlt.dms-2D vs QMat.dms-2D are named QEps.dms-2D. QEps.dms-2D explained 36.6% and 25.4% for flowering time under greenhouse and field conditions, respectively, 10.4% for maturity, and 8.6% for grain yield. RILs carrying the photoperiod insensitive mutant Ppd-D1a alleles from ‘Cutler’ at the two flanking markers for QEps.dms-2D have differed from those containing the photoperiod sensitive Ppd-D1b alleles from ‘AC Barrie’ for flowering time (p < 0.012), maturity (p < 0.050) and grain yield (p < 0.001), but not for plant height  and maturity (QMat.dms-4A.1), here referred as QEps.dms-4A.1. RILs carrying the ‘Cutler’ alleles at the two flanking markers for QEps.dms-4A.1 differed (p < 0.005) from those containing the ‘AC Barrie’ alleles for flowering time under field conditions and maturity, but not for plant height and grain yield  and plant height (QPht.dms-4D). RILs carrying the ‘Çutler’ alleles at the two flanking markers of the coincident QTL on 4D showed significant differences (p < 0.001) with those containing ‘AC Barrie’ alleles for maturity and plant height, but not for flowering time and grain yield , associated with both flowering time (QFlt.dms-7A.1) and maturity (QMat.dms-7A.1), with RILs consisting of the ‘Cutler’ alleles at the two flanking markers showing a reduction in flowering/maturity by 11.2–15.9 degree days than those RILs that had the ‘AC Barrie’ alleles , but not for maturity, plant height and grain yield (Four of the nineteen QTLs associated with the combined phenotype data of the four traits were common (coincident) for two or three traits. The first coincident QTL is the one that mapped at the proximal tip on chromosome 2DS, which is associated with flowering time both under greenhouse and field conditions , one QTL for maturity at 36 cM on 1B (QEps.dms-1B2) and one QTL for flowering time at 76 cM on 5B (QEps.dms-5B1). That study failed to identify any QTL for both plant height and grain yield across the combined phenotypic data of the four environments; only two environment specific QTLs were reported for grain yield. Our previous study was based on a total map length of 2,279 cM, with individual chromosome varying from 36 to 229 cM; the overall average map distance among adjacent markers  was 4.7 cM. We thought that the low marker density might have restricted our ability to identify more QTLs with larger phenotypic effect. The use of larger number of polymorphic markers provides a more accurate overview of informative recombinations and greater saturation of genetic linkage maps. The denser the genetic maps, the lower the chance of missing true QTLs [Ppd-D1a and Rht-D1b), which resulted in a total map length of 3996 cM and an overall average inter-marker interval of 2.2 cM. As compared with our previous study, therefore, the genome coverage in the present study increased by 78%, while average inter-marker interval decreased over two fold. Based on such higher genome coverage and reduction in map distance among adjacent markers, we expected to narrow down the confidence interval of the QTLs that we reported in our previous study and also uncover additional QTLs that may have been missed in our previous study. In the present study, we uncovered a total of nineteen QTLs associated with the combined phenotypic data, which includes five for flowering time in the greenhouse, six for flowering time in the field, five for maturity, one for plant height, and two for grain yield (QYld.dms-1B) and one QTL for flowering time under greenhouse on 5B, but we were not sure whether these two QTLs mapped at the same confidence interval as the three QTLs  reported in our previous study.Based on 488 SSR and DArT markers, we previously reported three QTLs associated with the combined phenotypic data across four environments [rue QTLs . Our prein yield . HoweverQEps.dms-1B2 was different between the two studies (which is expected with addition of large number of SNPs into DArT and SSRs), one of the flanking DArT markers (wPt-2694) remained the same. However, the position of the QTL associated with the combined grain yield data across 5 environments (QYld.dms-1B) was 52 cM distal to wPt-2694, which suggests that the QTL for maturity is different from that of the QTL for grain yield. For the QTL on 5B, the analysis using combined DArT, SSR and SNP markers identified QEps.dms-5B1, which has been reported in our previous study [QEps.dms-5B1 was associated with flowering time in the 2007 and maturity in the 2008 early planting environments  identified one of the QTLs for maturity on 1B between 74.5 and 80.5 cM interval, which accounted for 9.7–13.1% of the phenotypic variance for maturity in degree days across the combined data plus the 2007 and 2011 environments . Althougronments . This QTt height .Ppd-D1a and Rht-D1b).However, the integration of the SSR and DArT markers with the SNPs had two limitations. First, it reduced the number of RILs with complete genotypic and phenotypic data from 158 to 131. Secondly, the SSR and DArT markers affected locus order for many SNPs, which was difficult to tell due to lack of physical positions or consensus linkage maps for the different types of markers. Third, a number of SNPs remained either unlinked or fall into several smaller linkage groups, which significantly reduced the number of markers integrated in the linkage maps. We suspected an error in the DArT genotypic data, either mislabeling and/or data coding errors during linkage map construction and QTL analyses. The second possible reason may be the use of large numbers of DArT markers in our previous study, which are primarily dominant in inheritance . We therVrn loci  of which Vrn-A1, Vrn-B1 and Vrn-D1 mapped on the long arm of chromosomes 5A, 5B and 5D, respectively [Vrn-1 genes directly influence both flowering and maturity [Vrn-1 genes, because (i) direct comparison of the genetic map positions across different studies is not possible without having either common set of markers or physical positions; (ii) “Cutler’ and ‘AC Barrie’ were monomorphic for the VRN1 loci, both having the dominant Vrn-A1a and recessive vrn-B1 and vrn-D1 alleles [The QTLs for flowering time in the combined environments both under greenhouse and field conditions mapped on chromosomes 2D, 3B, 4A, 5A, 5B, 6B and 7A, each explaining between 6.3 and 36.6% of the phenotypic variance . For matectively , 50. Thematurity , 52. How alleles .QFlt.dms-2D), maturity (QMat.dms-2D) and grain yield (QYld.dms-2D). This coincident QTL is flanked by the well-known photoperiod response Ppd-D1a locus, and accounted from 19.6 to 36.6% for flowering time, from 10.4 to 11.2% for maturity, and 8.6% for grain yield  reduction on flowering/maturity, but suffered highly significant (p <0.001) yield penalty . Coincident QTLs have been reported in several other studies [Ppd-D1a and wsnp_CAP11_c3842_1829821) for the coincident QTL on 2D  is 22.6 cM, which is too large. It is, therefore, highly likely that the chromosomal segments associated with this coincident QTL on 2D carry two or more genes or QTLs, which could be determined by screening larger numbers of recombinants to break up the linkage [In the present study, we found a major effect and coincident QTL on 2D for flowering time both under greenhouse and field conditions  and medium effect QTL for maturity (QMat.dms-4D.2) on chromosome 4D , 2012 and combined environments  for the coincident QTL on 4D (QMat.dms-4D.2 and QPht.dms-4D) are 5 cM apart, which possibly contain two or more tightly linked genes or QTLs. Additional study is needed to explore whether such major effect coincident genomic region is due to tight linkage or pleotropic effect.The QTL on 4D that reduced plant height also increased days to maturity. As discussed above for the coincident QTL on 2D, coincident QTLs on 4D could also be due to either tight linkages between genes or QTLs or pleiotropic effect . For exavailable . The autPpd-D1 gene and reduced flowering and maturity time up to 5 days but showed yield penalty by 436 kg ha-1. The QTL on 4D mapped adjacent to a well-known height reducing Rht-D1 gene and reduced plant height on average by 13 cm, but increased maturity by 33 degree days. The coincident nature of the QTLs on 2D and 4D is very likely due to linkage, which may be determined by screening large numbers of recombinants to break up the linkage.There were discrepancies between our QTL results from the present and previous studies. First, we were not able to clearly confirm the flowering time and maturity QTLs on both 1B and 5B that were identified in our previous study using DArT and SSR markers. Second, the SNP and two functional markers provided us a better opportunity to uncover eight moderate effect and two major effect QTLs along with several other minor effect QTLs that were not identified in our previous study using SSR and DArT markers. The two major effect QTLs mapped on both chromosomes 2D and 4D. The QTL on 2D mapped adjacent to a well-known photoperiod response S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Nuclear genes of euglenids contain two major types of introns: conventional spliceosomal and nonconventional introns. The latter are characterized by variable non-canonical borders, RNA secondary structure that brings intron ends together, and an unknown mechanism of removal. Some researchers also distinguish intermediate introns, which combine features of both types. They form a stable RNA secondary structure and are classified into two subtypes depending on whether they contain one  or both  canonical spliceosomal borders. However, it has been also postulated that most introns classified as intermediate could simply be special cases of conventional or nonconventional introns.tubB, hsp90 and gapC genes from six strains of Euglena agilis were obtained. They contain four, six, and two or three introns, respectively (the third intron in the gapC gene is unique for just one strain). Conventional introns were present at three positions: two in the tubB gene  and one in the gapC gene. Nonconventional introns are present at ten positions: two in the tubB gene , six in hsp90 , and two in the gapC gene.Sequences of gapC gene an extended direct repeats at the intron-exon junctions are present, suggesting that double-strand break repair process could be the source of new nonconventional introns.Sequence and RNA secondary structure analyses of nonconventional introns confirmed that their most strongly conserved elements are base pairing nucleotides at positions +4, +5 and +6/ -8, −7 and −6 (in most introns CAG/CTG nucleotides were observed). It was also confirmed that the presence of the 5' GT/C end in intermediate/nonconventional introns is not the result of kinship with conventional introns, but is due to evolutionary pressure to preserve the purine at the 5' end. However, an example of a nonconventional intron with GC-AG ends was shown, suggesting the possibility of intron type conversion between nonconventional and conventional. Furthermore, an analysis of conventional introns revealed that the ability to form a stable RNA secondary structure by some introns is probably not a result of their relationship with nonconventional introns. It was also shown that acquisition of new nonconventional introns is an ongoing process and can be observed at the level of a single species. In the recently acquired intron in the The online version of this article (doi:10.1186/s12862-016-0620-5) contains supplementary material, which is available to authorized users. Pyramimonas . Sequences of all the primers used in this study are listed in  and EA9F1/R1  covering almost the entire gene; (2) EA9sF0/R0  and EA9sF1/R1  covering the fragment of gene with introns; (3) EA9sF0/R2  and EA9sF1/R3  covering the first part of the gene; (4) EA9sF2/R0  and EA9sF3/R1  covering the second part of the gene. Based on the cDNA sequence of the gapC gene from E. agilis ACOI 2970 [GenBank: KT962995], two pairs of primers were designed: EAgCF0/R0  and EAgCF1/R1  which cover the fragment of the gene where three introns were identified previously in the sequence of E. gracilis. Conditions of nested PCR: in the first PCR reaction, a 25 μl reaction mixture contained 0.5 U Phusion High-Fidelity DNA Polymerase (Thermo Scientific), 0.2 mM dNTPs, 3.5 mM MgCl2, 10 pmol of each primer, reaction buffer GC (Thermo Scientific), Q-solution (Qiagen), 1.2 μg Taq Single-Stranded DNA Binding Protein (EURx) and 10–50 ng DNA. The PCR protocol consisted of 2 min at 98 °C, followed by seven initial cycles comprising 30 s at 98 °C, 30 s at 55–68 °C (depending on the set of primers) and 2.5 min at 72 °C, then by 35 cycles comprising 15 s at 98 °C, 15 s at 55–68 °C and 2.5 min at 72 °C. The final extension step was performed for 5 min at 72 °C. In the second PCR reaction, a 25 μl reaction mixture contained 0.5 U Phusion High-Fidelity DNA Polymerase (Thermo Scientific), 0.2 mM dNTPs, 1.5 mM MgCl2, 10 pmol of each primer, reaction buffer GC (Thermo Scientific), Q-solution (Qiagen), 0.6 μg Taq Single-Stranded DNA Binding Protein (EURx) and 1 μl of undiluted mixture from the first step. The PCR protocol consisted of an initial 2 min at 98 °C, followed by 35 cycles comprising 15 s at 98 °C, 15 s at 55–60 °C and 2.5 min at 72 °C. PCR products were cloned in a pGem-T Easy vector and sequenced as described previously , hsp90 [GenBank: KU041762-7], gapC [GenBank: KU041768-76]. GenBank accession numbers for the tubB sequences from E. agilis strain ACOI 2970 are [GenBank: KC907661-3].The data set supporting the results of this article is included within the article and its Additional file


The approach presented here for the design of anodic alumina cantilever arrays by the combination of anodic oxidation and photolithography enables easy control over porosity, surface area, geometric and mechanical characteristics of the cantilever arrays for micromechanical sensing.We present here the behavior of the resonance frequency of porous anodic alumina cantilever arrays during water vapor adsorption and emphasize their possible use in the micromechanical sensing of humidity levels at least in the range of 10–22%. The sensitivity of porous anodic aluminium oxide cantilevers (Δ The last two decades have seen a surge in resonant micro- and nanomechanical engineering raised by the creation of high-performance and cost-effective detection systems. Highly elastic silicon and, in contrast, soft polymer cantilevers were implemented to chemical sensors with resonance shift assay –3. To da1) and before (ƒ0) adsorption as follows [The adsorption of chemicals on the surface of the cantilever manifests itself by the shift of the resonance frequency in dynamic vibration mode. The loading mass can be calculated from the change of resonance frequencies after (ƒ follows –10:k is given by k = (E·w·t3)/(4·l3), where w, t and l are the width, thickness and length of the cantilever and E is the Young’s modulus of the cantilever [Here, the spring constant ntilever . AccordiIn the present work we concentrated on the elaboration of experimental methods for the fabrication of anodic alumina cantilever arrays and their testing for adsorption of water vapors.The proposed procedure for the fabrication of cantilever arrays combines anodic oxidation of aluminium foil with a conventional photolithography process .Using the lithography approach for AAO pattern formation mostly becomes possible due to selective and anisotropic etching of anodic alumina films on aluminium in basic solutions. On the other hand anodic oxidation itself enables easy control over channel diameter and interpore distance of the porous film over a wide range  . MoreoveQ factor for both types of beams significantly increases after change air media to vacuum. Natural shifts to high-frequency region are observed for the porous cantilever as well for the Si cantilever.To predict the behavior of the cantilever in the gas/liquid phase we investigated alterations of the amplitude–frequency characteristics when changing pressure and humidity. The frequency response of elastic beam is strongly dependent on the fluid it is vibrating in . In vacuFor comparison we also studied the effect of the water content on the vibration of the cantilever from the AAO membrane obtained at 40 V in 0.3 M oxalic acid. The resonant frequency decreases from 117.7 kHz at 10% to 116.5 kHz at 21% humidity. Changing the humidity from 21 to 10% increases the resonant frequency by 1120 Hz and quality factor of the AAO cantilever increases from 61 to 82, respectively .m = 20 pg at a sensitivity Δf/Δm of 56 Hz/pg. On the other hand, the amount of absorbed water can be estimated from the Langmuir monolayer absorption isotherm. Taking into account the total area of the cantilever of 1.74·10−6 m2 (1.58·10−6 m2 interior and 1.63·10−7 m2 exterior area) and the density of the active sites of 4.8 nm−2 the maximal mass of a water monolayer adsorbed onto cantilever surface is 250 pg. This results in an increase of the experimental water surface coverage of about 8% within the experimental humidity levels.Using From the Langmuir sorption isotherm a humidity change from 10 to 22% should result in growth of the surface coverage by 8.9%, which fits well to the obtained value. However, despite the good agreement between the estimations we should stress that they can be far from being realistic due to a strong chemical interaction of water molecules with AAO surface and pore curvature.f = 118.99·(1 + 0.00198H)−1/2 (red line), which correlates well with After each humidity decrement the system was left to reach equilibrium for 10–30 min. Equilibration criterion was resonant frequency creep below 1 Hz/min. The curve indicates a linear behavior approximated by The proposed combination of anodic oxidation and photolithography processes enables the successful formation of porous alumina cantilevers with desired geometric characteristics. Because of the high surface area of the pores the cantilevers exhibit an exceptional sensitivity to absorbed mass. The resonance frequency shift in water vapor absorption experiments over a humidity range of 10–22% fits well to the monolayer adsorption isotherm. A strong response to environmental change enables the use of AAO cantilever arrays in microsensors with ultra-low detection limits. The study has to be considered as an initial experiment to focus on the profound understanding of micromechanical behavior of anisotropic porous materials having a high specific surface area, such as anodic alumina films.2C2O4  at a constant voltage of 40 V. The electrolyte was pumped through the two-electrode cell by a peristaltic pump, and its temperature was kept constant (2 °C) during anodization. The films with a thickness of 2 μm were obtained by controlling the total electric charge. To define the shapes of the cantilever array the pattern of the photolithographic mask was directly transferred onto the AAO surface . The size of the obtained microcantilevers equals 800 × 100 × 2 µm3 (AAO layer formation was carried out in 0.3 M Hadiation ). Then u × 2 µm3 .One should note that these dimensional characteristics can be simply tuned by choosing lithographical mask with the desired cantilever shapes and varying of anodization charge. Films were characterized by optical (Nikon Eclipse 600pol) and scanning electron (LEO Supra 50VP) microscopes. The density of anodic alumina was determined by measuring sample hydrostatic weight.Mechanical measurements with air, vacuum and wet media were carried out by AFM (NTEGRA Aura) in the range of 0–600 kHz. The experimental setup used in this study is shown on 2 dryer. Saturated salt-water was used to reach high relative humidity in the microscope chamber. After stabilization of humidity equilibrium in the system we start measurements of the resonance frequency over a CaCl2 dryer down to the lowest possible humidity level. The microscope determined the upper limit of humidity level. Typically around 10 min have been needed to stabilize the cantilever to a fixed baseline. Humidity levels were varied in the range of 10–22%. An example of the resonance spectrum of porous alumina cantilevers detected with the AFM is shown in The desired moisture level was obtained by controlling the humidity at 25 °C with saturated salt-water and CaClThe arrays were supported by Si tips before measurements. The reflectivity of the surface of anodic aluminium oxide is sufficient for detecting vibrations without additional metal reflective coating. According to the exhibited behavior, adequate level of sample reproducibility has been demonstrated by the fabrication procedure described. The frequency does not shift for different points along the one cantilever. In contrast, intensity and a uniform vibration were indicated along the whole cantilever independently from where the AFM beam was detected. The magnitude of the maximal resonance frequency (about 545 kHz) predictably decreases when the optical beam of AFM moves to base of array. Experimentally we established the excitation of mechanical vibrations perpendicular to the cantilever surface as torsion. The thickness of cantilever arrays influences on stiffness and may also cause an increasing in resonance frequency: from 500 kHz for 2 μm thick cantilevers to 670 kHz for 30 μm thick cantilevers.


Immune thrombocytopenic purpura (ITP) is a clinical syndrome in which a decreased number of circulating platelets (thrombocytopenia) manifests as a bleeding tendency, easy bruising (purpura) or extravasation of blood from capillaries into skin and mucous membranes (petechiae). The diagnosis of ITP can be made clinically on the basis of symptoms, we need to see if ITP can be confirmed in patients by quantification of residual RNA containing immature platelets (megakaryocytic mass) or immature platelets fraction (IPF) using automated hematology analyzers (Sysmex XE-2100).In order to check the efficacy of IPF% parameter of Sysmex XE-2100 a total of 231 patients of thrombocytopenia were included in this study. Complete blood count (CBC) was estimated. The data was statistically analyzed by SPSS version 17.About 62 patients were diagnosed as ITP and 169 patients were diagnosed as non ITP on the basis of clinical history. The mean IPF % value of ITP patients was 16.39% and the IPF % value of Non ITP patients was ~7.69% respectively. There was no significant difference in IPF% values with respect to time between sampling and acquisition of complete blood count. The diagnostic sensitivity of IPF% as biomarker for ITP and non-ITP was 85.71% (95%CI: 84.04% to 85.96%) and 41.76% (95% CI: 39.87% to 43.65%).The mean IPF % value by Sysmex XE-2100 can be used to predict ITP. The platelets are the key player in controlling bleeding problems. Abnormally low/ decreased number of circulating mature platelets (thrombocytopenia) may lead to excessive bleeding, easy bruising (purpura), or extravasations of blood from capillaries into skin and mucous membranes (petechiae).15 children/year; for adults the estimate is 3.3 per 105 adults/year.2Major cause of thrombocytopenia in ITP is accelerated platelet destruction and impaired platelet production. Exact etiology is unknown. The global incidence of acute ITP in children is between 1.9 and 6.4 per 10Following an episode of acute blood loss reticulated platelets (RP) containing remnants of RNA are generally observed. In case of ITP the number of such platelets is markedly high due to excessive peripheral platelet destruction.This study was conducted at National Institute of Blood Diseases & Bone Marrow Transplantation (NIBD), Karachi, from September 2012 to January 2013. About 227 patients with different age groups of both genders diagnosed as having thrombocytopenia were included. Patients with other causes of thrombocytopenia (inherited thrombocytopenias), bone marrow failure syndromes, lymphoproliferative disorders, myeloproliferative disorders, thrombotic microangiopathies, megaloblastic anaemia and pseudothrombocytopenia were carefully excluded according to the findings in history, physical examination and investigations.9/L. ITP was diagnosed on the basis of the patient’s medical history, isolated thrombocytopenia without other underlying diseases, and additional laboratory tests, including anti-platelet antibody tests and bone marrow examination, where necessary. The ITP positive cases were confirmed by morphological examination of stained peripheral blood smears. For the determination of diagnostic efficacy of the biomarker (IPF%) about 94 healthy individuals of either sex were also included as control groupThe diagnosis of the patient groups included ITP with platelet counts of less than 150×102EDTA  were analyzed for IPF% and all routine (full blood count) parameters, including platelet counts. All samples were kept at room temperature and were analyzed within 8 hrs after collection.Peripheral blood samples (3 mL) collected in KThe IPF% was measured using Sysmex XE-2100 . The IPF% results were available at the same time as the full blood counts were done. The IPF% of each individual sample was acquired twice right after collection (within 15 minutes.) and results were recorded as mean of two independent runs. The platelets count and mean platelets volume (MPV) were also recorded as comparative parameters. Total 40 samples; 20 each from confirmed ITP cases and non-ITP cases were re-analysed after 7.5 hrs of collection to observe any change in IPF% with time.The data was statistically analyzed by SPSS version 17. The descriptive parameters were described in terms of mean±standard deviation or median whereas inferential parameters for ITP, non-ITP and normal healthy individuals were statistically analyzed by Chi square test at significance level 95%.3/ul and platelet count of non ITP patients was 54.62±39.64x103/ul summarized in A total of 62 patients were diagnosed as ITP and 169 patients were diagnosed as non ITP on the basis of clinical history and laboratory tests. The median age was 23 years, 32 years and 33 years for the ITP and non-ITP patients and healthy controls respectively . There wITP may be diagnosed on the basis of clinical findings but laboratory confirmation is often required.109/l had a raised IPF%. There was significant inverse correlation of platelet count with IPF%; the lower the platelet counts, the higher the IPF%. The IPF% value reflected the severity of platelet destruction. With a poor sensitivity of about 41.76% for non-ITP thrombocytopenias IPF% may not be considered as a good biomarker for these cases. Further studies are indeed required to justify this conclusion.It was observed that IPF% was higher or >7% in ITP than in the normal population, and correlates with bone marrow platelet production or thrombopoiesis. These observations were in line with other studies. The reproducibility of the method of duplicate analysis was good for most parameters. Beside IPF% the MPV is another parameter used to make inferences about platelets production in bone marrow or platelets destruction problem . Higher MPV values associated with increased platelets destruction is a common observation in inflammatory bowel disease, ITP and myeloproliferative diseases.ITP is primarily a disease of increased peripheral platelet destruction, and this study demonstrates a fully automated, rapid method for the determination of the IPF, using the Sysmex XE-2100 equipped with upgraded software to analyze the highly fluorescent platelets. The results are available at the same time as the full blood count and the IPF is reproducible and stable in an EDTA sample stored at room temperature for at least 8 hrs. The IPF% provides a valuable diagnostic method to clearly establish the diagnosis of idiopathic (or immune) thrombocytopenic purpura in patients. Hence it can be emphasized that the IPF should become a standard routine parameter in the diagnosis and serial monitoring of the thrombocytopenic patient.


Di-peptide repeat proteins are translated in a non-canonical fashion from sense and antisense transcripts of the G4C2 repeat . DPR inclusions are abundant in the cerebellum, as well as in the frontal and temporal lobes of ALS and FTLD patients and some are neurotoxic in a range of cellular and animal models, implying that DPR aggregation directly contributes to disease pathogenesis. Here we sought to quantify inclusions for each DPR and TDP-43 in ALS cases with and without the C9ORF72 mutation. We characterised the abundance of DPRs and their cellular location and compared this to cytoplasmic TDP-43 inclusions in order to explore the role of each inclusion in lower motor neuron degeneration.Cytoplasmic TDP-43 inclusions are the pathological hallmark of amyotrophic lateral sclerosis (ALS) and tau-negative frontotemporal lobar dementia (FTLD). The GC9ORF72 repeat expansion (ALS-C9+ve) and five cases that were not were probed by double immunofluorescence staining for individual DPRs and TDP-43. Inclusions immunoreactive for each of the DPRs were present in the spinal cord but they were rare or very rare in abundance . TDP-43 cytoplasmic inclusions were 45- to 750-fold more frequent than any DPR, and fewer than 4 % of DPR inclusions colocalized with TDP-43 inclusions. In motor neurons, a single cytoplasmic DPR inclusion was detected (0.1 %) in contrast to the 34 % of motor neurons that contained cytoplasmic TDP-43 inclusions. Furthermore, the number of TDP-43 inclusions in ALS cases with and without the C9ORF72 mutation was nearly identical.Spinal cord sections from ten cases positive for the C9ORF72 mutation status. The near absence of DPR inclusions in spinal cord motor neurons challenges their contribution to lower motor neuron degeneration in ALS-C9+ve cases.For all other neurodegenerative diseases, the neurotoxic protein aggregates are detected in the affected population of neurons. TDP-43 cytoplasmic aggregation is the dominant feature of ALS spinal cord pathology irrespective of The online version of this article (doi:10.1186/s40478-015-0218-y) contains supplementary material, which is available to authorized users. Cytoplasmic inclusions containing the transactive response DNA-binding protein TDP-43) are the neuropathological hallmark of ~95 % of ALS cases and ~60 % of frontotemporal dementia (FTD) cases . Expansi3 are theC9ORF72 expansion are unknown but three mechanisms have been proposed: loss of C9ORF72 protein function; toxicity due to RNA aggregation and toxicity due to repeat associated non-ATG (RAN) translation of dipeptide repeat (DPR) proteins. Although the G4C2 expansion does cause a decrease in C9ORF72 mRNA, it is not clear that this leads to loss of the C9ORF72 protein and no null alleles or missense mutations in C9ORF72 have been identified in ALS or FTD cases to support the loss of C9ORF72 protein function hypothesis. We, and others have shown that G4C2 repeats generate length-dependant RNase-resistant intranuclear RNA foci, which can sequester RNA binding proteins potentially leading to defective RNA processing [4C2 repeat RNA form stable G-quadruplex secondary structures that can recruit the translation machinery [4C2 repeat strand and poly-PR, poly-PA and poly-GP from the antisense G2C4 repeat strand [The neurotoxic mechanisms of the ocessing . Finallyachinery  and genet strand . This laC9ORF72 mutation, DPR protein deposition and TDP-43 mislocalization is unknown. In the present study, we conducted a detailed comparison of the abundance and cellular location of the five DPR proteins and TDP-43 in the spinal cord of ten ALS-C9+ve cases. Using double immunofluorescence staining, we show that DPR protein aggregates are uncommon in the spinal cord of ALS-C9+ve cases, rarely co-localise with TDP-43, and are almost absent from motor neurons. The abundance of TDP-43 inclusions in ALS cases with or without a C9ORF72 repeat expansion was broadly similar. Given that TDP-43 mislocalization is a recognised cause of lower motor neuron neurodegeneration, and given the lack of DPRs in motor neurons, it is difficult to implicate DPRs in this process.Antibodies to specific DPR proteins detect cytoplasmic aggregates of poly-GP, poly-GA and poly-PA and intranuclear aggregates of poly-PR and poly-GR in the cerebellum and frontal cortex of ALS and FTD patients ,10. DPR C9ORF72 intronic expansion (ALS-C9+ve) and 13 sections from five ALS cases negative for C9ORF72, FUS and SOD1 mutations . Sections of frontal lobe and cerebellar cortex from a case of C9FTD with abundant p62 pathology were chosen as a positive control for DPR staining. All cases were provided by the Medical Research Council London Neurodegenerative Diseases Brain Bank . Samples were collected and distributed in accordance with local and national research ethics committee approvals. Expansion carriers were identified using repeat primed PCR [C9ORF72 mutations.A total of 30 spinal cord sections were analysed for the presence of TDP-43 and all DPR inclusions by double immunofluorescence from ten ALS cases positive for imed PCR ,12. DetaHistological examination was performed on 7 μm sections prepared from formalin-fixed, paraffin –embedded tissue from spinal cords of all ALS cases. Sections were stained with hematoxylin and eosin to perform motor neuron counts, and sequential sections were processed for double label immunofluorescence for the DPRs and TDP-43. Prior to double immunofluorescence staining, paraffin was removed with xylene and all sections were rehydrated in an ethanol series  for 3 min per step. Slides were incubated in 0.3 % Sudan black for 5 min to quench autofluorescence and washed with water. Antigen retrieval was carried out by microwaving for 6 min at maximum power and 12 min at medium power in 100 mM sodium citrate buffer (pH 6.0). Non-specific binding sites were then blocked for 20 min using 5 % normal donkey serum in PBS.For double immunofluorescence staining, spinal cord sections were incubated with primary rabbit antibodies against the five different DPRs  at a dilution of 1:100 together with TDP-43 ) at a dilution of 1:100 overnight in a humid chamber at 4 °C. The DPR antibodies were obtained from Dr. Leonard Petrucelli and colleagues and were previously characterised . After 3The presence of DPR and TDP-43 inclusions were scored in the anterior horn of the spinal cord using a previously published semi-quantitative grading scale , in whicThe appearance and subcellular location of each type of DPR inclusion within the spinal cord was recorded Fig. -h. Poly-C9ORF72 mutation (2.8 ± 0.2) when compared to those carrying the C9ORF72 mutation (2.9 ± 0.1). Thus, the presence of DPR inclusions in the spinal cord does not appear to be correlated with the abundance of TDP-43 inclusions.The frequency of each DPR inclusion was initially scored using a semi-quantitative system for each case Table . DPRs trIn order to provide greater detail, the number of TDP-43 aggregates in the anterior horn of the spinal cord in each case was quantified in representative sections to explore any correlation between the abundance of DPR and TDP-43 inclusions. An average of 122 ± 8 TDP-43 aggregates was identified per section per case, which is between 45- and 750- fold greater than the number of any DPRs inclusions  contained cytoplasmic TDP-43 inclusions Fig. , which i4C2 repeat mutation in the C9ORF72 gene is the most common cause of ALS and FTLD known to date. A leading mechanistic hypothesis is that neurotoxicity is due to the expression and aggregation of DPRs, particularly poly-GR and poly-PR that form intranuclear inclusions [C9ORF72 mutant positive and negative ALS cases.The Gclusions –17. In tC9ORF72 mutation positive cases (Additional file Inclusions for all five DPRs were detected in the spinal cord of ALS-C9+ve cases but they were rare or very rare and it was uncommon to see DPR and TDP-43 inclusions in the same cell. Poly-GA inclusions, present in the cytoplasm and occasionally the nucleus, were the most common. Poly-GP inclusions were purely cytoplasmic, which is consistent with published reports ,14,18,194C2 repeats were expressed in the central nervous system of mice, has shown that nuclear RNA foci, DPR inclusions and TDP-43 pathology were present in the cortex and hippocampus of mice, suggesting that the repeat itself, possibly through foci formation, RAN translation or another mechanisms entirely, caused aberrant TDP-43 deposition [Several groups have studied DPR protein toxicity in various cellular models but these have generated conflicting results –17,20,21position . We haveC9ORF72 mutation are nearly identical we can find no evidence that dipeptide repeat proteins are playing a pathogenic role in ALS-C9+ve cases.While we cannot entirely exclude the possibility that DPR aggregation and toxicity occurs at the sub-microscopic level, through toxic DPR oligomers, or that all motor neurons containing DPR inclusions were lost, this explanation seems unlikely as neurotoxic protein aggregates are detected in the affected neurons for all other neurodegenerative diseases. As the number of TDP-43 inclusions in ALS cases, with and without the


The As comparison, pristine Bi2WO6 nanoparticles have poor specific capacitance of 574 F/g at a charge and discharge currents of 2 A/g with weak cycle life. Though analyzing the Cyclic voltammetry curves, it is found that there are two oxidation reaction occurring in the materials: oxidation of Bi (III) to Bi (IV) and Bi (III) to Bi (V). The oxidation of Bi (III) to Bi (IV) is reversible while Bi (III) to Bi (V) will cause nonreversible destroy on structure. In this nano-Bi2WO6/reduced graphene oxide composite, graphene with well conductivity will enhance the electrically conducting as charge transfer channel, so that electrons will be transfer much faster in oxidation and most Bi (III) is oxidized to be Bi (IV) which ensure larger specific capacitance and long cycle life. This nano-Bi2WO6/reduced graphene oxide composite has application prospect in high-performance pseudo-capacitors.The nano-Bi Energy storage and conversion from semiconductor and alternative energy sources have stimulated a large of research due to the urgency demand for energy. Photovoltaic cells, batteries, fuel cells, and supercapacitors are mainly Energy storage and conversion devises1234678910121315161718192022232WO6 is a kind of Aurivillius phase oxide with a layered structure of the perovskite layer (WO4)2− lies between (Bi2O2)2+ layers2WO6 has excellent physical and chemical properties such as oxide anion conducting, a nonlinear dielectric susceptibility, ferroelectric piezoelectricity, pyroelectricity, catalytic behavior and luminescent properties3839402WO6-grphene has been prepared and the photocatalytic performance was shown42432WO6-grphene as pseudocapacitor electrodes has not been reported.Graphene is a two-dimensional material with an ideal single-atom-thick or several layers of limited structure, and it has attracted much attention recently due to its many potential applications in electronic, optical, catalytic and energy fields25262729303233343533342WO6-graphene composite which demonstrate larger specific capacitance, higher energy density and longer cycle life. As comparison, plain nano-Bi2WO6 has poor specific capacitance and weak cycle life due to the low electric conduction resulted in most Bi (III) will be over oxidized to be Bi (V) which causes nonreversible destroy on structure. In this nano-Bi2WO6-graphene, graphene will render the electrically conducting as charge transfer channel. In this case, Bi (III) is more inclined to be Bi (IV) in charging instead of Bi (V) which ensure the material has long cycle life.In this work, we reported a simple method to obtain nano-Bi2WO6 phase with orthorhombic russellite structure. The lattice parameter values deduced from the XRD peaks are in concordant with the standard JCPDS data file no 73-2020. No other matter is found confirmed the hydrothermal reaction occurred completely. Moreover, except there is little enhancement of crystallinity (about 10%), two XRD patterns of the BWO and BWO/RGO are almost the same which indicates that although under an extra hydrothermal progress, the BWO nanoparticles in the BWO/RGO composites have less change compared with pristine BWO. It seems that when exceeding 24 h, extra time for hydrothermal reaction has little influence on the structure of BWO.The BWO was obtained by a hydrothermal route while the BWO/RGO was synthesized via a two-step method with two hydrothermal progress. The crystal structure of BWO and BWO/RGO were studied by XRD analysis in 2WO6.The TEM and HRTEM images of BWO and BWO/RGO are shown in −1, D peak at 1360 cm−1. In general, the value of Id/Ig ratio reflects the surface defects density of graphene, which also give an information about the degree of reduction. From d/Ig = 1.08) than RGO (Id/Ig = 0.91). The results could confirm that the GO was reduced greatly after hydrothermal treatment.As compared, another composite was obtained by a hydrothermal reaction between reduced graphene oxide (RGO) and BWO nanoparticles. As 2WO6 in the curves which indicates the pseudo capacitive behavior and these redox peaks are due to the oxidation and reduction reactions occurred in the material. The background signal due to the Ni foam was negligibleCyclic voltammetry (CV) is the most important measurement in electrochemistry which provides the qualitative information regarding the electrochemical processes that takes place in the material. The CV curves of the BWO at various scan rates are shown in 2WO6/reduced graphene oxide composite material for high-performance pseudo-capacitors.The BWO/RGO composites were carried out similar electrochemical measurements. In the CV curves , the ave46The BWO/RGO exhibits excellent electrochemical characteristics and high cycling stability, making that potentially useful for high performance supercapacitor material. In same CV or galvanostatic method in three-electrode systems, the BWO/RGO composites show higher stable specific capacitance at higher charge/discharge rates than pristine BWO nanoparticles. There are several features lead to higher capacity and faster energy storage and releasing of BWO/RGO. First, the BWO nanoparticles are directly grown and anchored on reduced graphene oxide sheets after the hydrothermal reaction. The interactions between BWO nanoparticles and RGO could be both covalent chemical bonding and van der Waals interactions. This intimate binding offers facile electron transport between individual nanoparticles and RGO. Although BWO nanoparticles are electrically insulating, the RGO is well conductor and can transfer the electrons of BWO in redox which is key to both high specific capacitance and rate capability of the BWO/RGO. Most of the BWO nanoparticles in the system are electrochemically active through the graphene network. Rapid charge transport from BWO nanoparticles to the underlying RGO provides fast redox reactions. The better conductivity of BWO/RGO can be confirmed by the Electrochemical impedance spectroscopy (EIS) . In the 7/2 and 4f5/2 statesThe decline of columbic efficiency for BWO in the cycle of charge and discharge can be attributed to the irreversible oxidation in the redox reactions. A comparison of CV curves for BWO and BWO/RGO is shown in 2WO6/reduced graphene oxide composite which demonstrate larger specific capacitance, higher energy density and longer cycle life. As comparison, pristine Bi2WO6 nanoparticles have poor specific capacitance and weak cycle life due to the low electric conduction and over oxidation of Bi (III) to Bi (V) which causes nonreversible destroy on structure. In this nano-Bi2WO6/reduced graphene oxide composite, graphene with well conductivity will enhance the electrically conducting as charge transfer channel. In this composite, electrons will be transfer timely in oxidation and most Bi (III) is oxidized to be Bi (IV) which ensure long cycle life.In this work, we reported a simple method to obtain nano-Bi2WO6 (BWO) nanoparticles were prepared via a solvothermal method. A typical process is as following: 0.97 g Bi(NO3)3·5H2O and 0.33 g Na2WO4·2H2O were dissolved in 40 ml ethylene alcohol, after being stirred for 6 h, the transparent solution was added into a 50 ml Teflon-lined autoclave up to 80% of the total volume. Then the autoclave was sealed in a stainless steel tank and heated at 180°C for 24 h, in which the heating rate is 1°C/min. Subsequently, the reactor was cooled to room temperature naturally, the products were washed by de-ionized water and alcohol in turn and then the BWO nanoparticles were obtained after being dried at 80°C in air.The Bi2WO6/reduced graphene oxide composite is labeled as BWO/RGO. As compared, reduced graphene oxide (RGO) was obtained under a similar hydrothermal progress without adding of BWO.Graphene oxide (GO) was prepared from natural graphite  by a modified Hummers method−1), and the exposure time was 2 s.The crystal structures of the samples were characterized by using an X-ray diffractometer (XRD)  with Cu-Ka radiation. The microstructures were observed by using a high-resolution transmission electron microscope (HRTEM) . X-ray photoelectron spectroscopy (XPS) measurements were made on a Kratos AXIS Ultra DLD  spectrometer with a charge neutralizer to gain information on the chemical binding energy of the samples. The C 1s peak at 284.6 eV of the adventitious carbon was referenced to rectify the binding energies. Raman measurement was carried out using a Raman spectroscopy . The power of the laser was 10 mW, and the laser excitation was 488 nm. Scans were taken on an extended range , Galvanostatic discharge and Electrochemical impedance spectroscopy (EIS) measurements on a commercial electrochemical workstation with three electrode system. Electrodes made from carbon materials worked as the working electrode, Ag/AgCl electrode as the reference electrode, and platinum network electrode as the counter electrode, the electrolyte is the 6 M KOH solution.The working electrode was prepared by coating the active material onto nickel foam (1 cm × 1 cm) followed by pressing with a pressure of 0.5 tons. While the active material was the mixture of BWO or BWO/RGO, carbon black and PDFE binder (ratio 7:2:1). Before the coating, the mixture was ground in a mortar using a pestle, and the Grinding time was one hour. Then, the samples were uniformly distributed upon the Ni foam and allowed to dry in a vacuum oven for 2 hours. The end loading of active material for each electrode was 3 ~ 5 mg cmJ.Z. proposed and designed the experiments. P.L., G.X., Z.L., X.W.,Y.W., L.Y. and X.T. carried out the experiments  and characterizations. J.Z. wrote the paper. Y.Z., H.W., E.Z. and Z.J. provide scientific advice. J.X. conducted the SEM and HRTEM measurements, respectively. All authors read and approved the final manuscript.Supporting Information