Here, we elucidate the immunological features of both bronchoalveolar lavage fluid (BALF) and vitreous lavage fluid (VLF) samples from patients with histopathologically verified sarcoidosis. In addition, we assess the safety of vitrectomy in sarcoidosis patients by investigating the occurrence of complications and the recovery of visual acuity.Twenty-two eyes of 22 patients with tissue-proven sarcoidosis were enrolled in this study. BALF and VLF samples were obtained and compared in each patient, and the clinical course  was followed. The presence of sarcoidosis was assessed with a flow cytometric analysis of T-lymphocytes in the BALF and VLF samples.Our results indicated that the CD4 T-cell population and the CD4/CD8 ratio were significantly higher in the VLF T-lymphocytes than the BALF T-lymphocytes. On the other hand, the CD8+ T-cell population was significantly lower in the VLF T-lymphocytes.Therefore, our findings suggest that VLF samples have a high diagnostic value  for sarcoidosis. Moreover, we found that the sample collection did not affect visual acuity and that there were no adverse events after surgery. A flow cytometric analysis of a VLF sample may therefore be a useful adjunct in the diagnosis of sarcoidosis. The most common signs of sarcoidosis in clinical examinations are therefore usually related to the respiratory system. Abnormal chest radiography findings are especially common, including findings of bilateral hilar lymphadenopathy and/or pulmonary infiltration. Previous reports have shown that fiber optic bronchoscopy can be used to obtain transbronchial lung biopsies for the histological diagnosis of sarcoidosis.,5 The basic diagnostic criteria for sarcoidosis include the histopathological verification of noncaseating epithelioid cell granulomas and the exclusion of other diseases with granuloma formation reactions (such as tuberculosis).Sarcoidosis is an idiopathic inflammatory disorder characterized by noncaseating epithelioid cell granulomas in multiple tissues and organs, including the eye. It has been reported that an increased CD4+ helper T-cell lymphocyte subset or a CD4/CD8 ratio greater than 3.5 in bronchoalveolar lavage fluid (BALF) is helpful in the diagnosis of sarcoidosis. BALF lymphocytosis  and other laboratory features highly consistent with sarcoidosis, such as high serum levels of angiotensin-converting enzyme (ACE), can be reliable indicators when diagnosing sarcoidosis in cases where histology is not available.,8 Moreover, our previous results indicated that the T-lymphocyte population in the vitreous lavage fluid (VLF) also has a high diagnostic value for diagnosing ocular sarcoidosis.Immunological findings from clinical examinations are also important for diagnosing sarcoidosis.–13 and that bilateral anterior or posterior uveitis are also common. The clinical appearance of sarcoidosis uveitis is characterized by iris nodules, mutton-fat keratic precipitates, and tent-shaped peripheral anterior synechiae in the anterior segment. In the posterior segment, phlebitis and vitreitis with snowball-like vitreous opacities are common findings in sarcoidosis uveitis. Moreover, persistent ocular chronic inflammation can cause epiretinal membrane and cystoid macular edema, leading to severe visual impairment.,16 Internationally acknowledged criteria have been established for the diagnosis of sarcoidosis with ocular involvement. Although it is generally not difficult to diagnose ocular sarcoidosis with typical clinical findings, a considerable number of sarcoidosis cases present with nonspecific vitreous opacities. In these cases, differential diagnosis can be difficult. Moreover, analysis of VLF samples from sarcoidosis patients is a safe and useful way of diagnosing sarcoidosis uveitis with vitreal opacities. Flow cytometric analysis has also been reported to be useful in diagnosing uveitis, especially in sarcoidosis. Therefore, in the present study, we attempted to determine whether the diagnostic value of vitreous-infiltrating T-lymphocytes for sarcoidosis could be considered to be as high as that of BALF lymphocytosis.It has been reported that 30% to 60% of sarcoidosis patients develop ocular lesions2Patients who visited the uveitis clinic at Tohoku University Hospital or Kyoto Prefectural University from 2008 to 2015 and met the inclusion criteria were invited to participate in this study. This prospective study was approved by the Institutional Review Board of Tohoku University Graduate School of Medicine and Kyoto Prefectural University. All experimental procedures were conducted in accordance with the tenets set forth in the Declaration of Helsinki. The present study was registered in the University Hospital Medical Information Network Clinical Trial Registry. The purpose of the research and the experimental protocols were explained in detail to all patients, and their informed consent was obtained before participation in this study.2.1 The patients included cases of uveitis with visual disturbance due to epiretinal membrane. All patients agreed to undergo pars plana vitrectomy (complete vitrectomy) and take part in the study. Patients were excluded if they had any prior history of intraocular surgery, especially vitreous surgery.A consecutive series of 22 eyes with uveitis of 22 patients were enrolled; all eyes had received a definitive, tissue-proven, diagnosis of sarcoidosis, confirmed according to international diagnostic criteria.The patients in this study who were diagnosed histologically had positive biopsy results with suggestive intraocular signs and 2 positive investigational tests . Most patients underwent BALF examinations before the ocular surgery. In all cases except 2, the patients underwent BALF examinations before receiving any steroid treatment.2.2 A 3-way stopcock was attached at the point the suction tube line connects to the cutter probe, and a 10-mL syringe was then attached to the free end of the 3-way stopcock. Dry vitrectomy was then performed without balanced salt solution  perfusion. A cutting rate of 500 cpm was used to avoid damaging the cells infiltrating the vitreous. After a 1.5- to 3-mL sample of pure vitreous fluid was collected, the normal cutting rate vitrectomy (5000 rpm) continued with BSS perfusion. All of the obtained samples were promptly stored at 4 °C and brought to the clinical laboratory of the University hospital for cell analysis (including flow cytometry). All samples were immediately analyzed using the method described below.At the start of a conventional 25-ga pars plana vitrectomy, a vitreous specimen was obtained from each patient. Typical presentations included severe vitreous opacity with retinal vasculitis Fig. A and B, 2.3At the beginning of the procedure, first the upper and then the lower airways were anesthetized. After confirming the effect of the anesthesia, bronchoalveolar lavage was performed by instilling and immediately retrieving 3 50-mL aliquots of warmed normal saline. Lavage was performed in only 1 site, either the right middle lobe or lingula. The quantity of recovered fluid was generally about 50% of the quantity of the initial injected fluid. This method follows previous reports.2.4 the VLF and BALF samples were filtered with a 70-μm cell strainer . The samples were then washed and resuspended with phosphate-buffered saline  containing 2% bovine serum albumin  and 0.1% sodium azide  at a final volume of 0.5 mL. For the flow cytometric analysis, 0.1 mL of each sample was incubated with one of the following monoclonal antibody mixtures: fluorescein isothiocyanate (FITC)-conjugated mouse Immunoglobulin (Ig)G1 /phycoerythrin (PE)-conjugated mouse IgG1 (Dako)/PE-Cy-Chrome5-conjugated anti-CD45 , anti-CD3-FITC /anti-CD19-PE /anti-CD45-Cy5 , or anti-CD4-FITC /anti-CD8-PE /anti-CD3-Cy5 . After a 15-minute incubation period at room temperature in complete darkness, the samples were again washed and resuspended at a final volume of 0.5 mL. Lymphocyte subsets of the VLF and BALF samples were examined with an Epics XL-MCL flow cytometer (Beckman Coulter). CD45+ cells were initially counted on CD45 versus side-scatter plots combined with forward scatter and side scatter. If the counts determined that more than 100, CD45+ cells were contained in 1 test tube, further analysis was then performed to determine the T-cell /B-cell (CD19+ and CD45+) ratio, as well as the CD3+, CD4+ T-cell/CD3+, CD8+ T-cell ratio. The remaining portion (0.2 mL) of the resuspended VLF and BALF samples were prepared for cytology with a cytocentrifuge .Using a method we have previously reported,Flow cytometry was also used to study the BALF lymphocyte population. The specimens were washed and then resuspended at a final volume of 0.5 mL/tube.2.5All patients underwent vitreous surgery; visual acuity was analyzed both before and after surgery. All patients were followed at the outpatient clinic of our facility for at least 1 month postoperatively. Visual acuity was measured at every visit as the logarithm of the minimum angle of resolution (log MAR). Previtrectomy and postvitrectomy measurements were compared. Postoperative visual acuity was recorded as the last measurement obtained in the outpatient clinic (the average observation period was 20.5 ± 14.8 months). The patients were also monitored for adverse events after vitreous surgery.2.6+ and CD8+ T-cell population and of the CD4/CD8 ratio were carried out with a nonparametric analysis based on the Mann–Whitney test (significant differences were defined as P < 0.05). Analyses were performed with Prism software, version 5.0.1 . Visual acuity before and after vitrectomy was analyzed using the paired t test. These analyses were performed with Prism software, version 5.0.1 (GraphPad Software).Between-group statistical comparisons of the CD433.1+ cells. It is commonly known that the vitreous cavity contains only 4 to 5 mL of VLF in adults, meaning that the volume of the VLF samples was necessarily less than the BALF samples. This risked affecting the validity of the comparisons, prompting us to use a perfusion system in the vitreous cavity to collect a sufficient number of cells during vitrectomy. Both the VLF . The mean age was 67.6 ± 5.4 years. We analyzed flow cytometry data from the BALF and VLF samples in order to search for differences in the cell populations of these 2 fluids (Table 3.2P = 0.0006) than the ratio of the lymphocytes in the BALF samples  than the BALF samples.Flow cytometric data for the CD4 lymphocytes, CD8 lymphocytes, and CD4/CD8 ratio are summarized in Table les Fig. A. Flow cP = 0.0018) than the BALF samples.Flow cytometric data for the CD8 lymphocytes are summarized in Fig. 3.3P < 0.003)  than before  into the vitreous cavity, after which the cavity was washed and the redundant triamcinolone removed. Finally, a sub-Tenon injection of 20 mg of triamcinolone was performed to prevent complications. Vitrectomy has thus become a worthwhile and safe diagnostic tool for uveitis. Indeed, our present results indicated that most of the patients who underwent vitrectomy had improved visual acuity and no severe complications, with only a single patient experiencing a loss of visual acuity due to continuous cystoid macular edema before surgery. Therefore, the clinical course was favorable in most patients involved in this study, with no adverse events resulting from any of the surgical procedures, including the collection of the vitreous samples. Therefore, modern 25-ga vitrectomy, such as that used in the present study, can be considered a safe way to perform sample collection in uveitis patients, especially in sarcoidosis patients.According to the American Thoracic Society, bronchoalveolar lavage causes no complications or adverse events in up to 95% of cases. However, complications and adverse events such as cough, transient fever, transient chills or myalgia, transient infiltration, bronchospasm, transient reductions of lung function, and transient decreases in baseline partial pressure of O At the present time, a diagnosis of sarcoidosis with ocular manifestation requires additional systemic features for confirmation. However, a systemic search reveals considerable numbers of suspected ocular sarcoidosis cases with negative results. Transbronchial lung biopsies and bronchoalveolar lavage are said to be relatively less-invasive procedures, yet there is ongoing disagreement as to whether they should be performed on patients without clinical or radiological pulmonary presentation. The present results show that an elevated CD4/CD8 ratio and CD4 lymphocyte population represent focal lymphocytosis characteristic of ocular sarcoidosis. This immunological resemblance between VLF and BALF should become an important future part of the diagnosis of sarcoidosis since it is not observed in other types of uveitis.Previously, it was reported that in Japanese patients, ocular sarcoidosis symptoms are the most frequently seen symptom at first presentation.It remains important to distinguish between pulmonary tuberculosis and sarcoidosis, because the CD4/CD8 ratio in the BALF of tuberculosis patient has also been reported to increase. Clinically, the symptoms of tuberculosis uveitis resemble those of sarcoidosis uveitis, including such features as retinal vasculitis and retinal exudate. In the present paper, we used a tuberculin test to screen for tuberculosis in all patients and obtained negative results. However, the results of a tuberculin test are not enough by themselves to rule out tuberculosis. Therefore, we also performed a PCR analysis for tuberculosis DNA in the vitreous samples. Moreover, in patients with relatively high positive results in the tuberculin test, we also performed a T-spot analysis. The Japanese setting of this study provided few opportunities to examine tuberculosis uveitis patients undergoing vitrectomy. Nevertheless, if possible, we would like to include such patients in the future research in order to report their CD4/CD8 ratio.In conclusion, we believe that the testing methodology and immunological information reported here constitute an effective new way to diagnose sarcoidosis in cases with an active lesion localized in the eye and that in the future this method should enable earlier and less-invasive diagnosis of this ocular inflammatory disorder.We would like to thank Dr Wendy Chao for editing and critical reading of this manuscript. The authors thank Drs Hiroshi Kunikata, Kenji Nagata, Kazuhito Yoneda, and Kentaro Kojima for the helpful sample collections.


We study how double-stranded DNA translocates through graphene nanogaps. Nanogaps are fabricated with a novel capillary-force induced graphene nanogap formation technique. DNA translocation signatures for nanogaps are qualitatively different from those obtained with circular nanopores, owing to the distinct shape of the gaps discussed here. Translocation time and conductance values vary by ∼ 100%, which we suggest are caused by local gap width variations. We also observe exponentially relaxing current traces. We suggest that slow relaxation of the graphene membrane following DNA translocation may be responsible. We conclude that DNA-graphene interactions are important, and need to be considered for graphene-nanogap based devices. This work further opens up new avenues for direct read of single molecule activitities, and possibly sequencing. Graphene nanogaps are a promising candidate for such a sequencing device [Solid-state and biological nanopores hold great promise as analytical single-molecule tools . They eng device –20.Here, we present the first studies of double-stranded DNA (dsDNA) translocating through graphene nanogaps. The translocation signatures differ significantly from those found in other solid-state and biological nanopores, owing to the unique properties of these graphene nanogaps, and unique DNA-graphene nanogap interactions. Our nanogap formation procedure is based on capillary-force-induced breaking that can be controlled down to a few nanometers.We fabricate graphene nanogaps and demonstrate that DNA is able to translocate through them .2 wafers as described before [2 layer that has been treated with a 5 min long O2 plasma to promote graphene adhesion. A single-layer graphene sheet is identified by its contrast in an optical microscope.Graphene is deposited on Si/SiOd before . A 5–10 2 wafers with trenches and buried metal alignment markers are fabricated using electron-beam lithography (EBL), processing, and etching. Briefly, PMMA is patterned by writing with an electron beam and subsequent development in a 1:3 mixture of Methyl Isobutyl Ketone (MIBK) and Isopropyl Alcohol (IPA). We thereby created a 20 × 20 μm2 square mask, which we etch for 30 s in buffered oxide etchant . A 25 nm thin layer of Au is evaporated into the squares after a 3 nm Cr adhesion layer, followed by liftoff in 80°C acetone. A second layer of PMMA resist is applied, followed by EBL-definition of 1 × 100 μm2 trenches and development. The trenches are made ∼ 150 nm deep by subsequent etching in BHF.Si/SiOet al. [2 gas. The capillary forces of the drying IPA break the graphene sheet inside of the trench, thus forming a graphene nanogap [2 substrate, and can be controlled down to a few nanometers, as we describe below.Exfoliated single-layer graphene sheets are transferred onto the trenches using a wedge-transfer technique  developeet al. . Graphen nanogap . The sizTo create a narrow channel to guide the DNA to the nanogap, and to block any other potential holes that may have been accidentally created, a ∼ μm-size pore in PMMA, “micropore”, is fabricated on top of the graphene nanogap . The cooThe assembly is mounted in a fluid cell with a saline solution of 10 mM Tris acetate, 1 mM Ethylenediaminetetraacetic acid (EDTA) and 0.1 M KCl. Ag/AgCl electrodes are mounted on either side, and a ∼ 20 mV bias voltage is applied. The nanogaps used here can be expected to have a larger cross section and a corresponding larger conductance than the graphene nanopores reported earlier –15. In oDouble-stranded DNA  is introduced on the cis side, and upon translocation through the nanogap, causes abrupt changes in ion-current.EB with a surface binding energy gain ES . A linear fit of zSEM vs zOPT yields a best fit linear transform zSEM = azOPT + b. This fit simultaneously records offset b, scale |a|, and rotation arg(a). The error after the fit zSEM − azOPT − b is analyzed  is used to make a micropore in the PMMA above the graphene sheet. In order to locate the sheet with high accuracy, we calibrated the SEM beam deflection as a function of applied deflector voltage. A pattern of prefabricated Au square markers was imaged in an optical microscope  and the analyzed . A smallσ is calculated. Candidate events are determined as either positive or negative peaks exceeding 5σ. The area under the peak is calculated to determine the statistical significance of the candidate event. The threshold for calling it an ‘event’ is adjusted until the control experiments do not yield false positives anymore. We include particularly noisy control traces such as the black trace in unfiltered candidate events are fitted by a least squares method to both an exponentially decaying function and a rectangular function. We fit the unfiltered data to avoid distortions to the event shape by the FFT filtering and background subtraction. The squared difference between fit and data for both is used to determine whether the event better fits a rectangular or exponential event, and it is classified as such. The resulting fits are recorded and zoomed plots are generated  = exp(std(log(x))) in both ΔG and τ of σg ∼ 2. For all rectangular events, we find that the conductance increases during the event, i.e. ΔG > 0.We observe two types of events. Firstly, we observe rectangular events, where the current briefly changes from the baseline current , top. Th= 0.7 ms . These eτ ranging from ∼ 1 to 100 ms  to the low salt concentration that we employed in these experiments. In Si-based semiconducting nanopores, a crossover between ΔG > 0 to ΔG < 0 occurs at a salt concentration of 0.4 M [G > 0 at a concentration of 0.1 M is consistent with that picture and leads us to conclude that the conductance change is dominated by counterion current along the DNA molecule.We attribute the positive nature of these translocation events , γ in the viscous damping acting on the graphene by water, k is the equivalent spring constant of the graphene, and t is time.We will show that the long relaxation times measured in our experiments are consistent with the dynamics that would occur for a graphene sheet returning to equilibrium after being initially displaced by the DNA passing through the gap. We represent the motion of the fundamental mode of oscillation of the graphene membrane as a simple harmonic oscillator asFor this system, it is straightforward to show that the Reynolds number of the fluid flow is very small Re ≪ 1 which indicates the dominance of viscous effects over inertia. In light of this, we expect the graphene membrane to act as an overdamped oscillator after being displaced which has been demonstrated for nanoscale cantilevers in fluid .x0 = F0/k where F0 is an applied force, the return to equilibrium of the graphene membrane can be expressed as [Q = mfωf/γ is the quality factor and ωf is the resonant frequency of the graphene membrane when immersed in the fluid. For nanoscale oscillating objects in fluid ωf ≪ ω0 where ω0 is the resonant frequency in a vacuum.If we assume that the initial displacement of the graphene membrane is essed as x(t)=x0λQ we have |λ−| ≫ |λ+| where λ+ ∼ −ωfQ and λ− ∼ −ωf/Q and the large-t response is dominated by λ+. The solution can then be represented as x(t) = x0eλ+t = x0et/τ− where τ is the relaxation time and is given by τ = (ωfQ)−1.For small et al. [Q, ωf (and therefore τ) given only the values of frequency parameter R0, mass loading parameter T0, and the resonant frequency in vacuum ω0.If we assume that the membrane dynamics are similar to that of a wide elastic cantilever in fluid we can make the following analytical predictions to suggest the order of magnitude of the expected response for the graphene membrane. Using the approach described in Paul et al.  we can pE = 1 × 1012 N/m2 and a density of ρg = 2000 kg/m3. For water we will use a density of ρf = 1000 kg/m3 and a dynamic viscosity of μf = 1 × 10−3 kg/m ⋅ s. We next assume the membrane can be represented as a cantilever with a length L ≈ 1 μm, width W ≈ L/2, and a thickness of H ≈ 0.3 nm. Using these values yields an equivalent spring constant of k ≈ 3.4 μN/m and a resonant frequency in vacuum of ω0 ≈ 6.8 × 106rad/s.For the graphene membrane we will use a Young’s modulus of R0 = ρfω0W2/(4μf) = 0.43 and a mass loading parameter of T0 = πρfW/(4ρH) = 655. Given these values for R0, T0, and ω0 the analytical expressions of Paul et al. [Q ≈ 0.17 and ωf ≈ 1694 rad/s. Using this with our expression for the decay time yields τ ≈ 3 ms. This is commensurate with the long relaxation times that are measured in the experiments. We emphasize that these estimates are approximate and a more accurate analysis would require numerical simulations for the precise conditions of the experiment. In addition, the predicted value of the relaxation time from our approximate analysis is sensitive to the chosen geometry of the membrane. For example, as the length or width of the membrane becomes larger the decay time will increase.These values yield a frequency parameter l et al.  yield Q We have demonstrated translocation of DNA molecules through graphene nanogaps and observed signatures that have features different from those observed with circular solid-state nanopores. We argue that these derive from the DNA interacting with the unique nanogap geometry and we conclude that DNA-graphene gap interactions are important and need to be included in a realistic design of graphene-nanogap based sequencing devices. This study opens up new advancement in single molecule genomic screening devices, and DNA sequencing.S1 FileThis is the data described in the manuscript.(TGZ)Click here for additional data file.


The advantages conferred by the physical, optical and electrochemical properties of graphene-based nanomaterials have contributed to the current variety of ultrasensitive and selective biosensor devices. In this review, we present the points of view on the intrinsic properties of graphene and its surface engineering concerned with the transduction mechanisms in biosensing applications. We explain practical synthesis techniques along with prospective properties of the graphene-based materials, which include the pristine graphene and functionalized graphene , reduced graphene oxide (RGO) and graphene quantum dot (GQD). The biosensing mechanisms based on the utilization of the charge interactions with biomolecules and/or nanoparticle interactions and sensing platforms are also discussed, and the importance of surface functionalization in recent up-to-date biosensors for biological and medical applications. A biosensor is an analytical device which can detect a biomolecule-related element with an appropriate transducer to generate a measurable signal from the sample ,2. The b2-bonded carbon atoms occurring within a carbon material structure” [2/g) [The emergence of graphene, a two-dimensional (2D) nanomaterial, has played a tremendous role in the electronic and sensor communities. Graphene is defined as “a single-atom-thick sheet of hexagonally arranged, spructure” . The nane” [2/g) ,29 beinge” [2/g) . Graphene” [2/g) ,31.The uses of graphene-based materials for biosensing involve two points of view. One is based on charge-biomolecule interactions at π-π domains, electrostatic forces and charge exchange leading to electrical variations in the pristine graphene. The other uses are the effect of defects, disorder, the chemical functionalization to immobilize the molecular receptors onto the surface of graphene oxide (GO), reduced graphene oxide (RGO) and graphene-based quantum dots (GQDs). Recently, several excellent reviews have focused on the interactions of graphene, GO and RGO-based biosensors with their biomolecular targets ,32,33,342-hybridization of pure carbon atoms arranged in a hexagonal lattice with covalent bonds. Meanwhile, functionalized graphenes are achieved by synthesis and preparation, for examples, the carbon core structure can be oxidized forming GO, the reduced structure with vacancy defects is RGO, structures a few nanometers in size with quantum phenomena are GQDs. Interestingly, many types of the GQDs have attracted interest due to their outstanding optical characteristics in the presence of photoluminescence involving specific binding capability to a wide range of biomolecules. Such morphological and intrinsic characteristics should serve the analytical transduction of biosensors on the limit of detection (LOD), sensitivity, selectivity, repeatability, and biocompatibility.The synthesis of graphene-based materials in a presence of different methods can be controlled to confer properties for specific and desirable applications. To provide an understanding of the physics, chemistry, and engineering of graphene-based structures, and how these methods have crucial influences on their characteristic and properties, we will first discuss how graphene and related materials are prepared and synthesized.2 [6 m/s. The GFET can even be retained under ambient circumstances without any protection of the graphene. The charge carriers exhibit ultra-high mobility, found to be 200,000 cm2/Vs, at temperatures between 10–100 K, which can be induced by applying gate voltage, a strong ambipolar electric field effects in charge carrier concentrations at the range of 1013 cm−2 with a long mean free path of 400 nm, high thermal conductivity, very low resistivity (less than that of silver at room temperature), low spin-orbit coupling, high mechanical strength (200 times greater than stainless steel), high elasticity, transparent (98% transmittance) and biocompatibility [A monocrystalline graphitic film composed of a single one-atom-thick carbon layer can be prepared by peeling it from a small platelet of highly oriented pyrolytic graphite (HOPG) in a mechanical exfoliation approach with a Scotch tape, as seen in 2 . When thtibility ,37,38. A2. Several synthetic procedures have been established that possess scalability in their different characteristics and are more effective for matching applications.However, to obtain graphene by the Scotch-tape method, the maximum dimensions of the pristine graphene that can be isolated for fundamental studies and basic research that require high quality at only 1 mmChemical vapor deposition (CVD) is one of the most popular methods for industrial-scale fabrication of graphene at this moment. Even though graphene provided by this method is very high quality and the number of layers can be precisely controlled, the electronic and structural properties has remained poorer than those of the Scotch-tape graphene. Excellent applications of CVD graphene have been performed in many fields, including electronic transistors ,44, tranN-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), γ-butyrolactone (GBL) and 1,3-dimethyl-2-imidazolidinone (DMEU) [Liquid exfoliation is a low-cost top-down process for fabricating graphene in solution using an ultrasonic energy source to generate microcavitations and to blast the raw bulk graphite into smaller fragments and thinner layers of graphene. A number of hours are needed to effectively extract the graphite into individual layers. The approach of the liquid exfoliation is carried out in organic solvents such as e (DMEU) ,57,58,59e (DMEU) . The dis2/Si wafer. The resulting graphene layer however, has high amounts of defects with very low mobility of 100 cm2/Vs [A different method starts with silicon carbide (SiC) heated up to 1250–1450 °C under ultrahigh vacuum (UHV) as seen in 0 cm2/Vs . This me0 cm2/Vs ,62. The 0 cm2/Vs ,64. Ther0 cm2/Vs . The oth0 cm2/Vs ,67. The iodicity . The utiiodicity . AlthougChemical synthesis is a cost-effective top-down nanotechnique which utilizes oxidation and reduction reactions to indirectly synthesize graphene. In fact, chemical synthesis was the first method to yield a single layer hexagonal carbon structure, but the existence of a 2D layer has not been reported. In 2004, the experimental breakthrough of graphene allowed exploration of the evidence of its extraordinary properties. Since then, the chemical method has come back to be realized in large throughput synthesis. Graphene can be facilitated via the process involving oxidation of fine graphite to graphite oxide (GpO), which is then exfoliated and reduced  or irradiated to RGO. (1)Brodie method (1859) , the GpO(2)Staudenmaier method (1898) , this me(3)The Hummers and Offeman method (1958)  was deveThere are three original methods used to synthesize graphene, including:13C-NMR [As a result of the oxidation of graphite there is an increase in the interlayer spacing. This means that the separation between graphene interlayers is expanded by the intercalation of oxidized contents or the polar liquids such as sodium hydroxide  as seen 13C-NMR . The int13C-NMR . The amo13C-NMR ,76.2-hybridized carbon structure. The molecular structure is formulated as C2O. In 1947, Ruess [3-hybridized carbon structure as the basal plane whose surface is decorated with the hydroxyl (C-OH) groups. 1,3-Epoxides were placed at 1/4th of the cyclohexanes. Scholz and Boehm [2F)n. The model showed hydroxylated graphite intercalation along the c-axis. There were carbonyl (C=O) and hydroxyl (C-OH) groups which were bonded to the basal carbon structure. The molecular formula was intermediate between C8(OH)4 and C8O2 with C0 = 8.22 × 2 Å and C0 = 5.52 × 2 Å, respectively. The increase in the dehydration was influential in decreasing the distance along the c-axis. This model was deduced from the elemental analysis, X-ray diffraction and chemical reactivity studies. The most well-known and widely used model is the one proposed by Lerf and Klinowski in 1996 [13C and 1H magnetic-angle spinning nuclear magnetic resonance (NMR) data of GpO. The evidence suggested aliphatic 6-membered rings containing hydroxyl (C-OH), epoxide (C-O-C) and double-bonded carbon (>C=C<) which indicated a basal plane structure made up of aromatic islands. The basal carbon structure was not only separated by the C-OH attachments leading to wrapping, but the graphite oxide structure was reconfigured to be tetrahedral. This leads to the strong interaction with water molecules. In this model, the 1,2 epoxide and hydroxyl groups are at the basal plane while the carboxyl and hydroxyl groups are mostly at the edge of the plane. Observations [2 hybridized structure is found as a core of the graphite oxide sheet; (2) holes, a kind of defect, are present in the structure; (3) C-OH, 1,2-C-O-C groups are doped onto the structure; (4) the C=O groups are found along the edge of the holes; and (5) the oxidized regions are randomly located and no superstructures are formed. Another model was proposed by Dékány [13C-MAS NMR were used to reveal the structural details. The Dékány model was built upon the Ruess and Scholz-Boehm models. The graphite oxide contained trans-linked cyclohexyl chairs, having 1,3-epoxides and tertiary hydroxyls, and corrugated networks of keto/quinoidal groups. In addition, phenolic groups were inserted into this model to explain the acidity of graphite oxide. Two years later in 2008, Ruoff and colleagues [13C-labelled graphite oxide. Using 13C-MAS NMR analysis, hydroxyl and epoxide groups were found bonded to the carbon structure. Also, the carbonyl groups were found mostly bonded to the edge of defects. The carbon structure had non-protonated functionality as detected near 100 ppm. This was narrowed down to the possible structure of graphite detected by NMR. In 2009, Gao and Ajayan [13C-MAS NMR signals. The functional groups on graphite oxide included the 115 (hydroxyl and epoxide), the 3 , the 63 (sp2 hybridized carbon), the 10 (lactol:ester:acid carbonyl) and 9 (ketone carbonyl). Moreover, the GpO and GO are seen to have the acidic properties due to the ketones at the edge of carbon plane in a presence of incompletely hydrolyzed covalent sulphates, containing salts or esters of sulfuric acid. In contrast to that of usual acid properties, it originates from the presence of carboxyl groups. In fact, the acidic functional groups, i.e., vinylogous acid, on GpO and GO are originated gradually due to the interaction with water. This points out the instability of the oxide functional groups in the presence of water, leading to constantly evolving GpO and GO structures. Therefore the latest model has been proposed in terms of the dynamic-like structure.Understanding of the GpO structure is needed in order to develop modifications which would improve the mechanisms in any applications. Primarily, the GpO is composed of C, O and H atoms where the C-to-O ratio for complete oxidation is in a range of 1.5–2.5 . To mode7, Ruess  proposednd Boehm  suggestend Boehm  proposed in 1996 ,79. Theirvations  supportey Dékány  as recenlleagues  synthesid Ajayan  offered 2H4) at 1.0 mL (Mw 32.1 mmol). The reduction was carried out at 100 °C through a one-step refluxing process overnight. After the reduction, the GO turns into a black solid and precipitates gradually. The RGO is then repeatedly rinsed with water and ethanol and filtered, then air dried to get the solid product. The exfoliated GpO or GO in water exhibits hydrophilic behavior due to the oxygen contents as mentioned previously, but the RGO aggregates together due to the fact that the chemical linkages inside the interlayers are removed, thus the RGO sheets become highly hydrophobic and shrink together. More importantly, the structural characterization can be verified by Raman microscopy for the first-order scattering of E2g mode (G) and the extensive oxidation in-plane sp2-domains. It should be pointed out that not only the hydrazine hydrate is used in the reduction of GO, but the number of reducing agents known to date has increased, as well. The reducing agents can be categorized for creating good supports and for doping purposes. For instance, borohydrides, aluminium, hydride and hydrohalic acid are reducing agents which are used for good-support purposes while hydrazine, isopropanol, and l-ascorbic acid are used for doping purposes. The list of these reducing agents for chemical reduction of graphite oxide is also discussed elsewhere [The chemical synthesis brings the opportunity to produce graphene at a mass-scale level from GpO or GO in solution. The GpO is usually exfoliated via mechanical stirring or ultrasonication. Ultrasonication is an effective and fast exfoliation method, but it may break apart and tear off the GO sheets into small fragments. In the chemical reduction procedure , the GO lsewhere .Regarding the chemical reduction of GO, the advantages of this method are as follows: (1) the technique is a cost-effective and simple process using cheap chemical solutions; (2) the method provides a high yield of graphene dispersion scalable for industry; (3) the chemically-derived graphene forms highly stable colloids; and (4) large-size graphene layers can be produced and this facilitates macroscopic fabrication.This new material is called “carbon-based dots”. The discovery of carbon-based dots happened accidentally during purification of single-walled carbon nanotubes (SWNTs) from arc-discharge soot. The fluorescent carbon obtained had different color emissions under UV light when several sizes and concentrations of chopped SWNT dispersions were irradiated. These carbon particles were subsequently called carbon quantum dots (CQDs) . For con2-sp3 hybridized domains of carbon materials in which the structure of graphene exists as a core. Moreover, the term quantum dot is also reserved for the carbon-based dots that satisfy the nanometer-size and functionalized structures with the quantum confinement.The GQDs can be made up of a single atom of graphite layer or graphene chopped into fragments in a range of 2–20 nm. The disk feature of GQDs is very tiny, analogous to the dot ,95. Upon2 and C=N on the surface of the carbon-based dots causing an enhancement in quantum yield. Natural and artificial aromatic carbon substances have also been investigated enabling a fine tuning of crucial optical properties, such as tunable photoluminescence. These aromatic carbon substances can be employed as effective labeling molecules as recently reported, for example, polycyclic aromatic hydrocarbon (PAH) [3) and diethylamine (DEA) [2- and sp3- hybridized structures leading them to be modified by the oxygen/nitrogen functionalized groups and protein modification. The fluorescence properties and their mechanism arises from the localization of electron-hole (e-h) pairs which form excitons analogous to conventional QDs. The conjugated π-domains in the carbon-core structure arising from modifying surface chemistries and defects are also additionally promoted [The nano- to micro-sized fragments of GO from hydrothermal cutting were passivated by placing -C=O, -OH and -COOH groups on the surface . Suvarnaon (PAH) , chlorofne (DEA) , orange ne (DEA) , potato ne (DEA) , citric ne (DEA) . The mulpromoted . The strpromoted ,90,95, tpromoted ,104,105,promoted .Previously, the structures and outstanding properties of graphene-based materials using different synthesis methods have been exploited in biosensing applications since graphene is a semi-metal with ultra-high charge mobility giving excellent electronic properties, having large surface area, being capable of being functionalized on its surface. There are many possible approaches to engineer the receptor for targeting biomolecules. In the biomedical field, pristine graphene is not only referred to as an oxide-free graphene presenting π-π stacking, non-covalent interactions and high electrostatic force, but it also offers an infinite surface at a molecular level. Therefore, graphene provides for a high possibility of active sites for charge-biomolecular interactions due to the large specific surface area leading to a sensing enhancement as well as supporting the desired functionalization to target biomolecules to improve the selectivity. In biomedical applications, graphene-based nanostructures have been recently reported with highly sensitive and selective performance as biosensors as listed in 2+ ions in children’s blood, in which the blood matrix is very complicated. The mechanism to distinguish Pb2+ ions from common ions in the blood, including Na+, K+, Mg2+ and Ca2+ at lower 0.1 M/L, is intrinsically p-doping on the CVD graphene and the surface engineering by G-quadruplex, Thrombin binding aptamer (TBA), and 8–17 DNAzyme. The lowest concentration of GFET aptasensor is at 37.5 ng/L, which was approximately one thousandth of the safety limit (100 μg/L) for Pb2+ in blood. A GFET construction shows a similar Pb2+ detecting platform but using G-quadruplex as the receptor [In the graphene-based biosensors, graphene is able to enhance the sensitivity and LOD as well as the performance of the biosensor device by improving the charge or electron transfer between graphene and the biomolecules due to its extraordinary properties. For example, as seen in receptor . The mecreceptor . The surreceptor . The conIn the use of electrochemical properties of graphene material, a novel paper-based biosensor for human papillomavirus (HPV) detection was reported . The grad-dimers [On the development of electrochemical technology, graphene microelectrodes integrated with bilayer lipid membranes (BLMs) have shown promising results in both static and stirred experiments . Moreoved-dimers , urea [1d-dimers  and chol2+ improves the LOD down to 0.01 μM. In recent complex platforms, FET biosensors using RGO combined with PtNPS and anti-BNP have been explored as a brain natriuretic peptide (BNP) detector at the early stage level [Biosensors based on the subtype of graphene materials or functionalized graphene GO, RGO, and GQD are widely used in medicine, biomedical, and bioimaging regimes. This is due to their extremely large surface area and ability to interact with various types of molecules. In addition, the outstanding properties of solubility, biocompatibility, and functionalization play an important role in sensing mechanisms. Currently, as shown in ge level . The BNPSurface plasmon resonance (SPR) is a widely used technique to investigate biochemical reactions in scientific research and medical diagnosis . In partCurrently, fluorescence biosensors based on the GQDs have gained much attention as an alternative choice due to their ease of the synthesis, good stability, fast tissue internalization, and biocompatibility. The fluorescence biosensor relies on the energy transfer between the electron donor and acceptor which is powerful for drug delivery and biomolecular interactions at the nanoscale. Fluorescence resonance energy transfer (FRET) is a mechanism to describe the energy transfer between two fluorescent molecules, where one is a donor being in an excited state and ready to transfer to the other one or an acceptor via a non-radiative dipole-dipole coupling .2 for the detection of epithelial cell adhesion molecule (EpCAM), which is a glycosylated membrane protein expressed on the surface of circulating tumor cells (CTCs) [2 having a good quenching ability is the acceptor. When the PEG is conjugated onto GQD, the PEGylated/GQD exhibits a stronger fluorescence emission because of the quantum confinement. Then the PEGylated/GQD is conjugated with the aptamer via the van der Waals binding causing a proximity of GQD and MoS2 and quenching GQD. When the EpCAM protein with a strong binding affinity is introduced, the GQD could label on EpCAM aptamer and restores its fluorescence. Therefore, the EpCAM target protein can be monitored by the fluorescence emission.This simple technique has been reported in a novel FRET based on GQD-PEG aptamer/MoSs (CTCs) . In the In this review, we discuss our points of view on the intrinsic properties of graphene and its surface functionalization concerned with the transduction mechanisms in biomedical applications. We also explain several well-known techniques used for the synthesis of graphene-based materials and their properties. A variety of graphene-based materials have been made consisting of pristine graphene and the functionalization of graphene oxide, reduced graphene oxide and graphene quantum dot. The mechanisms are discussed with respect to the most recent biosensing devices for drug delivery, biosensors, healthcare sensors, bioimaging, and other novel techniques.Graphene is one of the most well-known 2D materials. The major characteristic and properties of graphene are outstanding, i.e., zero-bandgap semiconductor, linear-like at the Dirac point, relativistic-like charge velocity, ultra-high charge mobility, transparency, large surface area, non-toxicity, having proximity induced ability, high tensile strength and high thermal conductivity, etc. However, to maintain those properties, graphene has to be perfectly proper. Alternative techniques by chemical synthesis that serve for ambient circumstances are also presented. In the synthetic procedures, the graphite is modified and functionalized by various oxygen-containing groups. In addition, the functionalized graphene is always contaminated by impurities, defects, and disorder. Hence, the structure of graphene would be importantly changed, especially the electronic properties will be distorted. On the other hand, the presence of the oxygen-containing groups and the ability to functionalize onto the graphene-based structure are important in the electrochemistry of the biosensing applications such as for labeling biomolecule recognition, for enhancing the sensing signal, for increasing the number of active sites and active area, and for probing biomolecules in an imaging application. A novel variety of the graphene-based biosensors is presented in the last section. The engineering of biosensing platforms, the mechanisms and techniques are discussed. Many new approaches are discussed in detail in this review.Since graphene material has been very well established, other 2D materials have now been explored. This opens up a wide range of possibilities and plays a crucial role in sensor and biosensor applications utilizing the largest surface area. In the upcoming future, these 2D novel materials will be further developed and tailored for specificity of bioreceptors. These materials can be employed and integrated in different sensor and biosensor platforms giving an ultra-high sensitivity and may provide a solution to some challenges, such as early stage cancer detection.


Graphene is a promising candidate for the replacement of the typical transparent electrode indium tin oxide in optoelectronic devices. Currently, the application of polycrystalline graphene films grown by chemical vapor deposition is limited for their low electrical conductivity due to the poor transfer technique. In this work, we developed a new method of preparing tri-layer graphene films with chemical modification and explored the influence of doping and patterning process on the performance of the graphene films as transparent electrodes. In order to demonstrate the application of the tri-layer graphene films in optoelectronics, we fabricated the organic light-emitting diodes (OLEDs) based on them and found that plasma etching is feasible with certain influence on the quality of the graphene films and the performance of the OLEDs. Transparent conductive materials play a significant role in optoelectronic devices such as solar cells –6, senso2 atmosphere for 3–10 min in a tube furnace. Then, the source gas of CH4 was infused with a flow rate of 0.5–10 sccm by keeping the same temperature for 3–10 min. Finally, the copper foils were rapidly cooled to room temperature. The graphene films grown on the surface of the foils were transferred onto the target substrates by the following procedure:The solution of polymethylmethacrylate (PMMA) in anisole was spin-coated on the copper foils with graphene for 30–60 s at 3000–6000 rpm (the thickness of PMMA films is about 200 nm).3 aqueous solution to remove the copper foils.The samples of Cu/graphene/PMMA were floated on FeClThe PMMA/graphene film was washed with deionized water and moved onto the glass substrate.Finally, the PMMA/graphene films were immersed in acetone to remove the PMMA.Single-layer graphene (SLG) were synthesized by CVD on a 25 μm polishing copper foil . The foils were annealed at 1000 °C in H3 and HCl solution with the volume fraction of 20% as doping reagent. Then, we use the copper foils with graphene to pick up the PMMA/graphene from the dopant to form the PMMA/double-layer doped graphene/copper foils. After the dissolution of the copper, the multilayer graphene films with chemical modification can be obtained by repeating the above steps.The procedure used to prepare the tri-layer graphene films is schematically illustrated in Fig. N,N′-diphenyl-N,N′-bis (1-naphthyl)--4,4′-diamin (NPB) (60 nm)/tris-(8-hydroxyquinoline) aluminum (Alq3) (60 nm)/lithium fluoride (LiF) (1 nm)/Al (80 nm), where NPB was used as the hole transport material, Alq3 was used as both the host material and the electron transport material, and LiF and Al were used as the electron inject material and the cathode material, respectively.The tri-layer-doped graphene film was patterned by photolithography technology and etching. OLED devices were fabricated with a structure of graphene/I–V) characteristics of the fabricated OLEDs were measured with an experimental setup including a Keithley 2400 source meter. A spectroradiometer (PR750) was also employed to measure the electroluminescence spectrum of the 3 × 3 mm2 emitting area of the devices. The reference OLEDs with the same layer structures, except that the graphene film was replaced by a conventional ITO layer (20 Ω/□), were also fabricated for comparison.To characterize the graphene films, field-emission scanning electron microscope , transmission electron microscope, and atomic force microscope (NT-MDT) were used to examine the surface morphology. Raman measurements were performed using Thermo Scientific DXR Raman microscope spectrometer with a laser wavelength of 532 nm. For sheet resistance measurements, we used a semiconductor analyzer  combined with a four-probe station . The optical transmittance in the wavelength range of 300–1100 nm was obtained by a PV Measurements QEX10. The current–voltage  was also carried out to confirm the quality of the graphene. From Fig. Figure 3, respectively. Through the lamination and chemical modification, we can see that the sheet resistance of the pristine graphene and the tri-layer graphene modified with HCl and AuCl3 are 350, 258, and 150 Ω/□, respectively. The stacking of the graphene layers cannot only reduce the sheet resistance but also lower the transmittance of the films. To maintain the transmittance above 90%, the sheet resistance of the tri-layer graphene is still too high for application. Thus, the chemical modification is necessary to further reduce the sheet resistance. The transmittance of tri-layer graphene is 92.5%, and it dropped to 92.2 and 91% when the tri-layer graphene was doped with HCl and AuCl3. The conductivity stability is also an important index to evaluate the graphene film. The significant advantage of AuCl3 doping can be demonstrated in Fig. 3 showed more stability than those doped with HCl due to the volatility of HCl. HCl doping can provide extra holes for the graphene films, which increase the carrier concentration and improve the conductivity [3.Transmittance and sheet resistance are two important indicators for evaluating the transparent conductive electrode. Table uctivity . Therefouctivity  reported3. The reason for this is that the formation of the gold particles between the graphene layer and layer can cause some damage to the structure of graphene film while improving the conductivity of the films at the same time. Moon et al. [The pattern process of the graphene film is critical for the preparation of the OLEDs. We used lithography combined with plasma etching to achieve the pattern of the tri-layer graphene film. Figure n et al.  reported3. Fethullah et al. [3 can form Au clusters to affect the RMS (root mean square) of the graphene films, which is similar with our results. The RMS of the tri-layer graphene doped with AuCl3 is 5.2 nm, which is higher than the pristine tri-layer graphene after patterning. From Fig. The roughness of the film surface is one of the important criteria for judging the OLED anode. Figure h et al.  reported3(~150 Ω/□) composite films than that of ITO (~20 Ω/□). The current efficiency of the two devices are lower than those of the ITO-based device. The best device in our experiment based on the tri-layer graphene doped with AuCl3 anode exhibits a current efficiency of 0.263 cd/A. There are four main reasons for the poor performance of the devices: (1) the most important factor is that the work function of graphene (about 4.4 eV) is lower than that of the ITO (about 4.8–5.0 eV) so that the hole injection barrier of tri-layer graphene is larger than that of the ITO and finally affected the carrier injection; (2) the conductivity of the graphene films is very poor, which affects the injection of carriers; (3) the process of the patterning compromised the quality of the graphene films; and (4) the influence of surface roughness on the graphene anodes. The simple device structure is also a possible reason of the poor device performance. Figure 3 anode at 150 cd/m2. The hole injection layer (HIL) can enhance the current of holes and hole injection efficiency toward the emission zone [The OLEDs with the anode of the tri-layer graphene and tri-layer doped graphene have the structure of anode/NPB (60 nm)/Alq3 (60 nm)/LiF (1 nm)/Al (80 nm). The OLEDs based on the ITO anodes were also fabricated for comparison. Due to the electrical performance of tri-layer graphene doped with HCl and pristine tri-layer graphene tending to be consistent after a long time from Fig. ion zone –27. In o3 is higher than that of the devices based on the tri-layer pristine graphene film due to the contribution of high conductivity. These results suggest that the tri-layer-doped graphene films are a promising candidate as the transparent conductive electrode in optoelectronic devices.In conclusion, we developed a new method to prepare tri-layer graphene films with chemical modification and explored the influence of doping and patterning process on the performance for the graphene films as transparent electrodes. We have demonstrated that the tri-layer graphene films can be used as transparent and conductive anodes in the OLEDs. The performance of the devices based on the tri-layer graphene films doped with AuCl


We found that we can successively fabricate GO (oxidation) and graphene (reduction) from rGO by controlling the oxidation or reduction procedure time and then clearly comparing the critical properties among them as we perform various oxidation and reduction activities.In order to compare the catalytic activities of reduced graphene oxide (rGO), graphene oxide (GO), and graphene, we conducted oxidation of 2-aminothiophenol (2-ATP) and reduction of nitrobenzene (NB) in their presence by using high-resolution photoemission spectroscopy (HRPES). In addition, we determined conversion rates of CO to CO Carbon-based materials such as reduced graphene oxide (rGO), graphene oxide (GO), and graphene are the most extensively utilized supports for metal-based catalysts and are used on their own as oxidative or reductive catalysts because they provide high surface areas, are resistant to acidic and basic media, and their surfaces can be readily engineered by introducing functional groups1234567910111214151719202122232 by using residual gas analyzer (RGA) in an ultra-high vacuum (UHV) condition, Orange II and methylene blue degradations by using UV-vis spectrophotometry, and in electrochemistry (EC) under an aqueous solution.With this aim in mind, by commencing with an rGO sample, we successfully fabricated GO through an oxidation process and graphene through a reduction process (see the data and results section). We then assessed the capacities of GO, rGO, and graphene to oxidize 2-aminothiophenol (2-ATP) and to reduce nitrobenzene (NB) under UHV conditions with 365 nm UV light illumination to enhance the photocatalytic reaction. Moreover, we also compared their catalytic activities in the conversion of CO to COHerein, we precisely compared the morphologies and electronic properties of the rGO, GO, and graphene samples, and assessed their catalytic capacities (oxidation and reduction) by using scanning electron microscopy (SEM), tunneling electron microscopy (TEM), Raman spectroscopy, HRPES, RGA, UV-vis spectrophotometry, and EC measurements.SEM and TEM were used to characterize the morphologies of the three distinct carbon-based materials . The rGO sample in Additionally, in order to confirm that the GO and graphene is fabricated from rGO, we also acquired X-Ray diffraction (XRD). As shown in D/IG values of rGO and GO are 1.761 and 0.912 respectively, and the D and G bands at 1347.6 and 1592.1 cm−1 are typical of GO and rGO related bands19−1) and D-peak (1320 cm−1) are due to the E2g phonon at the Brillouin zone center and the breathing mode of sp2 carbon atoms respectively27s core-level spectra of the rGO , hydroxyl groups , carbonyl groups , and carboxyl groups s core-level spectrum is evident at 284.7 eV, which corresponds to the binding energy of surface sp2 hybridized states the rGO  and GO , –OH (hydroxyl group at 533.1 eV: marked as O2 peak), –C=O (carbonyl group at 532.0 eV: marked as O3 peak), and –COOH (carboxyl group at 531.2 eV: marked as O4 peak) bonding features. O 1s core level spectra of rGO shows two distinct oxygen features (hydroxyl group marked as O2 and carbonyl group marked as O3). This interpretation is consistent with Lerf’s modelWe also acquired O 12 by using residual gas analyzer, Orange II and methylene blue degradations by using UV-vis spectrophotometer, and their oxidation activities in aqueous solution by performing EC measurements.Through the Raman and HRPES results, we confirmed that they are consistent with previous results for GO and graphene, and thus we could confirm that the oxidation and reduction of rGO were successful. Next, we determined the oxidation (of 2-ATP) and reduction (of NB) activities of our rGO, GO, and graphene by using HRPES, their activities in the conversion of CO to COp peak due to 2-ATP, which was obtained at a photon energy of 230 eV. p core level spectra after the co-exposure of 360 L 2-ATP to the same amount of oxygen on three distinct samples under 365 nm UV light illumination. These spectra contain three distinct 2p3/2 peaks at 161.5, 162.9, and 168.6 eV, each respectively corresponding to the C–SH unbounded state (denoted S1), the bounded state (denoted S2), and the sulfonic acid group (–SO3H) (denoted S3)312 react on GO or rGO under UV illumination.We performed the photocatalytic oxidation of 2-ATP with the same amount of molecular oxygen and the reduction of NB with the same amount of molecular hydrogen adsorbed on the three distinct samples; both the oxidation and reduction reactions were carried out with exposure to 365 nm UV light. The oxidations of 2-ATP were monitored by determining the change in the intensity of the S 2s core-level spectra of NB on rGO, GO, and graphene on silicon substrates, obtained with HRPES at a photon energy of 460 eV. The N 1s core level spectra was obtained after introducing 360 L NB onto the rGO, GO, and graphene samples. As shown in 2), is evident, which indicates that no reduction occurs on GO as expected. In contrast, in the cases of rGO and graphene as shown in 2) and the aniline peak (N2: -NH2). This indicates that the reduction reaction occurs on these samples. The ratios of the intensities of N2 to N1 were also plotted  in the presence of the three samples. As mentioned above, the bottom panels of 2. Hence, we monitored the oxidation of CO to CO2 in the presence of the three distinct carbon materials by using RGA for various substrate temperatures in the range 300 K~700 K under UV irradiation. 2 exposures at 300 K to the three carbonaceous materials . Clearly, the rate of conversion of CO to CO2 on GO is larger than those on rGO and graphene, as expected because the various oxygen functional groups on GO facilitate CO oxidation. In addition, to determine the variations of substrate temperature in the CO oxidations of the three samples, we measured the conversion rates for various substrate temperatures (in 50 K increments from 300 K to 700 K) as shown in A standard test for catalysts is the conversion of CO to CO2 is dependent on that of CO even though the oxygen carriers on the carbon materials can assist CO oxidation; the low conversion rate on graphene confirms this phenomenon. Second, the conversion of CO to CO2 occurs stably in the range of 300 K to 600 K, as shown in 2), the conversion process from CO and CO2 and the decomposed process can occur together! Hence, above 600 K, the conversion rate looks like decrement.Two interesting points emerged from these measurements. First, the variation in the intensity of CO2 result. Consequentially, GO is better than others.The catalytic oxidation of Orange II and methylene Blue was employed to probe the efficiency of the as prepared samples. As show in CVs were obtained to measure the electrocatalytic properties of rGO, GO and graphene with regards to the oxidation of 2-ATP. As shown in Further, there is a high oxidation current at 0.15 V in the CV results for rGO see  and grap2 in the presence of these catalysts by performing residual gas analyzer (RGA), Orange II and methylene blue degradation and electrochemistry (EC) measurements in aqueous solution, as well as by obtaining cyclic voltammograms and determining the change in the condition of the electrodes before and after the oxidation of 2-ATP. We found that we can successively fabricate GO (oxidation) and graphene (reduction) from rGO by controlling the oxidation or reduction process time. Furthermore, we characterized the morphologies, electronic properties, and electrochemistry of the catalysts by using SEM, TEM, Raman spectroscopy, HRPES, RGA, UV-Vis spectrophotometry, and EC measurements. We confirmed that GO can act as a catalyst for oxidation reactions and graphene can act as a catalyst for reduction reactions.In conclusion, we have successively fabricated GO (oxidation) and graphene (reduction) from rGO by controlling the reaction times. In order to compare the catalytic activities (in oxidation and reduction processes) of the three carbonaceous materials, we conducted oxidation of 2-aminothiophenol and reduction of nitrobenzene in their presence and monitored the reactions by using high-resolution photoemission spectroscopy (HRPES). In addition, we also determined the rates of conversion of CO to CO4, H2SO4, and 0.5 M NaNO3 as we controlled the reaction time. Graphene fabricated from rGO was carried out by adding hydrazine (1.0 mL) into the dispersion of rGO (50 mg of rGO in 100 mL of water)s core-level spectroscopy  gas were purified by turbo-pumping to remove impurities prior to dosing onto the GO, rGO, and graphene samples grown on silicon substrates. A direct dozer, controlled by using a variable leak valve, was used to dose the substrates.2-aminothiophenol  and nitrobenzene , Orange II , methylene blue , carbon monoxide  and molecular oxygen (O+ ion laser (Spectra-Physics Stabilite 2017) as an excitation source, a spectrometer (Horiba Jobin Yvon TRIAX 550), and a CCD detector (Horiba Jobin Yvon Symphony) cooled to −133 °C. The wavelength of the incident excitation beam was 514.5 nm. HRPES experiments were performed at the 8A2 beamline at Pohang Accelerator Laboratory (PAL) which is equipped with an electron analyzer . The S 2p, C 1s, and N 1s core level spectra were obtained by using photon energy of 230, 340, and 460 eV respectively. The binding energy of the core level spectra was determined with respect to the binding energy of the clean Au 4f core level for the same photon energy. All spectra were recorded in the normal emission mode. The photoemission spectra were carefully analyzed by using a standard nonlinear least squares fitting procedure with Voigt functions2, and CO2 gas intensities. The experiments for degradation of orange II and methylene blue (MB) in water by advanced oxidation technology were conducted in a 500 mL beaker under 25 °C thermostat water baths with constant magnetic stirring. Typically, with Orange II (0.15 mM) and MB (0.15 mM), Intensity variation of GO, rGO, and graphene was obtained at regular intervals, joined immediately with 1 mL methanol as the inhibitor, and then filtered. The real-time concentration was measured in an UV−vis spectrophotometer  at 484 nm. The electrochemical experiments were performed by using a CHI617B potentiostat  with a three-electrode cell placed in a Faraday cage. A 2 mm diameter glassy carbon electrode was used as the working electrode and a 1 mm diameter Pt wire was used as the counter electrode. The reference electrode was Ag/AgCl (3 M KCl). All working electrodes were polished with alumina (0.05 μm) paste on micro-cloth pads  prior to use. The drop casting method was performed on glassy carbon electrodes to measure the electrocatalytic properties of GO, rGO, and graphene. The samples (5 μL) were placed on glassy carbon electrodes, and then were stored in an oven at 40 °C for 6 h. All cyclic voltammograms (CVs) were obtained in 50 mM tris buffer (pH 9.0) at a scan rate of 100 mV/s39Scanning electron microscopy (SEM) images of the samples were obtained by using a field-emission scanning electron microscope (JEOL JSM-7600F) operated at an acceleration voltage of 15 kV and a field-emission transmission electron microscope (JEOL JEM-2100F(HR)) operated at an accelerating voltage of 200 kV. Raman spectra of the samples was collected by using a home-built system equipped with an ArHow to cite this article: Lee, H. et al. Comparative Study of the Catalytic Activities of Three Distinct Carbonaceous Materials through Photocatalytic Oxidation, CO Conversion, Dye Degradation, and Electrochemical Measurements. Sci. Rep.6, 35500; doi: 10.1038/srep35500 (2016).


Using core genes, we evaluated the distribution and architecture of Fusarium circinatum spliceosomal introns, and linked these characteristics to the accuracy of the predicted gene models of the genome of this fungus. We also evaluated intron distribution and architecture in F. verticillioides, F. oxysporum, and F. graminearum, and made comparisons with F. circinatum. Results indicated that F. circinatum and the three other Fusarium species have canonical 5′ and 3′ splice sites, but with subtle differences that are apparently not shared with those of other fungal genera. The polypyrimidine tract of Fusarium introns was also found to be highly divergent among species and genes. Furthermore, the conserved adenosine nucleoside required during the first step of splicing is contained within unique branch site motifs in certain Fusarium introns. Data generated here show that introns of F. circinatum, as well as F. verticillioides, F. oxysporum, and F. graminearum, are characterized by a number of unique features such as the CTHAH and ACCAT motifs of the branch site. Incorporation of such information into genome annotation software will undoubtedly improve the accuracy of gene prediction methods used for Fusarium species and related fungi.Removal of introns from transcribed RNA represents a crucial step during the production of mRNA in eukaryotes. Available whole-genome sequences and expressed sequence tags (ESTs) have increased our knowledge of this process and revealed various commonalities among eukaryotes. However, certain aspects of intron structure and diversity are taxon-specific, which can complicate the accuracy of  Our specific aims were threefold: (i) to compile a dataset consisting of the sequences for the standard set of eukaryotic core genes  for the automatically annotated introns; and (iii) to manually compare and contrast among the four fungi the distribution, length, phase, and cis-element structure of introns in the core gene set. Apart from providing information regarding the accuracy of the F. circinatum genome annotation and potential approaches to improve gene models, our results will significantly broaden current knowledge regarding the organization and sequence characteristics of fungal spliceosomal introns.In this study, we evaluated the accuracy of the automated gene predictions utilized to annotate the genome of the fungus F. circinatum EST data used in this study were obtained from two independent studies. In the first study, a pathogenic strain of F. circinatum (FSP34) was used to generate two cDNA libraries from carbon- and nitrogen-starved mycelia as described previously  were used for the construction of two cDNA libraries on complete medium. One library included cDNAs from strains GL 57 and GL 100, and the other library cDNAs from strain GL 101. The libraries were generated by growing the F. circinatum isolates in potato dextrose broth (Difco laboratories) and extracting total RNA from the cultures using TRIzol (Invitrogen). Invitrogen’s Dynabeads mRNA Purification Kit was used to obtain mRNA. For cDNA synthesis, the Universal RiboClone cDNA Synthesis System  was used. Following purification using the MinElute PCR Purification Kit (QIAGEN), cDNAs were submitted for sequencing to the DNA Technology Facility at the University of California, Davis (U. C. Davis) using the Illumina GAIIx Sequencing platform.In the second study, three F. graminearum, F. verticillioides, and F. oxysporum were accessed from the Broad Institute’s Fusarium Comparative Database (http://www.broadinstitute.org/annotation/genome/fusarium_group), while the genome sequence for F. circinatum (accession number PRJNA41113) was available from a previous study  and used in BLASTp searches against the Fusarium Comparative Database at the Broad Institute. FASTA files were subsequently compiled with the F. verticillioides, F. oxysporum, and F. graminearum nucleotide sequences for the core genes. The F. verticillioides, and in a few cases the F. oxysporum and F. graminearum, core gene sequences were then used to identify and retrieve the corresponding sequences from the F. circinatum genome by making use of BLASTn, CLC Genomics Workbench version 3.7.1 (CLC bio A/S), and BioEdit version 7.0.9.0  of the National Center for Biotechnology Information . We also included the EST data for two additional strains of F. circinatum used in an ongoing study . The F. graminearum default transition matrix  was used because it is the only Fusarium species for which a matrix is available in Augustus. In addition to resolving incongruent intron positions, the EST data of the four genomes was also useful for identifying unusual cis-elements (see below).ClustalW alignments of all core genes with apparently incongruent (nonconserved) intron positions were analyzed in Augustus version 2.4  hypothesis was that all the mean lengths of the introns in different positions are equal  with 6 (numerator) and 500  d.f. . Critical values for Tukey’s HSD (q) were determined using the d.f.  and the number of treatments (7), which is the number of intron positions compared.The dependence of intron length on its position in the coding sequences (CDS) of a gene was tested using ANOVA  and hypore equal . For thily) d.f. . To testt-test, where Ho and Ha were, respectively β1 = 0 and β1 ≠ 0 (β1 is the slope of the regression line)  . When β1 Add-ins  were use− 2 d.f. .The 226 core gene dataset described above was used to analyze the intron phase. Intron phases were examined manually in BioEdit , using tcis-elements  of introns from all the genes in the dataset were examined. To achieve this, the annotated introns were extracted from each core gene alignment using the “Extract Sequences” and then the “Extract Annotations” functions in the CLC Genomics Workbench. In all cases, an additional five nucleotides in the upstream exon and five additional nucleotides in the downstream exon were extracted with the introns. The extracted introns were then aligned using the “Create Alignment” function after which they were manually annotated relative to the annotations previously produced for fungal introns . A minimal definition of six consecutive nucleotides with at least three 2′-deoxythymidines  and no 2′-deoxyadenosine was used for this purpose   and are currently available from the Broad Institute fungal ftp site: ftp://ftp.broadinstitute.org/pub/annotation/fungi/fusarium.All the genome sequences and EST data used in this study are available without restriction. Fusarium Comparative Database at the Broad Institute. We intended to use the identified F. verticillioides genes in BLASTn searches to identify homologs in the genome of F. circinatum. However, homologs for 12 of the 458 genes were absent in the F. verticillioides genome and, to compensate for these, we instead used genes from F. graminearum (nine) and F. oxysporum (three). These searches allowed for the identification of the full set of 458 core gene homologs in the F. circinatum genome, although 22 were separated over multiple contigs and thus excluded from further analyses. The final core gene dataset utilized in this study thus consisted of 436 core gene sequences.The use of a set of 458 core genes common to all eukaryotes  has been determined previously  using the available EST data. The analysis revealed that 54% (82) of the 152 genes were wrongly annotated. In part, this was due to the fact that dissimilar gene prediction software was used for the four Fusarium genomes. Also, Augustus does not recognize GC–AG introns as was previously also shown for Armillaria mellea  erroneously had very short introns of 5–23 nucleotides (nt) in length, which were subsequently resolved using the EST data. After this process of EST-based position resolution, introns with nonconserved positions were present only in 69 of the 436 core gene alignments. Therefore, the large majority (84%) of the core genes examined harbored introns with conserved positions.Of the 436 core genes examined, 152 appeared to be nonconserved with respect to intron position. To ensure that this lack of conservation was not due to the different gene prediction methods used during annotation of the four genomes, all 152 genes were reanalyzed with Augustus . F. oxysporum had one case of a GT to GA substitution. Seventeen gene alignments had nonconserved intron positions due to intron insertions/deletions (indels) . A number of gene alignments had shorter  or longer predicted ORFs (two in F. circinatum). Eleven gene alignments contained at least one truncated  homolog of the gene in one of the species examined. All the above-mentioned gene alignments were excluded from the core gene dataset used in subsequent analyses.For the 69 genes with introns that were apparently not conserved with respect to position, EST data were available for 23 genes only. Examination of the 46 gene alignments lacking EST support revealed five genes for which positional incongruences among introns were due to nucleotide substitutions. In one case, F. graminearum had whole-intron deletions, while one had a 2′-deoxythymidine to 2′-deoxyguanosine (T > G) substitution in the second nucleotide of the 5′ splice site. Of the six intron position incongruences observed in F. oxysporum, three were as a result of whole-intron deletions, one had a shorter ORF, while two were divergent in nucleotide sequence when compared to the other Fusarium species. Of the three genes with nonconserved intron positions in F. circinatum, one gene had a whole-intron deletion and two had shorter ORFs compared to the other three species.For the remaining 23 genes for which intron positions were supported by EST data, the apparent lack of positional conservation was due to one of three factors. Of the 23 genes, 15 from Fusarium species we examined. We made alignments and manually annotated all the genes. However, for manual in-depth analyses we scaled the number of genes down to a manageable set of 226 genes for each species. Within each of these datasets, the number of introns ranged from 0 to 15 introns per CDS , with an average density of 2.53 introns per CDS. However, consistent with what has been observed for Neurospora crassa . Within this dataset, the gene encoding CTP synthetase had the highest number of introns , followed by that encoding glutamine-dependent NAD+ synthetase with 14 introns in F. verticillioides, F. circinatum, and F. oxysporum, and 12 in F. graminearum . Of the 226 examined core genes, seven harbored no introns in any of the four Fusarium species and these included genes encoding pre-mRNA splicing factor clf, amino methyltransferase-mitochondrial precursor, seryl tRNA synthetase, DNA pantothenate metabolism flavoprotein 2, Sol1 family protein, uridylate kinase, and the DNA repair helicase RAD25.For the analysis of intron length, frequency and distribution, a subset of the CDSs for 226 core genes were used. Our initial dataset consisted of ∼436 genes for each of the four Fusarium species was on average 75.4 nt and ranged from 42 to 529 nt with a median of 57 nt , 76.3 (42–520), 75.8 (42–529), and 75.7 (43–525) nt, where the numbers in parentheses denote intron size range. In almost all alignments, the lengths of introns in the core sequences of F. graminearum were different from those of F. verticillioides, F. circinatum, and F. oxysporum (see Table S2 in File S4).Among the 226 CDSs examined, intron length in the four of 57 nt . The meaFusarium species was ∼93 nt (42–529). The rest of the introns at positions 2–7 had mean intron lengths between 54 and 72 nt . A similar trend was observed for all four species when analyzed independently  was compared to M2 through to M7  in a pairwise manner, were found to be greater than 6.67, 6.73, 6.73, and 5.71, respectively. All other comparisons that excluded the mean sizes of the first introns produced values < 1.94. The critical value at P = 0.05 was 4.17, indicating that the null hypotheses were only rejected when M1 was compared with M2 to M7.Longer introns were mostly located at the 5′ end of CDSs. This phenomenon was more pronounced when first introns were compared with the rest of the introns in the core dataset. The mean intron length of the first introns from all four endently . Based oFile S3). The Student’s t statistic values for F. verticillioides, F. circinatum, F. oxysporum, and F. graminearum were, respectively, 1.67, 1.98, 1.62, and 1.90. With P values of 0.10 and 0.05, and n − 2 d.f., the t critical values were 1.65 and 1.96, respectively. The null hypothesis that there is no correlation between CDS and intron length was rejected for F. verticillioides and F. graminearum at the 90% confidence level, and for F. circinatum at both confidence levels. Only in the case of F. oxysporum could the null hypothesis not be rejected at either confidence level because of the low t statistic values.A statistically significant negative correlation between CDS length and intron length was observed, where longer genes had shorter introns . In a few cases all introns were located in the middle of CDSs, in few others there was a 3′ positional bias within the CDS, and in a limited number of instances  there was an approximately even distribution of introns across the CDS. The distribution of introns in the CDSs of the four Fusarium species followed the same trend when they were analyzed separately.Analysis of the distribution of introns within the CDSs of the 226 core genes revealed a 5′ region positional bias . The larFusarium species, albeit at different frequencies. In the set of CDSs for 226 core genes, most introns were phase 0, which appeared to be true also for the one, three-, four-, five-, seven-, eight-, nine-, and twelve-intron genes . Although the number of introns in phase 0 was lower than the number of phase 1 and phase 2 introns added together, the number of phase 0 introns was the highest when all three phases were compared individually. In addition, the 5′ regions of CDSs contained more phase 0 introns than phase 1 and 2 introns . The highest proportion of introns in the first and the fourth quarter of the genes were in phase 0. Although negligible, this was also the case for introns in the second quarter of the genes .Our analysis showed that introns of phases 0 (when an intron is located between two codons), 1 (an intron that is located between the first two nucleotides of a codon), and 2 (when an intron is located between the last two nucleotides of a codon) were present in all four F. verticillioides, 212:171:143 for the 526 introns examined in F. circinatum, 212:171:141 for the 524 introns of F. oxysporum, and 211:172:137 for the 520 introns in F. graminearum. For genes with the same number of introns, the phases were conserved among the four Fusarium species. The only two exceptions were in the CDS of the gene encoding glutamine-dependent NAD + synthetase in F. graminearum, where the eighth intron was phase 1 and not phase 0, and the fourteenth intron was phase 0 and not phase 2, in comparison to the other Fusarium species.The respective ratios of the three intron phases (0:1:2) in the four fungi were 215:172:147 for the 534 examined introns of F. circinatum , and one each in F. oxysporum (protein phosphatase PP2A regulatory subunit A) and F. graminearum (glucose 6 phosphate 1 dehydrogenase)], each of the sequences had an extra nucleotide that, when deleted, generated an intron phase similar to those in the other species. Another potential sequencing error involving a pyrimidine substitution was observed in F. circinatum (AP 2 adaptor complex subunit α).Our data further showed that the analysis of intron phase could potentially aid in the identification of potential sequencing errors. This was primarily in terms of errors associated with mononucleotide tracts. In four instances [two in 38A38G53|G100T99A74A42G93T66 . However, the presence of two variants (RAG|Y or RAG|R) in a small proportion of the introns  examined was also revealed (File S4). With regard to the branch site motif, > 90% of the sites had the consensus CTRAY, while three additional variants  were also detected in a small number of introns. The same motifs were observed for the each of the four Fusarium species.Analysis of the 2022 introns also allowed refinement of the 3′ splice and branch site motifs . AlignmeFusarium species (File S4). In a dataset containing 166 introns from 10 genes, the length of the predicted polypyrimidine tracts ranged from 6 to 25 nt. The number of predicted polypyrimidine tracts per intron also varied considerably. In some cases, as many as nine were predicted, while in 24 instances no sequence resembling this element could be detected. For more than half (53%) of the introns analyzed either one or two polypyrimidine tracts could be predicted. Our analyses also showed that the predicted polypyrimidine tracts occupied two intron regions, the 5′ region and the 3′ region, where the branch site was the reference point . Only 14–20% of the predicted polypyrimidine tracts occupied the 3′ region. Of the latter, F. graminearum had the most predicted polypyrimidine tracts in the 3′ region, while F. oxysporum had the fewest. In total, 61% of the examined introns had predicted polypyrimidine tracts exclusively in their 5′ region, 35% exclusively in their 3′ region, and 5% in both the 5′ and 3′ regions (Table S3 in File S2).The polypyrimidine tract  in gene models involving nonconserved intron positions during the 2012–2015 period. A similar scenario was observed for F. graminearum. During their study of alternative intron splicing in this fungus, Fusarium Comparative Database are incorrect. In a subsequent study , as they contain additional regulatory elements (A50/C23) G94 (T76/C15) . However, as with the study in N. crassa, our study provided more information on this consensus sequence  was highly similar to that found in As. fumigatus, Ca. albicans, Cr. neoformans, Sc. pombe, and S. cerevisiae, with their 3′ splice site consensus motif being Y89A100G100|R58 (N. crassa (Y92A100G100|Y88) in the first nucleotide of the 3′ exon, where pyrimidines are more frequent than purines  and CTHAH (3.96% of introns), as well as an ACCAT motif that occurred in 0.05% introns. Of these, the TTRAY motif has been reported for other fungi. S. cerevisiae, Sc. pombe, As. nidulans, N. crassa, and Cr. neoformans, and it has also been observed in 1 out of 72 genes examined in N. crassa  were done using GENEid , the 3′ splice site that should contain a minimum of four nucleotides with the canonical AG dinucleotide at the second and third positions, and the 5′ splice site that should contain a minimum of nine nucleotides: three from the upstream exon, the intron dinucleotide, which can either be a GT or a GC, together with the four nucleotides following it. Our data on the minimal requirements for splicing of the introns could also be used to restrict intron length in gene prediction programs (Incorporation of the findings presented here into gene finding procedures would undoubtedly increase the accuracy of annotations for programs . Consistwww.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300344/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.