A strong electric field enhancement produced in the 3D framework nanohybrids of graphene nanosheet/Ag-nanoparticles is responsible for this great enhancement of catalytic reaction, due to larger electron collective oscillation in the composite system. Especially the oxygen adsorbed on the graphene and Ag nanoparticles can be excited to triplet excited states, and the electrons on the graphene and the nanoparticles can be effectively transferred to the oxygen, which plays very important role in molecular catalytic reactions. Our results demonstrate the contribution of graphene in plasmon-driven catalytic reactions, revealing a co-driven reaction process.This excellent SERS substrate can be used for future plasmon and graphene co-catalytic surface catalytic reactions, graphene-based surface plasmon sensors and so on.Three dimensional (3D) plasmonic nanostructure is perfect for the surface-enhanced Raman scattering (SERS) and also very suitable for surface catalytic reaction, but how to design and fabricate is still a robust task. Here, we show a 3D plasmonic nanohybrid of vertical graphene-nanosheet sandwiched by Ag-nanoparticles on the silicon nanocone array substrate for enhanced surface catalytic reaction. By SERS detection, we find that this hierarchical nanohybrid structure is highly efficient in the enhancement of catalytic reaction, even at a very low concentration of 10 Noble metallic nanoparticle catalysts and their use in catalytic processes have recently been widely investigated experimentally and theoretically due to their advantages in nanoscience and nanotechnology. Since the first report of plasmon-driven catalytic reaction of p-aminothiophenol (PATP) oxidizing to dimercaptoazobenzene (DMAB) revealed by surface-enhanced Raman scattering (SERS)13456789111213141517181920Graphene, as an atomic-scale honeycomb lattice made of carbon atoms, conducts heat and electricity with great efficiency. Recently, graphene has also been used as the substrate of SERS162324252728Recently, the graphene-nanoparticle hybrids were reported to be able to tune the enhancement of Raman scattering3133−11 M. A mechanism of graphene enhancing plasmon-driven catalytic reaction is proposed and discussed here. This excellent 3D SERS substrate can be used for future graphene and plasmon co-driven surface catalytic reactions, graphene-based surface plasmon sensors and so on.In this work, we fabricated a 3D hierarchical nanostructure of vertical flower-like graphene nanosheets (FGNSs) sandwiched by Ag nanoparticles (Ag-NPs) grown on silicon nanocone arrays substrate, in which silicon nanocone arrays can further optimize the 3D hierarchical hybrid structure and greatly improve its surface-to-volume. Firstly we verified that this hierarchical hybrid structures have a great SERS ability to be a perfect SERS substrate. Further, to demonstrate that this excellent SERS substrate is very suitable for graphene enhancing plasmon-driven catalytic reactions, PATP and 4NBT were separately oxidized and reduced to DMAB. Our results demonstrate that this hierarchical hybrid structure is very efficient in graphene enhancing plasmon-driven catalytic reaction, even at the level of 109/cm2. The FGNSs were uniformly grown on the silicon nanocone array by hot filament chemical vapor deposition using the mixture of H2 and CH4. Furthermore, the structural details of the as-grown graphene nanosheets are acquired from TEM images and EELS, respectively, as shown in The Ag nanoparticles are coated on both sides of graphene nanosheet grown on the Si nanocone arrays, and the distribution of Ag nanoparticles can be tuned by Ag nanofilm with different thicknesses from 2 to 8 nm, in which 6 nm thick Ag nanofilm with 30 nm in average particle diameter is selected as next tested sample because this thickness exhibits the best SERS ability among different Ag thicknesses /I2D(1335) = 1.66, I2D(1432)/I2D(1335) = 2.11, I3D(1396)/I3D(1335) = 3.10, I3D(1432)/I3D(1335) = 4.24. More than twice enhancement in the relative intensity ratio reveals that the 3D structure of graphene/Ag-nanoparticles is much more efficient for enhancing plasmon-driven catalytic reactions than 2D plane structure.In order to further demonstrate the significant effect of graphene nanosheets on this co-driven catalytic reaction, the SERS spectra of three kinds of substrate deposited by Ag nanoparticles using the same Ag film thickness of 6 nm – a pure planar silicon substrate, a CVD single-layer graphene (SLG) coated planar silicon substrate by transferring process and a silicon nanocone array coated by FGNSs – were measured under the same conditions for PATP, as shown in −1 over 1335 cm−1 is gradually decreased. Even at 10−11 M, the Raman profile is at 1432 cm−1 of DMAB can be observed in −11 M. This result is much better than our previous plasmon -driven catalytic reaction at the molecular concentration 10−71−5 M) at 10×10 μm. The ratio of Raman intensities of DMAB at 1432 cm−1 over Raman intensities of graphene at 1335 cm−1 can be seen in 2 at 1361 cm−1 can also be found. By increasing of laser power, Raman peaks of the graphene are significantly enhanced, but the Raman peak of DMAB at 1432 cm−1 only increases a little along with the increase of laser power. By increasing the laser intensity, it seems that the probability of such catalytic reaction is increased slightly. So, it seems that the reduced catalytic reaction is not as good as the oxidized catalytic reaction (from PAPT to DMAB) in this 3D hybrids nanostructure, and the reason for this will be discussed in the next section.It is known that DMAB can also be converted from 4NBT. It is interesting to study the laser power dependent probability and efficiency. The contribution of graphene nanosheet sandwiched by Ag nanoparticles see  to the p2 adsorbs on two Ag atoms of a cluster at the ground state, as shown 2 to the Ag cluster on the triplet electronic transition of O2 adsorbed on the Ag cluster, as displayed in 2, and the electrons on triplet excited O2 transfer to the Ag cluster. This is a very good process for the plasmon-driven catalytic reaction. The lowest energy of transition (S1) from singlet oxygen at ground state to the triplet excited oxygen is 1.7726 eV. The S2 transition (2.2234 eV) can also result in the triplet excited state for oxygen, since the related energy is also lower than the laser frequency (532 nm) that we used. So, it is occurred that plasmon and graphene co-catalytic surface catalytic reactions in our experiments, in which graphene play a key role in enhancing plasmon-driven catalytic reactions.It has also been shown that surface plasmon resonance can activate the oxygen to the triplet excited state, which plays very important role in plasmon-driven catalytic reactionsIt has been found that the reduced catalytic reaction (from 4NBT to DMAB) is not as good as the oxidized catalytic reaction (from PAPT to DMAB). The reason is that in the oxidized reaction, the hot electrons generated from plasmon decay play an important role only in decreasing the reaction barrier, where the hole electrons temperately attach to the molecules, and then the neutral potential energy surface (PES) being ion PES12345678911121314151112131415In summary, we designed and fabricated a 3D plasmonic hybrid nanostructure of flower-like graphene nanosheets sandwiched by Ag nanoparticles on silicon nanocone arrays, which has been applied for graphene enhancing plasmon-driven catalytic reactions and showed the more superiority than 2D graphene-nanoparticles hybrids. Our experimental results reveal that the sensitivity of the co-driven catalytic reaction in this 3D plasmonic hybrid nanostructure can be improved by fourfold over that of the plasmon-driven catalytic reaction. The mechanism of plasmon and graphene co-driven catalytic reaction has been theoretically revealed for the first time. Our results are very valuable for designing and fabricating excellent SERS substrates based on graphene and plasmon co-driven surface catalytic reactions, graphene-based surface plasmon sensors, and so on.2 and SF6 are used as the etching gas to fabricate the Si nanocones. Geometry of Si nanocones, for example, the height, and density as well as the aspect ratio, were modulated through changing etching parameters. Samples of the Si nanocone array were fabricated at the conditions: SF6/O2 = 22/7, pressure of 6 mTorr, ICP power of 800 W, and the etching time of 7 minutes. An as-formed silicon nanocone array served as the substrate to grow the few-layer graphene nanosheets by hot filament chemical vapor deposition (HFCVD). The Ar, CH4 and H2 were fed into the HFCVD system with the ratio of 40:10:1 for the growth of the petaloid graphene nanosheets (PGNSs). And, the growth processes were carried out at the temperatures of 1000 °C, where the pressure was maintained at 2 kPa. The positive bias voltage was added on the filament during growth. The bias current was measured with the ammeter. The structure, morphology and the chemical bond-state of the obtained samples were characterized by the scanning electron microscopy (SEM), and transmission electron microscopy (TEM), and high resolution transmission electron microscopy (HRTEM) (at 200 kV), the electron energy loss spectroscopy (EELS), and the Raman spectra, respectively. And then, the Ag nanospheres were prepared by the 6 nm thick Ag nanofilm deposited on the PGNSs using the E-beam evaporation.The silicon nanocone arrays are fabricated by the maskless etching in the inductively coupled plasma (ICP) system, where O−5 M PATP or 4NBT ethanol solution for 4 hours, and then which was rinsed with the ethanol to get rid of the non-adsorbed molecules. The 10−5 M solution was gradually diluted to 10−6 M and up to 10−11 M by Multiple dilutions.SERS measurements were performed with 532 nm laser with the different powers from 0.1 mW to 1 mW. The objective lens is used to focus the excitation laser on the samples, and to collect Raman spectra with the 1μm laser spot. The as-fabricated graphene nanosheets/Ag-nanoparticles hybrids structure substrates are immersed in 102/(V*s) and 1×106 m/s, respectively. The ground state geometry of graphene-O2 system was optimized with density functional theory (DFT)FDTD was used to demonstrate the plasmon intensity and field distribution in our substrate, which revealed the contribution of graphene to electronic field enhancement. The diameter of Ag nanoparticles is set at 30 nm and the graphene sheet is set at 1 nm (3 layers). Fermi energy, mobility and Fermi velocity of graphene in the calculation are 0.4 eV, 1500 cmHow to cite this article: Zhao, J. et al. Three Dimensional Hybrids of Vertical Graphene-nanosheet Sandwiched by Ag-nanoparticles for Enhanced Surface Selectively Catalytic Reactions. Sci. Rep.5, 16019; doi: 10.1038/srep16019 (2015).


Nanofluids with enhanced thermal properties are candidates for thermal management in automotive systems, with scope for improving energy efficiency. In particular, many studies have reported on dispersions of nanoparticles with long-term stability in the base fluid, with qualitative evaluations of the dispersion stability via either the naked eye or optical instruments. Additives such as surfactants can be used to enhance the dispersion of nanoparticles; however, this may diminish their intrinsic thermal properties. Here, we describe molecular dynamics simulations of nanofluids containing graphene sheets dispersed in ethylene glycol and water. We go on to suggest a quantitative evaluation method for the degree of dispersion, based on the ratio of the total number of nanoparticles to the number of clustered nanoparticles. Moreover, we investigate the effects of functional groups on the surface of graphene, which are expected to improve the dispersion without requiring additives such as surfactants due to steric hindrance and chemical affinity for the surrounding fluid. We find that, for pure graphene, the degree of dispersion decreased as the quantity of graphene sheets increased, which is attributed to an increased probability of aggregation at higher loadings; however, the presence of functional groups inhibited the graphene sheets from forming aggregates. Thermal management is a crucial factor in heat transfer equipment, and determines the energy efficiency. In automotive systems, working fluids such as water, ethylene glycol (EG), propylene glycol, engine oil, mineral oil, kerosene oil, and silicon oil are indispensable for effective thermal management; however, enhancements in the thermal conductivity of these fluids are desirable for improved energy efficiency . DispersThe concept of a “nanofluid” was first reported by Choi  at Argon2 nanotubes =90. Therefore, the degree of dispersion would be Gact/Ntotal=90/100=0.9.where Figure Chemical exfoliation is a promising method to form graphene sheets from graphite. During this process, various types of oxide functional groups are formed on the surface of graphene sheets, which can influence the dispersion stability in a base fluid. These functional groups can induce hydrogen bonding, stabilizing the dispersion, and providing active sites that enable the formation of heterogeneous structural composites. We investigated three functional groups at the surface of graphene sheets: alcohol and amide (amine) groups, as shown in Fig. Figure On the other hand, we compared a value of the mean square displacement (MSD), or mean square fluctuation between graphene sheets with and without functional groups. In statistical mechanics, it demonstrates the measure of the spatial extent of random motion measuring the portion of the system “explored” by the random walker, and is defined asT is the time for average, and x0 is the reference position of the particle. Typically, this reference position will be the time-averaged position of the same particle. Figure where We investigated the degree of dispersion of nanofluid formed of graphene sheets with and without functional groups at graphene loadings in the range 1–25 wt.%. Figure The degree of dispersion of the functionalized graphene sheets exhibited a much weaker dependence on the graphene loading (see the red triangles in Fig. The concept of a nanofluid was first suggested in 1995, and nanofluids are promising candidates for thermal management in automotive systems. We carried out MD simulations of nanofluids formed of graphene sheets dispersed in a 1:1 mixture of EG and water. In this study, we have suggested an evaluation method to quantify the degree of dispersion of nanoparticles in a base fluid based on the ratio of the total number of nanoparticles to the number of nanoparticles that exist in clusters.Furthermore, we clearly quantify that the functional groups at the surface of graphene sheets improved the degree of dispersion due to steric hindrance and chemical affinity for the surrounding fluid, without requiring additives. With non-functionalized graphene sheets, the degree of dispersion decreased as the graphene loading increased because of an increased probability of aggregation, whereas the functionalized graphene sheets did not tend to aggregate, even at high loadings.


In principle, accurate guideline recommendations should lead to optimal management based on a secure diagnosis. However, current IPF diagnostic guidelines do not meet the needs of a major sub-group (possibly the majority) of patients with idiopathic pulmonary fibrosis (IPF). A great many IPF patients have HRCT appearances of “possible UIP”. A surgical biopsy is very often impracticable due to age, disease severity, co-morbidities or patient refusal. A guideline-based diagnosis cannot be made in these patients, although the diagnosis is often obvious. Inflexible diagnostic criteria, although essential for treatment trials, must necessarily be structured around an inflexible diagnostic algorithm. With this approach, non-standardised information  must be omitted, including observed disease behaviour prior to and on treatment, findings on bronchoalveolar lavage, likelihoods in relation to age and a wealth of ancillary clinical information. However, when a diagnosis cannot be made using guideline criteria, a probable or highly probable “working diagnosis” of IPF can and should be made in most IPF patients by means of clinical reasoning, integrating all available non-standardised information. Idiopathic pulmonary fibrosis (IPF), the most progressive and most prevalent of the idiopathic interstitial pneumonias (IIPs), requires a radically different management from the other IIPs due to its prognostic implications and the advent of IPF-specific therapies. With regards to their management, the remaining IIPs can largely be conceptualised as forms of immune dysregulation; however, this model has failed in IPF .It might be supposed that the accurate diagnosis of IPF should depend upon the application of evidence-based diagnostic guidelines. Yet, in at least half of IPF patients, the diagnosis cannot be made using current American Thoracic Society/European Respiratory Society/Japanese Respiratory Society/Latin America Thoracic Association (ATS/ERS/JRS/ALAT) guideline criteria . The ‘faCurrently, in roughly half of IPF patients, ATS/ERS/JRS/ALAT 2011 diagnostic criteria for IPF are not satisfied and the disease remains unclassifiable. In this setting, clinicians must speculate on the best course of action – whether to manage the disease according to a diagnosis of alternative disorders or to apply IPF-validated anti-fibrotic therapies. In a nutshell, the ATS/ERS/JRS/ALAT 2011 IPF diagnostic guidelines are ‘broken’.How has it come to this? The pace of recent events is partly to blame. In 2009/2010, when the ATS/ERS/JRS/ALAT guidelines were formulated, there was no universally accepted therapy known to delay disease progression and it was generally agreed that best management consisted of participation in a novel treatment trial , was avaNevertheless, recent events are only partly to blame. There is a second issue related to the decline and fall of ‘eminence-based medicine’. Fortunately, the age of the opinion hegemony – a handful of experts driving best medical practice based on their anecdotal views – is over. However, we have fallen into a new and equally damaging trap – the belief that logic and common sense must be discarded and only data formalised within an ‘evidence base’ can be taken into account. It may seem surprising that the Evidence-based Renaissance Group recently published a perspective entitled, Evidence-based Medicine, a Movement in Crisis . HoweverWhatever else is said about the ATS/ERS/JRS/ALAT 2011 IPF guidelines, it cannot be doubted that, for the first time, an evidence-based ethos was energetically applied to the diagnosis and management of IPF. However, it was telling that nothing useful was said about that great desideratum, multidisciplinary diagnosis. Certainly, the phrase ‘multidisciplinary diagnosis’ makes cameo appearances from time to time, but what is described in the document as multidisciplinary diagnosis is unrecognisable to most practising clinicians, radiologists and histopathologists. The casual reader, focusing on tables and key conclusions, might be excused for imagining that the process of multidisciplinary diagnosis consists of amalgamating a histological grade and a high-resolution computer tomography (HRCT) category, each reached in isolation, providing a definition of possible, probable or definite IPF. In this model, the clinician has no particular role, apart from the exclusion of a primary cause of pulmonary fibrosis. The problem, here, is that diagnosis for trial purposes, enshrined in the 2011 guideline, differs radically from enlightened multidisciplinary diagnosis.In treatment trials, it is essential that diagnosis is rigorous and standardised in order to minimise diagnostic variation between treatment arms. In essence, this means the use of simplistic clinical exclusion criteria and HRCT data, and a requirement for a diagnostic surgical biopsy when HRCT appearances are inconclusive. Rigid diagnostic criteria require the exclusion of all information not available in all patients. Observed disease behaviour, on and off therapy, does not feature as a possible influence on the likelihood of IPF in the ATS/ERS/JRS/ALAT 2011 guidelines because, at enrolment into a trial protocol, observations of disease behaviour are not always available. Bronchoalveolar lavage is not a part of the diagnostic algorithm in all countries and therefore it was relegated to an undefined ‘weak negative’ role in the ATS/ERS/JRS/ALAT 2011 guidelines. This is relevant because, in clinical practice, hypersensitivity pneumonitis exposures are not, in reality, a simple ‘present/absent’ dichotomy. Often, there is real difficulty in assigning significance to exposure. The whole question of what constitutes occult connective tissue disease is too complex to summarise concisely, but frequently contributes to the warp and weft of multidisciplinary diagnosis.For trial purposes, diagnosis must necessarily be ‘dumbed down’. Multidisciplinary diagnosis is a different matter altogether. Trial diagnostic criteria require the consideration only of data standardisable in all patients. By contrast, multidisciplinary diagnosis requires the consideration of all potentially relevant information in every individual patient. Trial diagnostic criteria confine expert judgement to the isolated views of the radiologist and histopathologist, with subtle clinical interpretation disallowed. Multidisciplinary diagnosis consists of debate and the integration and reconciliation of a huge amount of ancillary clinical information. It is sometimes supposed that the major value of multidisciplinary diagnosis consists simply of broadening expertise. However, it can also be argued that the true value of a multidisciplinary team lies in bringing together trained minds accustomed to civilised disagreement and the scrutiny of logic and common sense. Above all, the use of logic, common sense and review of all data (which differs widely in its completeness in every patient) cannot be validated using an evidence-based approach in which inflexible diagnostic criteria are required.any fool can make a rule and any fool will mind it”.With the use of a multidisciplinary approach, a diagnosis of IPF can often be made, based on very high probability, in patients who lie outside guideline criteria. Anti-fibrotic therapy in this large patient subgroup can be instituted with the same confidence as in ‘definite IPF’, as defined in the 2011 guidelines. In other cases, in which IPF is merely the most likely of several possible diagnoses, an eventual working diagnosis of IPF and the introduction of anti-fibrotic therapy can be justified by subsequent disease progression despite immunomodulatory therapy. In this area, medicine is as much an art as a science. The evidence-base apparatchik need not despair – the time will come for the multidisciplinary approach in IPF to be subjected to evidence-based evaluation. The accuracy of a flexible approach to diagnosis, based on the integration of clinical reasoning and morphologic (CT and pathology) pattern recognition, can be examined against subsequent natural history and treatment course, given that the value of making a diagnosis is in the provision of this information. However, the answer does not lie in recreating new rigid diagnostic criteria for IPF to the detriment of clinical reasoning. As Thoreau, father of the pragmatic philosophy movement once said, “


This unique feature stems from the fact that the permittivity of doped graphene can be close to zero at a certain photon energy. The electrical controllability of evanescent coupling strength can enable novel design of optical devices. As a proof-of-concept, we designed a waveguide-type optical modulator of a novel operation principle: transmission modulation depends on the electrically controlled existence of a guided-mode of the waveguide, not the variation of the ohmic loss of graphene, resulting in a low insertion loss and a small device footprint.Electrically tunable permittivity of graphene provides an excellent tool in photonic device design. Many previous works on graphene-based photonic devices relied on variable absorption in graphene, which is naturally small in the optical region, and resonant structures to enhance it. Here we proposed a novel scheme to control evanescent coupling strength by inserting two graphene layers to a frustrated total internal reflection (FTIR) configuration. The resulting structure behaves in a drastically different way from the original FTIR: optical transmission though the structure can be electrically controlled from ~10 Recently, graphene29136Among the many graphene-based optical devices, optical modulators have attracted much interest because of their broad optical bandwidth and Si waveguide compatibility5614In this work, we propose a novel scheme to modulate optical transmission by combining the tunable permittivity of graphene, which can be very close to zero, and the frustrated total internal reflection (FTIR)EF) of graphene is properly controlled to make its permittivity negativeEF , yielding a reflection coefficient of approximately one, thus, greatly reducing the transmission. Because the EF of graphene can be controlled by applying a gate voltageWhen light from a high-index medium is refracted on the surface to a lower-index medium, total internal reflection will occur if the angle of incidence exceeds a certain limit known as the critical angle. If the low-index medium is very thin and another high-index medium is added to the end of the structure, part of the incident light can tunnel through the low-index medium and transmit to the other end; this is known as FTIREF = 0.9 eV and the thickness of the SiO2 layer tgap, was 10 nm. The spectra were calculated using the rigorous coupled-wave analysis (RCWA) method. As expected, the presence of graphene drastically modified the behavior of the FTIR. At the photon energy Eph of 1.304 eV (λ = 951 nm), reflection is approximately 100% over a wide incident angle range. This is attributed to the near-zero permittivity of graphene at that photon energy when EF = 0.9 eV, which is the result of the light incident at an oblique angle on the epsilon-near-zero (ENZ) material, as previously reportedEph ~ 1.215 eV (corresponding to λ = 1021 nm), transmission is high with little dependence on angle over a wide incident angle range. Eph > 1.304 eV, the real part of the permittivity of graphene is positive and approaches zero at Eph = 1.304 eV. This region is known as the locus of Brewster angle in the  planeEph < 1.215 eV, there is the leaky plasmonic mode due to the two graphene layers. These leaky modes can be excited by the plane wave incident from the high-index medium (Si) because their dispersion curves lie above the light line (black solid line). Using the dispersion curves of the leaky modes, we calculated the Eph that satisfies the phase-matching condition as a function of the angle of incidence. The plots are shown in Eph < 1.215 eV) is very flat near the light line due to the thinness of the graphene layerEph ~ 1.215 eV has little dependence on the angle of incidence over a wide angle range. k for the two leaky modes is near zero, which explains why the optical loss of the GA-FTIR structure is so small, as seen in To understand the behavior of the GA-FTIR, we calculated the dispersion curves of leaky modes in the GA-FTIR structure using the method previously described by J. Hu and C. R. MenyukEph = 1.304 and 1.215 eV are presented in Eph = 1.304 eV, transmission of <1% was obtained for angle of incidence θ > 15°, as demonstrated in Eph = 1.215 eV, transmission >98.5% was observed over an angle range of 0° < θ < 75°, where reflection and absorption were both <1% as shown in To illustrate the unique performance of the GA-FTIR structure for a wide range of angle of incidence, the reflection and transmission spectra for tgap = 40 nm but the same chemical potential used in the previous calculations (EF = 0.9 eV). The behavior of the structure is similar to that with tgap = 10 nm. High reflection occurred at the same photon energy, Eph = 1.304 eV, as that for the structure with tgap = 10 nm because EF was the same. However, the photon energy for high-transmission shifted closer to that of the ENZ point (Eph = 1.283 eV) because of the corresponding shift of the second leaky mode in the dispersion curve, as seen in tgap = 10 and 40 nm and different chemical potentials of graphene but with a fixed angle of incidence of θ = 60°. The choice of the angle of incidence was arbitrary. Changing EF shifts the wavelength  at which the minimum (near-zero) permittivity of graphene occurs and thus, the wavelength of the maximum reflectance (Rmax). Similarly, the wavelength of the edge of the second leaky mode, where the minimum reflectance (Rmin) occurs, also shifts with EF. Therefore, the reflectance of the GA-FTIR at a certain wavelength can be modulated by electrically controlling EF . Rmax tends to increase with EF because the minimum permittivity of graphene tends to zero as EF increases, according to the Kubo formulaRmin tends to decrease as EF increases. Consequently, the modulation depth, defined as Rmax/Rmin, increases as EF increases. For the structure where tgap = 10 nm, shown in Rmax ~ 98.9% and Rmin ≤ 10−5 are achieved at EF = 0.9 eV. In this case, the ratio of the modulation depth to the insertion loss, which is frequently used as a figure of merit (FOM) for modulators, is ~1040. To the best of our knowledge of graphene-based modulators, this FOM is higher than the best value reported in the literature28We investigated the tunability of the spectral behavior of the GA-FTIR. tgap = 40 nm, shown in tgap = 10 nm because of the wider gap. On the other hand, the change in chemical potential (ΔEF) that is required for full modulation of reflectance is smaller because the difference in photon energy between the ENZ point and the second leaky mode edge is smaller, as mentioned above. For example, if the operating wavelength is λ = 951 nm, then ΔEF = 0.07 eV (from 0.9 to 0.97 eV) is needed for the full modulation of reflectance when tgap = 10 nm. Whereas, a smaller ΔEF , e.g., ΔEF = 0.02 eV (from 0.9 to 0.92 eV), is required for the full modulation of reflectance when tgap = 40 nm.Changing the gap size does not fundamentally alter the behavior of the structure. For the structure where 2 layer, and a semi-infinite Si medium on the bottom. EF = 0.9 eV and an operating wavelength of 951 nm. The operating wavelength and EF were chosen to obtain wide-angle high reflectance from the GA-FTIR scheme. As a result, the top slab supports the guided mode, and thus a launched TM wave propagates along the top slab waveguide, resulting in a high-transmission state (on-state). EF = 0.97 eV at the same operating wavelength. In this case, the GA-FTIR scheme offers very low reflectance over a wide range of angle of incidences and, thus, the top slab no longer supports the guided mode, so a launched wave leaks into the bottom Si medium, resulting in a low-transmission state (off-state). For comparison, TM wave propagation in a structure in which the two graphene layers and the SiO2 gap are replaced by a Si medium, as shown in the inset of In this work, we propose a waveguide-type optical modulator based on the GA-FTIR scheme. The wide-angle reflectance modulation properties of the GA-FTIR structure can be utilized to control the existence of a guided mode if one of the two semi-infinite high-index (Si) media is replaced with a slab waveguide, as depicted in 2 layer was 10 nm thick, the Si optical waveguide was 400 nm high and 500 nm wide, and the device was 4 μm long. EF = 0.9 and 0.97 eV, and Building on the results of the 2D optical modulator calculations, we investigated a more practical device structure, shown in EF = 0.524 and 0.54 eV), which are appropriate for an operating wavelength of 1550 nm. The modulation performance of the device depicted in In this work, we proposed a novel scheme to control optical tunneling in a FTIR configuration by introducing two graphene layers doped to have the ENZ effect. In addition to the incident angle independent high reflection due to the ENZ graphene layer, very flat dispersion characteristics of the leaky mode supported by two plasmonic coupled graphene layers provides strong optical tunneling modulation over a wide incident angle range, without requiring any complex resonant structures. The electrical controllability of evanescent coupling strength provided by the proposed scheme, which is unique to this scheme to the best of our knowledge, can enable novel design of optical devices. As a proof-of-concept, we designed a waveguide-type optical modulator of a novel operation principle: transmission modulation depends on the electrically controlled existence of a guided-mode of the waveguide.F . In practice, the breakdown of a gap material, SiO2 in the gate structure limits the maximum achievable value of EF: the carrier density in graphene is related to the chemical potential EF as NG = EF2/(πħ2VF2), where ħ is the reduced Plank constant and VF is the Fermi velocity, and from the simple capacitive model, the electric field in the gap layer is given by qNG/εoεSiO2 , which should be smaller than a certain value to avoid the breakdown of the gap material. This limitation can be overcome with combining chemical doping and electrostatic doping induced by a gate voltage. Chemical doping of up to EF = 2.0 eV have been successfully demonstratedF = 0.9 to 0.97 eV, we can dope graphene chemically up to EF = 0.9 eV and apply varying gate electric field from 0 to ~8.3 MV/cm (assuming VF = 106 m·s−1), which lies within SiO2 breakdown electric field of ~13 MV/cmThe operating wavelength of the proposed scheme can be varied over a wide range by changing the chemical potential of graphene, E2O3 could increase the maximum achievable chemical potential. The relative permittivity of Al2O3 is 1.752 and the breakdown electric field of high quality Al2O3 film formed by the atomic layer deposition can be as high as 30 MV/cm2O3 adopted for the gap material, EF up to ~0.83 eV can be achieved by electrostatic doping alone, induced by a gate voltage, which is much higher than the required value of EF for device operation at λ = 1550 nm and will allow the operating wavelength as short as ~1182 nm.The use of a better insulator with higher permittivity and breakdown electric field such as AlVF =106 m·s−1 used for the calculation in this work is a conservative choice and likely represents the lower limit, corresponding to the case where electron - electron interactions are weakVF ~ 2.49 × 106 m·s−1 and ~1.49 × 106 m·s−1 were achieved with quartz and BN substrates, respectivelyVF ~ 1.19 × 106 m·s−1, electrostatic doping alone can achieve EF ~ 0.54 eV and enable device operation at λ =  1550 nm, avoiding breakdown of SiO2.Another way to achieve higher chemical potential for a given gate voltage is to increase the Fermi velocity: a two fold increase in Fermi velocity would reduce the required gate voltage by a factor of four to achieve the same chemical potential. The value of 2 gap material, the maximum achievable chemical potential by the gate voltage is ~0.45 eV even for the conservative value of Fermi velocity of VF = 106 m·s−1, which is large enough to allow device operation at λ = ~2 μm.As mentioned early, the operating wavelength of the proposed scheme can be varied over a wide range and the allowed shortest operating wavelength is limited only by the maximum achievable chemical potential of graphene. For the GA-FTIR scheme with SiO2 were assumed to be 3.42 and 1.452, respectively, and the permittivity of graphene was calculated using the Kubo formulaVF was 106 m·s−1; and the mobility μ was 1 m2·V−1·s−1.In our numerical analysis, the permittivity of Si and SiOThe spectral response of the scheme and its angle dependency were calculated by using the commercial R-Soft DFMOD RCWA software. The finite element method commercial software COMSOL was used to calculate the frequency domain field distribution of the 2D waveguide type structure. For 3D structure, the FDTD commercial package from Lumerical was used.How to cite this article: Tran, T. Q. et al. Tunable Wide-Angle Tunneling in Graphene-Assisted Frustrated Total Internal Reflection. Sci. Rep.6, 19975; doi: 10.1038/srep19975 (2016).


Using a Rhox3 short hairpin (sh) RNA with 100% complementarity to all 8 Rhox3 paralogs, expressed from a CRE-regulated transgene, we successfully knocked down Rhox3 expression in male germ cells in vivo. These Rhox3-shRNA transgenic mice had dramatic defects in spermatogenesis, primarily in spermatocytes and round spermatids. To determine whether this phenotype was caused by reduced Rhox3 expression, we generated mice expressing the Rhox3-shRNA but lacking the intended target of the shRNA—Rhox3. These double-mutant mice had a phenotype indistinguishable from Rhox3-shRNA-expressing mice that was different from mice lacking the Rhox3 paralogs, indicating that the Rhox3 shRNA disrupts spermatogenesis independently of Rhox3. Rhox3-shRNA transgenic mice displayed few alterations in the expression of protein-coding genes, but instead exhibited reduced levels of all endogenous siRNAs we tested. This supported a model in which the Rhox3 shRNA causes spermatogenic defects by sequestering one or more components of the endogenous small RNA biogenesis machinery. Our study serves as a warning for those using shRNA approaches to investigate gene functions in vivo.RNA interference (RNAi) is widely used to determine the function of genes. We chose this approach to assess the collective function of the highly related  A particularly useful application is to elucidate the functions of gene paralog families. As long as the family members are sufficiently related, a single siRNA can simultaneously knockdown all family members and thereby unveil the redundant function of the gene paralog family [RNA interference (RNAi) is a widely used approach to silence genes and thereby deduce their function. While primarily used for g family –3.reproductive homeobox (Rhox) gene cluster. This gene cluster harbors 33 homeobox genes that are selectively expressed in the male and female reproductive tract, suggesting that they encode transcription factors critical for reproduction [Rhox genes are expressed in reproductive tract, their spatial and temporal expression patterns are different. For example, RHOX10 is expressed specifically in spermatogonia and early spermatocytes [RHOX genes are expressed specifically in germ cells in the testes and are aberrantly methylated in infertile patients [We elected to take advantage of this approach to conditionally knockdown highly related gene paralogs in the oduction . Althougatocytes  and RHOXatocytes . RHOX5 iatocytes ,7,8. Thepatients ,10.Rhox3 gene paralogs, which comprise eight gene copies in mice that are highly homologous with each other—displaying 98.2–99.7% sequence identity [Rhox3 gene paralogs are expressed in the adult testes except Rhox3d , which is likely to be a pseudogene since it harbors a premature translation termination codon [Rhox3 gene paralogs appear to have expanded as a result of selection pressure exerted specifically in the mice lineage, as rats have only a single Rhox3 gene [Rhox3 paralogs have undergone purifying selection in the amino-terminal region and weak positive selection for changes in the homeodomain region [The subject of this report is the identity –14. Usinon codon . The Rhon region ,12,14. Tn region .Rhox3 paralogs are interspersed with other Rhox genes, a knockout strategy to determine their collective in vivo function is not feasible and thus we chose to use a RNAi approach instead. To provide specificity, we used a conditional RNAi approach in which the Rhox3 shRNA was selectively expressed in male germ cells, the cell type that we found normally express Rhox3. As described herein, this germ cell-specific shRNA approach successfully knocked down Rhox3 expression in the testis in vivo and it led to dramatic defects in spermatogenesis.Because these eight In vitro RNAi studies to determine gene function in cultured cells typically incorporate controls in their experiments to distinguish whether the phenotypic defects observed are the result of depletion of the target gene product or an off-target effect [in vivo rarely have such controls [in vivo studies, conditional/inducible shRNA expression methods have been developed that restrict the expression of the siRNA to specific cell types, developmental stages, and/or temporal windows [Rhox3 gene paralogs—reversed the phenotypic defects caused by the Rhox3 shRNA. An off-target effect seemed a distinct possibility given that we found that mice lacking the Rhox3 paralogs exhibited different defects from mice expressing Rhox3-shRNA. Indeed, we found that elimination of the intended target did not measurably reduce or change the defects caused by the Rhox3 shRNA, thereby demonstrating that the Rhox3 shRNA acts by a mechanism independent of its ability to knockdown Rhox3. We considered and tested different mechanisms by which the Rhox3 shRNA might cause off-target effects. This led to the discovery that Rhox3 shRNA mice have reduced production of endogenous (endo)-small interfering (si) RNAs. In contrast to microRNAs (miRNAs), which are expressed in most of mammalian cells, endo-siRNAs are only known to be expressed in germ cells and embryonic stem cells in mammals [t effect . In contcontrols . To redu windows ,19. Whil mammals –23. Our ad libitum. Euthanasia was performed by CO2 inhalation for 5 min followed by cervical dislocation. All efforts were made to minimize suffering.This study was carried out in strict accordance with the Guidelines of the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Diego. The protocol was approved by the IACUC at the University of California, San Diego (Permit Number: S09160). All animals were housed under a 12 h light: 12 h darkness cycle and provided with food and water Rhox3 paralogs. U6 promoter driven-shRNA expression vectors (G-1256 and G-1257) targeting Rhox3 were generated by subcloning PCR products that contained both the target sequence and the mouse U6 promoter into the pGEM-T Easy vector, as previously described [Rhox3 target sequences used were 5’-GGAGCAGATTCCTGAGCAT-3’ (G-1256) and 5’-GCATGTTGAAGGAGAGAGT-3’ (G-1257). The construction of the shRNA expression vector to target firefly luciferase (G-472) is previously described [Rhox3 shRNA transgenic construct (G-1029), oligos MDA-4212 and-4213, which contained shRNA sequences, were annealed and ligated into the ApaI and EcoRI sites of the pBS/U6-ploxPneo plasmid , as described elsewhere [The siDESIGN algorithm  was used to design siRNA target sequences that have 100% complementarity with all 8 escribed . The Rholsewhere .Rhox3 shRNA plasmids. A U6-driven Luciferase shRNA plasmid was used as a control in transfection experiments. To delete the loxP-flanked neomycin cassette from the U6-ploxPneo Rhox3 shRNA transgenic construct, a Cre recombinase (pMC-Cre) expression plasmid was co-transfected. Total cellular protein extracts were prepared 48 h after transfection.HeLa cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum and 50 mg/ml of penicillin and streptomycin and transfected with Lipofectamine-2000 following the manufacturer’s instructions. All cell culture reagents were purchased from Invitrogen Technologies . Cells were plated on 6-well culture plates and cotransfected with 500 ng of an expression vector encoding FLAG-tagged RHOX3 and various concentrations of U6-driven Rhox3 shRNA mice, a 2.7-kb DNA fragment containing the U6-ploxp-neo Rhox3 shRNA transgene in the G-1029 plasmid was excised using the restriction enzymes, AflIII and Asp718, and gel purified for microinjection. Positive transgenic mice were detected by PCR using tail DNA as a template and the primers MDA-4290 and-4291, which are specific for the U6 promoter and neomycin cassette. Four founder lines  containing the transgene were obtained. All lines were mated with Stra8-iCre mice to generate double transgenic lines (expressing the Rhox3 shRNA and CRE) to make germ cell-specific shRNA mice (Rhox3-shRNA mice) and the single transgenic littermates (either Rhox3-shRNA or Stra8-iCre) served as control (Control mice). Line 2 was subjected to detailed histological analysis and also bred with Rhox-c-KO mice.Transgenic mice were generated by pronuclear microinjection of FVB/NJ mouse embryos  at the M. D. Anderson Cancer Center transgenic core facility. To generate U6-ploxPneo Rhox-c-floxed mice, two targeting vectors were used to insert loxP sites at the beginning (upstream genomic region of Rhox1) and the end (the second intron of Rhox13) of the Rhox cluster. The targeting vectors were constructed from BAC clones . The two targeting vectors were electroporated sequentially into ES cells; in each case, cell clones that underwent homologous recombination were selected by Southern blot analysis. An ES cell clone containing both loxP sites was selected for blastocyst injection to obtain chimeric mice. Germline-transmitted Rhox-c-floxed mice were bred with EIIa-Cre mice to generate mice lacking the entire Rhox cluster (Rhox-c-KO or Rhox-c-heterozygote mice). The Rhox-c-heterozygote female mice were bred with Rhox3-shRNA transgenic mice to generate Rhox-c heterozygote; Rhox3-shRNA female mice and these mice were further bred with Stra8-iCre mice to obtain Rhox-c KO;Rhox3-shRNA;Stra8-iCre male mice (Rhox-c-KO/Rhox3-shRNA mice).To generate For quantitative RT-PCR (qRT-PCR) analysis, total cellular RNA was extracted from cell lines and tissues by the Trizol method  and converted to cDNA using iScript reverse transcription . Relative gene expression was determined by SYBR green incorporation in an Applied Biosystem StepOnePlus real-time PCR system, as described previously , with prCytoplasmic and nuclear protein extracts from adult testis were prepared as described elsewhere . WesternRhox3-shRNA mice  and control mice , using an Affymetrix GeneChip mouse exon 1.0 ST array. Raw data was normalised using robust multi-array averaging with correction for GC content of the probes (GCRMA) [Rhox3-shRNA testes were statistically tested using one-way ANOVA. All microarray data analysis was carried out using Partek genomics suite v6.3 . Microarray data is available from Gene Expression Omnibus under accession number GSE64324.Microarray analysis was performed at the University of California, San Diego (UCSD) GeneChip microarray core facility using total cellular RNA from P15 testis isolated from  (GCRMA) . Gene exin situ apoptosis detection kit , following the manufacturer’s instructions.For histology analysis, testes and epididymis were fixed in either 10% neutral buffered formalin or Bouin’s fixative and stained with hematoxylin-eosin or periodic acid Schiff-hematoxylin, as previously described . The sizRhox3 gene paralogs have the potential to function, we examined their developmental expression pattern during the first wave of spermatogenesis. Using qRT-PCR analysis with primers designed to amplify all eight Rhox3 paralogs, we found that Rhox3 mRNA is expressed at only trace levels during early stages of the first wave of spermatogenesis—postnatal day 5 (P5) to P18—when postnatal germ cells first proliferate and undergo meiosis. Rhox3 mRNA level is upregulated at P20  in testes nuclear lysates .. In addjsd mice testes, which lack virtually all germ cells except for undifferentiated spermatogonia [jsd mutant testes, implying that RHOX3 is expressed predominantly in germ cells (Rhox3 mRNA (at P20)  ., sugges(at P20) .in vivo, we elected to use a conditional RNAi approach developed by Shukla et al. [To determine the functional role of RHOX3 in male germ cells a et al. . In thisRhox3 genes in vivo, we first identified siRNAs exhibiting 100% complementary with all eight Rhox3 gene paralogs and then screened for those that efficiently depleted Rhox3 expression in vitro. This screen led to the identification of a siRNA sequence that reduced RHOX3 protein expression by ~90% in HeLa cells upon cotransfection with an expression vector encoding FLAG-tagged mouse RHOX3 (data not shown). We inserted this siRNA sequence (in a shRNA context) into the U6-ploxP neo construct  to create a Rhox3-shRNA transgene construct  [Stra8-iCre mice were crossed with mice from the four transgenic lines to generate double transgenic mice that carry both Stra8-iCre and the Rhox3-shRNA transgene. These Rhox3-shRNA;Stra8-iCre mice  were sacrificed at 6 weeks of age and analyzed for endogenous Rhox3 expression in the testis. The single transgenic mice that carry either the Stra8-iCre or Rhox3-shRNA transgene from same litter served as control mice. Testes from the 4 different Rhox3-shRNA mice lines displayed a ~50% to 85% reduction in Rhox3 mRNA level relative to control mice testes (Rhox3 mRNA level (Line 2) and observed a dramatic decrease in RHOX3 protein level (>95%) (The  and P7) . Stra8-ie testes . We examl (>95%) .Rhox3-shRNA mice lines 2, 12, and 13 decreased , compared to littermate control mice that had the Rhox3-shRNA transgene but lacked CRE  was decreased by ~50% . The majority of the tubules had vacuoles  increased numbers of apoptotic pachytene spermatocytes in seminiferous epithelial cycle stage IV, leading to missing generations of spermatocytes and/or spermatids in particular tubule areas in stages VI-VIII  fe VI-VIII ; (iii) m VI-VIII ; (iv) fe VI-VIII ; (v) a r VI-VIII . The redrol mice . We alsorol mice . Finallyrol mice .Rhox3-shRNA and control mice (Rhox3-shRNA adult testes relative to control adult testes. Some spermatocyte marker genes (Spo11 and Rec8) were also decreased significantly, but by less (~2- to 3-fold) than post-meiotic genes. Other spermatocyte marker genes (Sycp1 and Sycp3) did not exhibit a significant change in level. This confirmed that there was a defect in the progression of spermatocytes, probably at a mid or late phase. In contrast, spermatogonia markers (Stra8 and c-Kit) were increased in level by ~2- to 3-fold in Rhox3-shRNA testes, which is consistent with an enrichment of spermatogonia as a result of depletion of spermatocytes and spermatids. Similar increases were observed for Sertoli cell markers (Wt1 and Rhox5) and Leydig cell markers (Cyp450 and Lhgcr), suggesting that these cell populations were enriched too. Similar findings were observed in mid-postnatal (P20 and P25) testis samples . In brief, we introduced loxP sites at both ends of the Rhox cluster in ES cells (by homologous recombination), generated mice harboring these loxP sites, and mated them with EIIa-Cre mice (which express Cre from a ubiquitous promoter) to generate Rhox-cluster-KO (Rhox-c-KO) mice progeny. These Rhox-c-KO mice did not exhibit the defect in meiotic prophase we detected in Rhox3-shRNA mice, but instead had only a modest (~28%) reduction in testes size . If the Rhox3-shRNA mice phenotype is the result of depletion of Rhox3, Rhox-c-KO/Rhox3-shRNA mice would be predicted to show the same defect as Rhox-c-KO mice because Rhox3 is not there to be depleted by shRNA. Alternatively, if the defect from the shRNA is independent of the presence of Rhox3, Rhox-c-KO/Rhox3-shRNA mice would be predicted to show the same severe defect as Rhox3-shRNA mice. We found that the Rhox-c-KO/Rhox3-shRNA mice showed virtually the same phenotype as that of Rhox3-shRNA mice: 80% reduction in testis weight  that corresponds to mid-meiosis. The time point is ~10 days after Rhox3 shRNA would be predicted to be first expressed as a result of CRE-mediated transcriptional activation [Rhox3-shRNA mice at this developmental time point , we found that only one of the 8 down-regulated genes exhibit complementarity with the putative Rhox3 shRNA seed site—position 2–8. This gene, Cldn2, encodes a member of the claudin family of proteins, which are considered to be the most important components of tight junctions. While some claudins have been shown to function in spermatogenesis, Cldn2 does not appear to have such a role, as Cldn2-KO mice have only been reported to have kidney dysfunction; no fertility defects were observed [What is responsible for the severe spermatogenic defects in the mice expressing the tivation . We alsome point .. In conme point ., which me point . In totaentarity . Using Robserved .Rhox3 shRNA ties up a sufficient proportion of the small RNA processing machinery that it reduces the generation of small RNAs. We considered this to be an attractive possibility, as the histological defects we observed in Rhox3-shRNA testes were very similar to that of Dicer-conditional KO mice generated using the same Cre-driver mice as we used in our study—Stra8-iCre mice [Rhox3-shRNA mice  out of 78 known testicular endo-siRNAs [Rhox3-shRNA testes compared to littermate control . The line with no obvious defects (line 11) exhibited only modest Rhox3 depletion, suggesting a dose-response relationship rather than an insertional mutagenesis mechanism  reduced testes weight  res weight , (iii) fs weight , (iv) nus weight , (v) muls weight , (vi) ins weight ., and  RNA-binding proteins can change the substrate recognition and processing behavior of DICER in vitro [A surprising finding in our study is that siRNAs, but not miRNAs, were dysregulated by the x3 shRNA , as bothin vitro . For exain vitro . shRNA pin vitro , raisingRhox3-shRNA mice testes (T9 and T12) are not reduced in level in Dicer KO pachytene spermatocytes, suggesting that some siRNAs are processed in a DICER-independent manner [Rhox3 shRNA might sequester factors required for siRNA biogenesis other than DICER. Regardless of the precise mechanism, we suggest that the ability of shRNAs to inhibit the expression of endo-siRNAs in vivo can be used as a tool to study the biogenesis, stability, and/or function of these small RNAs.Another possibility is that shRNAs promote the destabilization of endo-siRNAs rather than inhibiting endo-siRNA biogenesis. This destabilization could be specific for endo-siRNAs, not miRNAs, explaining the selectivity we observed. For example, shRNAs might interfere with siRNAs forming RNA-induced silencing complexes (RISC), leading to their destabilization. In support of this, loss of AGO2, a key RISC component, results in decreased levels of siRNAs in mouse oocytes . Little t manner . This, iRhox3-shRNA mice may not be representative of all miRNAs; i.e., there may be a pool of shRNA-resistant miRNAs in male germ cells. We regard this is unlikely, as we tested endo-siRNAs with different expression characteristics  is consistent with them being subject to meiotic sex chromosome inactivation (MSCI), a transcriptional repression mechanism that transcriptionally silences the X and Y chromosomes during the meiotic phase of spermatogenesis [Rhox3 gene paralogs exhibit induced expression at a postnatal time point when round spermatids first appear   ., raisinia stage .in vivo highlights the need for controls to properly interpret shRNA-mediated knockdown studies. New strategies must be developed to specifically knockdown genes with reduced side effects.Other studies in addition to ours have identified unintended effects of shRNAs. For example, target-independent cellular toxicity and animal morbidity have been shown to result from the expression of some exogenous shRNAs ,62. SomeS1 FigRhox3 mRNA level  in total testicular RNA from mice (n = 3) of the age shown (Rhox3 mRNA level at P5 was arbitrarily set to 1). Values denote the mean fold change ±S.E. (B) Western blot analysis of adult testis lysate. The blot with lanes 1 and 2 was probed with purified RHOX3 IgG (α-RHOX3). The arrow points to the presumptive RHOX3 band (~18 kDa); the higher and lower migrating bands are proteins binding non-specifically to the RHOX3 IgG. As negative controls, blots were probed with IgG purified from the preimmune serum (Pre IgG) (Lane 3) or purified RHOX3 IgG that were preincubated with excess amounts of recombinant RHOX3 protein (α-RHOX3+recRHOX3) (Lane 4). Red boxes denote the lack of detectable RHOX3 in the negative controls. β-ACTIN is the loading control. N, nuclear protein extract; C, cytoplasmic protein extract.(A) qRT-PCR analysis of (TIF)Click here for additional data file.S2 FigRhox3-shRNA and control mice at 6 weeks of age. (B) Seminal vesicle weight of the Rhox3-shRNA and control mice lines at 6 weeks of age. Values denote the mean ± S.E. Rhox3-shRNA mice, Rhox3-shRNA;Stra8-iCre double-transgenic mice; Control mice, Rhox3-shRNA single-transgenic mice.(A) Body weight of (TIF)Click here for additional data file.S3 FigRhox3-shRNA mice testes have elevated levels of spermatogonial and somatic cell markers and reduced levels of some meiotic and all post-meiotic germ cell markers. qRT-PCR analysis of testes from Rhox3-shRNA and control mice at 6 weeks of age. Values were normalized to the mRNA encoding the ribosomal protein RPL19 and denote the mean ± S.E. Rhox3-shRNA mice, Rhox3-shRNA;Stra8-iCre double-transgenic mice; Control mice, Rhox3-shRNA single-transgenic mice.Adult (TIF)Click here for additional data file.S4 FigRhox3-shRNA and control mice from the indicated postnatal ages. Values were normalized to the mRNA encoding the ribosomal protein RPL19 and denote the mean ± S.E. Asterisk (*) indicates significant changes, p<0.05. Rhox3-shRNA mice, Rhox3-shRNA;Stra8-iCre double-transgenic mice; Control mice, Rhox3-shRNA single-transgenic mice.(A–D) qRT-PCR of testes from (TIF)Click here for additional data file.S5 FigRhox3-shRNA testes (P25). (B) Quantification of the percentage of seminiferous tubules containing TUNEL-positive cells at the indicated developmental stages. Asterisk (*) indicates significant changes, p<0.05. Rhox3-shRNA mice, Rhox3-shRNA;Stra8-iCre double-transgenic mice; Control mice, Rhox3-shRNA single-transgenic mice.(A) Representative images of TUNEL assay of control and (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Accurate cervical intra-epithelial neoplasia (CIN) lesion grading is needed for effective patient management. We applied computer-assisted scanning and analytic approaches to immuno-stained CIN lesion sections to more accurately delineate disease states and decipher cell proliferation impacts from HPV and smoking within individual epithelial layers. A patient cohort undergoing cervical screening was identified (n = 196) and biopsies of varying disease grades and with intact basement membranes and epithelial layers were obtained (n = 261). Specimens were sectioned, stained (Mib1), and scanned using a high-resolution imaging system. We achieved semi-automated delineation of proliferation status and epithelial cell layers using Otsu segmentation, manual image review, Voronoi tessellation, and immuno-staining. Data were interrogated against known status for HPV infection, smoking, and disease grade. We observed increased cell proliferation and decreased epithelial thickness with increased disease grade . Analysis within individual cell layers showed a ≥50% increase in cell proliferation for CIN2 vs. CIN1 lesions in higher epithelial layers . Higher rates of proliferation for HPV-positive vs. -negative cases were seen in epithelial layers beyond the basal/parabasal layers in normal and CIN1 tissues. Comparing smokers vs. non-smokers, we observed increased cell proliferation in parabasal (low and high grade lesions) and basal layers (high grade only). In sum, we report CIN grade-specific differences in cell proliferation within individual epithelial layers. We also show HPV and smoking impacts on cell layer-specific proliferation. Our findings yield insight into CIN progression biology and demonstrate that rigorous, semi-automated imaging of histopathological specimens may be applied to improve disease grading accuracy. Predicting outcomes for cervical intra-epithelial neoplasia (CIN) lesions remains a complex challenge. Some lesions progress to later disease stages while others do not, meaning some patients experience risks and costs of treatment unnecessarily. Further, HPV infection status for normal and early CIN tissues may be insufficient for stratifying progression risk. New tests are needed to accurately stratify patients presenting with CIN and to reduce the number of women treated unnecessarily for high grade squamous intraepithelial lesions (HGSILs).Multiple biomarkers have been tested to identify CIN lesions with a high risk of progression. P53, p16, and Ki67/Mib1 are amongst the best accepted for patient management It is well-accepted that smoking is a cofactor for development of CIN Herein, we report our analysis on the effects of HPV infection and smoking on cell proliferation for normal and neoplastic cervical epithelia. We sought to apply a rigorous semi-automated approach to quantify these effects in CIN lesions. To achieve this, we analyzed individual epithelial layers in a well-annotated, thoroughly reviewed patient cohort. Through this, we have gained insights into the impact of these factors on cell behavior for different disease stages. This work provides a rationale for wider evaluation of a combined approach involving clinical features  and automated analysis of protein expression in epithelial layers as a biomarker for managing CIN.Samples were chosen amongst 1850 patients (3735 biopsies) aged ≥18 that were collected during a multi-center study to evaluate Mib1 and p16 staining as a means of improving diagnosis of HGSIL Non-pregnant women ≥18 years were enrolled from 1999–2006. The study protocol was approved by the University of British Columbia/BC Cancer Agency Research Ethics Board (protocol #C02-0476) and written consent was obtained from all enrolled patients. Patient demographic features  were obtHistopathological specimens were reviewed at BCCA by both WHO and Bethesda criteria. Initial reviews were performed by one of the gynecological pathologists on clinical duty (the “pool”). There were seven pathologists in the pool, which also included study pathologists. A second blinded review was performed by one of two study pathologists. If the first and second readings agreed exactly in the WHO system, third reviews were not performed. Discrepancy of two grades based on WHO criteria mandated a third blinded reading by both study pathologists. Kappa scores for pathology readings have been reported Endocervical samples were collected and tested for HPV using the Hybrid-Capture II system. This assay detects both low-risk  and high-risk  HPV types. All samples were processed using funds from public grants.2O2 in phosphate-buffered saline (PBS). Sections were immersed in sodium citrate buffer and microwave heated at power settings of 1000 W for 2 min, then 160 W for 15 min. Before immuno-staining, slides were soaked in PBS. Sections were incubated with biotinylated swine anti-rabbit antibody (DAKO) at 1∶100 for 30 min. Visualization of the complex was realized using diaminobenxidine/H2O2 for 10 min (room temperature). Two washes in PBS were performed before counterstaining with Mayer’s Hematoxylin. Sections were dehydrated using graded ethanol and mounted with a mixture of distyrene, plasticiser, and xylenes.Paraffin sections (4 µm) were mounted on slides, dried overnight (37°C), deparaffinised in xylenes, and rehydrated in alcohol. Next, endogenous peroxidase activity was blocked by 3% H2, and the effective pixel sampling area was 0.116 µm2. In each case, areas corresponding to diagnostic areas selected by study pathologists were carefully located by expert technicians. Images of that region were then captured. Regions of interest (ROI) were interactively and carefully delineated. Both basement membranes and superficial apical surfaces were separately defined.Getafics A simple, automated thresholding method followed by an Otsu segmentation algorithm was applied to extract centers of gravity of all cell nuclei Voronoi cell layers were defined thusly: all cells whose corresponding Voronoi polygons touched the basement membrane were assigned to layer 0 . Layer 1  consisted of those cells which were not in layer 0 and had a neighbor in layer 0. Layer 2 consisted of those cells which were not in layer 1 or layer 0 and had a neighbor in layer 1. Higher number layers were defined similarly. Generally, layers from layer 2 to the penultimate layer observed were interpreted as “intermediate” epithelial layers, while final layers were termed the “superficial” epithelial layer.The percentage of Mib1-positive cells in each layer was calculated as the number of positive cells divided by the number of cells belonging in the layer. Voronoi diagrams and layers-based analyses were undertaken as follows: given a set of points S in a plane, a Voronoi tessellation for S is the partition of the plane which associates a polygon V(p) with each point p of S in such a way that all locations in V(p) are closer to p than to any other point in S All analyses were done with STATISTICA10. All p-values were two-sided, with a two-sided p-value<0.05 considered significant. For comparisons between groups, ANOVA testing was done. When testing showed a statistical difference across groups, Least Significant Differences testing or planned comparisons were used to compare groups two-by-two.Two-hundred sixty-one biopsies obtained from this diagnostic population  were stained with Mib1 . Based oThe mean and standard deviation of epithelial thickness, number of layers, and percentage of proliferating cells for four pathological grades were determined . There wVoronoi layers were calculated as described above and in In normal epithelia, >60% of cell proliferation occurred in the parabasal layer and layer 2 . ProlifeWe next returned to assessing proliferation on a layer-by-layer basis for the four diagnostic groups . The perThis relative proliferation pattern changed in upper layers. The percentage of Mib1-positive nuclei for CIN1 cases decreased by ∼10% from layer 1 to 2 but increased by ∼6% from layer 1 to 2 in CIN2 lesions. The decrease in the percentage of Mib1-positive nuclei in CIN2 and CIN1 lesions was almost identical from layer 2 to layer 6, with an 18% decrease in CIN2 lesions and 14% decrease in CIN1 lesions, respectively.We noted several HPV impacts on proliferation patterns . This anNovel methods described herein allowed quantification of cell proliferation on a per-epithelial-layer basis . This yiOne key observation was that differences in proliferation rates between CIN1 and CIN2 lesions occurred in specific epithelial layers . While pWe also observed HPV-driven proliferation rate differences within a given disease stage. Moreover, these differences were detected in specific epithelial layers. Given high prevalence of HPV infection in advanced disease stages, we were only able to derive statistically meaningful results related to HPV status for normal and CIN1 tissues. For both these tissue types, we noted higher rates of proliferation for HPV-positive cases relative to HPV-negative cases in epithelial layers above basal and parabasal layers . These dFinally, we noticed marked smoking-mediated differences in the distribution of proliferating cells in CIN lesions. When epithelia were analyzed at full thickness, negligible separation in the percentage of proliferating cells was observed for normal and LGSIL tissues from smoking vs. non-smoking patients  . NonetheOur analyses of cell proliferation in normal cervical and CIN tissues have identified epithelial layer-specific features that delineate disease grades. Our results also show that proliferation within cell layers varies depending on HPV infection and smoking status. We have demonstrated the utility of computer-assisted histopathological slide analysis for identifying novel cell features associated with disease grade. More accurate clinical diagnoses based on this technique could improve patient management by facilitating more accurate estimations of disease progression risk. This work bolsters the argument that research into HPV and smoking impacts on cell proliferation must account for spatial differences in proliferation based on individual epithelial layers . Independent analyses using the approaches described here are needed to determine whether cell proliferation in epithelial layers beyond the basal and parabasal layers has utility for discriminating CIN1 and CIN2 lesions. Further analyses are also needed to determine the biological processes causing the HPV- and smoking-driven cell proliferation changes we observe within epithelial layers.


The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results. Preparing biological material for electron microscopy (EM) involves harsh processing steps that can poorly preserve cellular ultrastructure. Here the authors apply a single layer of graphene onto wet cells to enable direct EM using low voltage, and correlate actin filaments and mitochondria using super-resolution microscopy. A major challenge in the application of electron microscopy to biological samples has been faithful preservation of cellular ultrastructure during the laborious dehydration and embedding/coating procedures required for sample preparation124568910111213Here we utilize graphene, a single-atom-thick honeycomb lattice of carbon atoms1617182023252619We report that monolayer graphene can hermetically seal and protect large areas of mammalian cells, cultured on conventional coverglass, from external environments, including the high vacuum typically encountered in an electron microscope. This protection, combined with the high electrical and thermal conductivity of graphene and its ultimate thinness, enables facile electron microscopy of wet and untreated cells with excellent contrast and resolution, as well as correlated super-resolution microscopy directly on the culturing substrate. In particular, individual actin filaments are resolved in wet cells through electron microscopy and well correlated with super-resolution results.2) areas of cells conventionally cultured on coverglass  growth on copper foil and wet-transferred to cover large  was readily reached during pump down.Having verified that graphene can provide a hermetic seal for cells, we moved forward to examine its applicability to electron microscopy of wet cells under high-vacuum conditions. Graphene sheets were deposited onto wet cells cultured on coverglass such that most of the coverglass surface was overlaid with graphene. Silver paint was used to contact a corner of the deposited graphene sheet to the sample holder for dissipation of electric charge during electron microscopy , ‘Ag'. TWe first examined fixed cell samples that were briefly stained with a 0.5% uranyl acetate solution but otherwise remained fully hydrated. Under SEM, the non-covered, non-conductive parts of the sample rapidly accumulated electric charge, leading to excessively bright and unstable signals . Zoomed-12V0) of 3 kV, substantial contrast was obtained for the internal structure of the graphene-covered, untreated cells (V0 (2 kV) led to less transparent images, but was helpful in outlining the overall cell morphology (V0 (5 kV), the untreated cells became overly transparent with only the nuclei providing contrast (V0 (>10 kV) to penetrate the relatively thick (>∼50 nm) membranes, thus providing limited contrast on unstained animal cells1123V0. The fact that graphene is an excellent thermal and electrical conductor further reduces damage by the electron beam so that the same unfixed cells can be imaged multiple times and under different conditions without noticeable structural changes , and then post-fixed for 20 min in 2% (v/v) glutaraldehyde in CB12−1) were used to label Tom20, respectively. For fluorescent labelling of actin filaments, samples were incubatedMammalian cells  were cultured on common glass coverslips  following standard tissue culture protocols. For live cell experiments , cells wFor data presented in CVD graphene on copper foil−7–2 × 10−5 torr) was readily reached during pump down. Calibration of magnification was verified with a replica of a 2,160 lines per mm waffle-pattern diffraction grating (Ted Pella 604-A).The graphene-covered coverslip was mounted on a standard metallic sample mount with carbon tape, and a small amount of silver colloid paint (Ted Pella 16031) was used to create a conductive bridge between graphene and the sample mount. SEM imaging was performed under standard secondary electron mode on a FEI Quanta 3D FEG system or a JEOL JSM-6340F system. Normal operational vacuum (5 × 102 triangular) at the centre of the coverslip, which was readily identifiable both under optical microscope and in SEM under the coverage of graphene. 3D-STORM imaging32−2; for STORM of AF647) or 560-nm laser  was used to excite fluorescence from labelled dye molecules and switch them into the dark state. Concurrent illumination of the 405-nm laser was used to reactivate the fluorophores to the emitting state. The power of the 405-nm laser  was adjusted during image acquisition so that at any given instant, only a small, optically resolvable fraction of the fluorophores in the sample were in the emitting state. For 3D-STORM imaging, a cylindrical lens was inserted into the imaging path so that images of single molecules were elongated in x and y for molecules on the proximal and distal sides of the focal plane (relative to the objective), respectively−1 glucose oxidase and 40 μg ml−1 catalase. After STORM imaging, the coverslip was stored in PBS before processing for graphene-based SEM imaging (as described above). To align the obtained STORM and SEM images, the STORM image was mapped to the coordinate system of the SEM image through a two-dimensional affine spatial transformation (MATLAB) on the basis of corresponding features (control points). About 20 control points were selected in each data set for inferring an averaged, global, affine transformation matrix.To facilitate location of the same cells under STORM and SEM, a diamond scribe was used to make a scratch mark .Supplementary Figures 1-12.


The demand for nanocomposites of graphene and carbonaceous materials decorated with metallic nanoparticles is increasing on account of their applications in science and technology. Traditionally, the production of graphene-metal assemblies is achieved by the non-environmentally friendly reduction of metallic salts in carbonaceous suspensions. However, precursor residues during nanoparticle growth may reduce their surface activity and promote cross-chemical undesired effects. In this work we present a laser-based alternative to synthesize ligand-free gold nanoparticles that are anchored onto the graphene surface in a single reaction step. Laser radiation is used to generate highly pure nanoparticles from a gold disk surrounded by a graphene oxide suspension. The produced gold nanoparticles are directly immobilized onto the graphene surface. Moreover, the presence of graphene oxide influences the size of the nanoparticles and its interaction with the laser, causes only a slight reduction of the material. This work constitutes a green alternative synthesis of graphene-metal assemblies and a practical methodology that may inspire future developments. Graphene sheets decorated with metal nanoparticles (MNPs) are excellent materials that can be used in several fields such as energy storage, optoelectronics, biosensors or catalysis, due to their physical and chemical properties13489101113The vast majority of graphene-MNPs are synthesized by the simultaneous reduction of metal salts and highly oxidized graphene derivatives15161820Ligand-free MNPs have been successfully prepared by laser irradiation using an alternative and easy top-down technique that fulfils the principles of green chemistry2224252628Decoration of ligand-free gold NPs created separately by PLAL and subsequently mixed with graphene derivatives has been reported3335In this work, we have focused on producing ligand-free gold NPs anchored to graphene oxide sheets in a single reaction step. The impact of the femtosecond radiation-based technique on the control of the production of ligand-free gold NPs in an environment filled with graphene oxide sheets has been studied. With this aim, AuNPs were produced in two different liquid environments for comparison purposes: (i) deionized water and (ii) a graphene-oxide homogeneous suspension. Our results demonstrate that it is possible to create ligand-free MNPs directly anchored to graphene oxide sheets in a single reaction process. This methodology has been proved to provide an effective way to obtain graphene metal assemblies that could potentially be used in several applications where undesirable cross chemical effects need to be avoided.Gold nanoparticles were obtained from the ablation of a gold solid target immersed in deionized water using an amplified Ti:Sapphire femtosecond laser source working at a maximum energy per pulse of 0.8 mJ, with a pulse duration of 30 fs full width half maximum and 1 kHz of repetition rate. The schematic drawing of the set-up is presented in 2. This is the characteristic MNP shape obtained through the PLAL process. During laser radiation, fragmented material is released from the bulk material and suspended in the liquid medium with a certain density. The average size of the obtained gold particles was 26 nm with a size distribution of 18 nm  files for cubic Au crystals (JCPDS: 4-784).The EDX spectrum shown in 2. The immobilization of ligand-free AuNPs happens in a natural way after particle formation. The particles are formed in an environment filled with the carbonaceous material, which becomes a support for the newly formed nanoparticles.The decoration of graphene oxide sheets with AuNPs was achieved through the PLAL process by radiation a gold disk in a graphene oxide (GO) suspension in deionized water (1 mg/mL). The experimental set-up and conditions were exactly the same as those previously described using water as the medium. As such, the fluence was set to ca. 1 J/cmThe size of the particles, the morphology and the purity of the AuNP/GO material were characterized by TEM, EDX, Fast Mapping with SDDs (FS Mapping) and Dark-Field Scanning Transmission Electronic Microscopy (DF-STEM). The micrographs depicted in A statistical analysis showed that the average size of the AuNPs in the final material is 3 ± 1.1 nm. The majority of the particles are in the average size, but a few particles of size >20 nm were also found in the sample. The average size distribution achieved is lower than that using only water as the solvent. When water was used as the medium, the average particle size was 26 . Under tThe FS Mapping measurements, obtained by means of the EDX system and the special HAADF detector, show that this laser-assisted fabrication route produces highly pure AuNPs anchored on GO with an heterogeneous distribution . The DF-The EDX spectrum shows that, when the irradiated area by the electron beam is large, some extra elements  can be detected, similarly to what was observed in the samples produced in water . The mosd inter-planar spacing for hexagonal graphite are included in the Joint Committee on Powder Diffraction Standards (JCPDS): 41-1487).The interaction between a high power electromagnetic field, such as the one associated to femtosecond laser radiation, and almost any kind of material promotes a considerable interchange of energy that can result in structural changes in the material. In order to study how femtosecond laser radiation centered at 800 nm modifies the graphene oxide sheets, the AuNP/GO material was further analyzed by powder X-ray diffraction. The XRD pattern of rGO is completely different, the [001] signal disappears; however, the peak corresponding to the [002] plane appears at 24.85°, and the peak for [101] is observed at 43.1°. The reduction of GO using hydrazine to obtain rGO removes many oxygen functionalities and restores part of the π-network of the materiali.e. deionized water and a graphene oxide suspension in water.Using PLAL, we have decorated graphene oxide sheets with ligand-free AuNPs in a practical and single step reaction. The laser-based method causes a slight reduction of graphene oxide but avoids the use of extra chemicals that can alter the properties of the gold nanoparticles. The irradiation of a gold disk containing a GO suspension in water leads to the immediate immobilization of the generated ligand-free AuNPs on the graphene surface. According to the experimental set-up , the lasComparison of the results of both experiments suggests that graphene oxide has an influence on the AuNP formation and in graphene oxide itself by i) decreasing the average particle size, ii) immobilizing the particles on the graphene surface, iii) heterogeneously distributing the anchored AuNPs at the graphene wrinkles, and iv) slightly reducing the graphene oxide.2 onto the surface of the gold target disk. The effect of graphene oxide as a density filter was evaluated experimentally by performing an additional experiment: AuNPs were produced using only water as the medium at a fluence of ~0.25 J/cm2 which is the fluence calculated to reach the gold disk through the GO dispersion  was then added to the resulting solution and the mixture was stirred for 12 h. TEM analysis revealed that all the AuNPs previously formed in water had been immobilized onto the graphene oxide surface , but also the peak corresponding to the [002] plane . These results are further confirmed by XPS. After laser radiation, we have observed an increase of C/O ratio indicating that there is a decrease of oxygen functional groups. In this work, we have shown the convenience of using a laser-based method for the production of ligand-free AuNPs that are directly immobilized on the surface of graphene oxide. The modulation of laser properties and the presence of the graphene material have an influence in the size of the AuNPs. The properties of graphene oxide are not altered considerably after laser irradiation and we have only observed a slight increase in the reduction degree of the material. Considering previously reported results, we can make a comparison of gold nanoparticles anchored onto graphene obtained by chemical methods and laser ablation. We have observed that chemical methods allow a better control of the size dispersion of nanoparticles and produce a complete reduction of GO44in-situ femtosecond laser radiation strategy known as PLAL. The particles obtained using this procedure are in agreement with previous reported literatureIn order to produce the nanomaterials and understand the influence of graphene sheets in the synthesis of AuNPs, two different experiments were performed. For the first experiment, deionized water (18 MΩ) was used as the liquid medium to fabricate gold NPs by the 2O and H2O + GO respectively) and the ejected material that comes from the ablation process was captured in the surrounding medium. The gold disk was placed at the bottom of a glass chamber filled with H2O for the first experiment and H2O + GO for the second. The thickness of the fluid layer above the gold target in all cases was set to be ~2 mm. Then the target was irradiated from the air-liquid interface as shown in In all cases, a gold disk of thickness 1 mm and diameter 6.5 mm (99.99% purity) was used as a target to produce ablation. The target was surrounded by the liquid medium  emitting pulses of 30 fs, with a central wavelength of 800 nm, a maximum energy per pulse of 0.8 mJ, and 1 kHz repetition rate. The delivered energy was monitored by means of an analogical power meter  and controlled using a set of calibrated neutral density filters. The diameter of the incoming laser beam was controlled using an iris of 6 mm of diameter placed before the focusing lens.2 in order to avoid the plasma breakdown of water, while having enough energy to promote ablation. For the complimentary experiment where GO was considered a neutral optical density filter, we replaced the absorption contribution of H2O + GO by a neutral optical density filter which promotes the same power decrement, achieving this way a fluence of ~0.25 J/cm2 in the target. The dispersion introduced by either of the liquids, and the optical elements in general, was partially compensated in a post-compression stage by changing the relative position of two fused silica Brewster prisms, which guaranteed the minimum pulse duration at the sample.The optimized fluence of the laser beam at the sample plane was set to ~1 J/cmThe samples were analyzed by X-ray diffraction and electronic microscopy. High Resolution Transmission Electron Microscopy (HRTEM) images and High-Angle Annular Dark-Field (HAADF)-STEM images of the samples were obtained using a Jem-2100 LaB6 (JEOL) transmission electron microscope coupled with an INCA Energy TEM 200 (Oxford) energy dispersive X-ray spectrometer (EDX) operating at 200 kV. The preparation of the samples for TEM measurements was carried out by deposition of a droplet of the colloidal suspension onto a carbon-coated copper-based TEM grid. The liquid content was removed with the help of absorbent paper and, then, the sample was air-dried for several hours. The solid material collected on the grid surface was used for analysis. This technique allows the preparation of dry and highly dispersed particles. The crystallographic structure of the samples was analyzed by X-ray powder diffraction (XRD) using a Bruker D4-Endeavor diffractometer with Cu-Kα radiation at a wavelength of 0.1542 nm. X-ray photoelectron spectroscopy (XPS) spectra were acquired on a Kratos AXIS ultra DLD spectrometer with a monochromatic Al Kα X-ray source (1486.6 eV) using a pass energy of 20 eV. The photoelectron take off angle was 90° with respect to the sample plane. To provide a precise energy calibration, the XPS binding energies were referenced to the C1s peak at 284.6 eV.How to cite this article: Torres-Mendieta, R. et al. In situ decoration of graphene sheets with gold nanoparticles synthetized by pulsed laser ablation in liquids. Sci. Rep.6, 30478; doi: 10.1038/srep30478 (2016).


Graphene-based carbon materials such as fullerenes, carbon nanotubes, and graphenes have distinct and unique electronic properties that depend on their dimensionality and geometric structures. Graphene wrinkles with pseudo one-dimensional structures have been observed in a graphene sheet. However, their one-dimensional electronic properties have never been observed because of their large widths. Here we report the unique electronic structure of graphene nanowrinkles in a graphene sheet grown on Ni(111), the width of which was small enough to cause one-dimensional electron confinement. Use of spatially resolved, scanning tunnelling spectroscopy revealed bandgap opening and a one-dimensional van Hove singularity in the graphene nanowrinkles, as well as the chemical potential distribution across the graphene nanowrinkles. This observation allows us to realize a metallic-semiconducting-metallic junction in a single graphene sheet. Our demonstration of one-dimensional electron confinement in graphene provides the novel possibility of controlling its electronic properties not by chemical modification but by ‘mechanical structuring'. Wrinkles in graphene can restrict the motion of electrons to one dimension. Here, the authors observe a one-dimensional van Hove singularity and bandgap opening in a five nanometre wide wrinkle, which enables them to create a metallic-semiconducting-metallic junction in a single graphene sheet. Graphene wrinkles, which are one-dimensional 1D) folded graphene structures, have generally been observed in graphene produced by chemical vapour deposition. These structures have been thought to be the result of the difference in the thermal expansion coefficient between graphene and its substrateD folded 345In the following, we demonstrate the 1D electron confinement in graphene nanowrinkle (GNW) by scanning tunnelling microscopy/spectroscopy (STM/STS), whose width is <5 nm. Moreover, spatially resolved electronic structures have been investigated, and the manipulation of graphene geometry by STM tip has been demonstrated. Our results imply that a semiconducting property can be realized by the mechanical deformation of the graphene geometry not by chemical modification, which is analogous to the case of a strain-induced pseudo magnetic field7Epitaxial graphene with GNWs was synthesized by dissociating acetylene on a clean Ni(111) surface. A rapid cooling process is necessary, which is the most critical step to synthesize GNWs. Most of the GNWs were observed in the region where the terrace width of the underlying Ni surface was as small as several tens of nanometres . These GVs) of 1 V and a feedback current (If) of 1 nA, whereas the centre region was scanned with a smaller tip–sample distance (Vs=0.05 V and If=1 nA), so that individual carbon atoms could be clearly imaged. If up to 6 nA on the same area as h) and width (w) of the GNW were precisely measured. The translation vector (T), chiral vector (Ch) and chiral angle (θ: angle between T and the armchair direction) were defined as shown in w=2.14 nm, h=0.42 nm and θ≈10°, we could estimate the indices of the Ch to be  for the GNW ; this behaviour is similar to the ‘band-gap opening' feature observed in a single-wall carbon nanotube (SWCNT)910121314I/dV images in Vs, including the voltages at peak positions (Vs=–0.6 V (v1) and 0.5 V (c1)) and between v1 and c1  as well At the peak positions, the measured dI/dV intensities, which are indicative of the LDOS, were higher on the GNW area than on the pEG area. In contrast, lower LDOSs were observed on the GNW at Vs values in the energy gap between v1 and c1 (ΔEg) (I/dV images at the second set of peaks (Vs=−1.6 V (v2) and 1.5 V (c2)), and Vs between the first and second peaks (Vs=−1.0 V and 1.0 V)). The dI/dV images clearly show the metallic-semiconducting-metallic junction across a GNW, which has never been observed in the dI/dV spectra previously measured on other graphene wrinkles6The electronic structures of GNWs located on the terraces of the underlying Ni111) were investigated by STS and d1 were inI/dV spectra in the present work were compared with Landau levels observed in monolayer graphene under magnetic fields18n=0) should be observed near Fermi level (EF), since electrons behave like a Dirac fermion. In addition, the position of the nth Landau level (En) follows the relationship v1 or c1) are too far from the EF to assign one of them as the zeroth Landau level, even when considering the doping effect. Also, the energy gaps between the peaks do not match the relationship, We considered two plausible origins of these peaks, a strain-induced magnetic field and a 1D electron confinement. An induced magnetic field due to the high curvature in graphene can result in Landau levels in the graphene, as observed in graphene nanobubbles291011ip  ref. . In our ΔEg, which should show a relationship to w (ΔEg∝1/w), if ΔEg originates from 1D electron confinement. I/dV spectra obtained from the other GNWs within the width range 1.67 nm (a) to 3.65 nm  is used rather than width of the GNW or the diameter of the SWCNT, because L is more directly related to ΔEg induced by 1D electron confinement. Hence, ΔEgs measured in GNWs, and calculated ΔEgs for both SWNCTs20L, that is, arc length of GNW, circumference of SWCNT and width of AGNR, respectively. The arc length of the GNW was estimated on the assumption that the GNW arc shape of its cross section is a part of a circle 131415π orbitals remain at the GNW owing to the freestanding feature of GNW from the Ni surface  can have the 1D electronic structure. The strong interaction between pEG and the Ni(111) substrate may play a critical role in making a 1D electron channel along GNWs. Owing to the strong interaction, the  surface . These det al.EF in the dI/dV spectra. This implies that the strong interaction at the pEG is essential for the 1D electron confinement. Because the graphene nanoribbons were physically deposited by a spin-coating method on a Au(111) substrateTao I/dV were measured at different positions across the GNW , the STM image became very unstable (scratched lines appeared), and the width of the GNW became broader  corresponding the threshold width showing vHS. Another possibility that cannot be excluded is possible modification of the tip apex during the manipulation that can affect the spectral shape to some extent.Finally, we attempted to manipulate the GNW by the STM tip . An STM  broader . Subsequ broader . To expl broader . The mec broader , the pEG broader . The preOur results constitute the demonstration of electron confinement in GNWs with dimensions <5 nm and the demonstration of an open bandgap, which implies that GNWs have semiconducting properties. Many efforts have been made to manipulate the electronic properties of graphene by chemical modification25+ sputtering-and-annealing cycles. EGs consisting of GNWs were synthesized in a ultrahigh-vacuum chamber (base pressure: 2 × 10–7 Pa) by dissociating C2H2 (–5 Pa) on Ni(111) at 650 °C for 5 min. For rapidly cooling, the sample was immediately removed from the heating stage after the reaction. All STM images were obtained in a constant-current mode. We measured the differential conductance (dI/dV) with a lock-in amplifier by applying an a.c. bias of 50 mV (797 Hz) to the tunnelling bias. We focused only on the GNWs on the terrace of the Ni(111) surface. A total of 27 individual GNWs were observed with atomically resolved STM images.The experiments were performed in an ultrahigh-vacuum, low-temperature STM (Omicron LT-STM) at 4.7 K. The Ni(111) single crystal was cleaned by repetitive ArHow to cite this article: Lim, H. et al. Structurally driven one-dimensional electron confinement in sub-5-nm graphene nanowrinkles. Nat. Commun. 6:8601 doi: 10.1038/ncomms9601 (2015).Supplementary Figures 1-12, Supplementary Table 1, Supplementary Notes 1-3 and Supplementary References