ZnO nanoparticles were synthesized by pulsed laser fragmentation in liquid to attain a simple, efficient, ligand-free method for nanoparticle fabrication. By combining simple fabrication processes with a promising device architecture, highly sensitive ZnO nanoparticle/graphene UV photodetectors were successfully demonstrated.By combining the enhanced photosensitive properties of zinc oxide nanoparticles and the excellent transport characteristics of graphene, UV-sensitive, solar-blind hybrid optoelectronic devices have been demonstrated. These hybrid devices offer high responsivity and gain, making them well suited for photodetector applications. Here, we report a hybrid ZnO nanoparticle/graphene phototransistor that exhibits a responsivity up to 4 × 10 The advantages of graphene in sensor applications are numerous, including high mobility >104 cm V1s−1 [22 V1s−1) , excelle2 V1s−1) ,4. While2 V1s−1) ,6, photo2 V1s−1) . Nonethe2 V1s−1) . These p2 V1s−1) , and Ti2etection ). 2 V−1s−1 at room temperature) [Because of its wide band gap (~3.3 eV) and high exciton binding energy (~60 meV), zinc oxide (ZnO) is a promising candidate for ultraviolet (UV)-sensitive hybrid photodetectors . In addierature) , its comerature) ,13,14,15erature) ,17,18,19erature) ,21,22,23erature) . At thisMany different techniques have been utilized to fabricate ZnO nanostructures for hybrid graphene photodetector applications, such as hydrolysis methods for nanoparticle fabrication  and hydrHere, we have demonstrated PLFL for the synthesis of ZnO nanoparticles with a bimodal size distribution (~18 nm and 46 nm). These nanoparticles were integrated into graphene-based hybrid phototransistors, which were then characterized to determine the optical and electrical performance, including wavelength selectivity and responsivity. 2 on the backside of the Si wafer was laser-micromachined to expose the highly conductive Si for device back-gating. Graphene on Cu foils  were spin-coated with poly resulting in a ~600 nm thick layer. The foils were then baked at 100 °C in air for 2 min on a hot plate. The graphene on the backside of the Cu foil was etched by floating the foil on a 10% HNO3 solution for 3 min followed by rinsing with deionized water. The Cu was remove by etching in a ferric chloride solution for 2 h. The remaining PMMA/graphene film was then floated on a dilute 2% HCl solution to remove any particulates introduced during the Cu etching process. The film was rinsed in deionized water before wet transfer. The PMMA/graphene film was then transferred onto the SiO2/Si substrate and allowed to air dry. A small droplet of PMMA was drop casted onto the surface of the PMMA/graphene to encourage flattening of the film, followed by air drying. The PMMA was then removed with acetone, followed by rinsing in isopropanol and then water. After processing the graphene, source and drain electrodes (5 nm Ti/150 nm Au) were deposited via electron beam evaporation using a shadow mask that was laser-micromachined from a thin (75 µm) polyetherimide sheet. After electrode deposition, isolation lines were laser-micromachined around each device on the chip, yielding active device areas of 2 mm × 1 mm.Phototransistor devices were fabricated using standard wet transfer ,36,37,38ZnO nanoparticles were produced using pulsed laser fragmentation in liquid (PLFL) . ZnO powThe optical properties of the fabricated graphene transistors before nanoparticle deposition were characterized via Raman spectroscopy  which revealed the quality of the graphene layer. Optoelectronic characterization was performed using UV illumination that was fiber coupled from a monochromator into a 10× objective, mounted on a probe station. The light intensity was adjusted by a computer-controlled attenuator, maintaining a uniform spot size of ~2 mm for all experiments. Electrical characterization, including the drain and gate sweeps as well as temporal measurements, was conducted using a semiconductor characterization system .Because of the unique properties afforded by nanoscale materials, different methods have been developed for simple and efficient fabrication, including wet chemical synthesis , sol gelThe mechanisms responsible for nanoparticle formation via PLFL and the effect that initial size, concentration and material have on the resulting particle size have been studied extensively ,32,33,34There are several advantages to this technique, including simple experimental set-up and the ability to maintain complex stoichiometries with narrow particle size distributions . The mecsmall/rlarge ≈ 0.42) is potentially advantageous for the final device design because a more efficient packing factor becomes possible compared to the unimodal particle distribution. For bimodal spheres, ideal packing can be efficiently achieved up to a particle size ratio of ~0.41 [Both the original ZnO powder particles before PLFL and the resulting smaller nanoparticles after PLFL can be seen in of ~0.41 , where tof ~0.41 . To give insight into the quality and defect content of the generated nanoparticles, photoluminescence measurements (λ = 343 nm) were conducted. The photoluminescence of ZnO nanoparticles has been studied extensively and typically reveals two distinct emission bands, one in the UV and one in the visible spectrum. The emission peak observed in the UV region is a result of near-band-edge emission which is mediated through exciton–exciton interactions. As the particle size decreases, the fluorescence is blue-shifted due to an increase in transition energy ,56. The The as-received ZnO powders, which can be seen in In addition to the insight into the quality and defect density induced in the nanoparticles during laser processing, the optical transmission spectra of the ZnO nanoparticle solutions were collected to help understand the effect of laser-processing on the particles optical bandgap, which is shown in The mechanism of photoconduction in ZnO nanoparticle/graphene phototransistors is schematically illustrated in 2 up to 1.4 mW/cm2. Drain current as a function of drain voltage can be seen in To understand the photoresponse of the devices, electrical transport properties were measured with UV illumination (λ = 365 nm), from 34 µW/cmThe responsivity and gain as a function of incident UV power were measured to determine the performance of the phototransistors. The responsivity of the devices is the ratio of the measured photocurrent to the UV illumination, where:phI and darkI are the induced photocurrent under UV illumination and the dark current, respectively. The responsivity at a drain current of VD = 5 V with no gate voltage applied can be seen in 4 AW−1, where experimentally at P = 34 µW/cm2, the responsivity is measured to be 2 × 103 AW−1. The photoconductive gain G can be described as the ratio between the number of electrons collected per unit time and the number of absorbed photons per unit time [h is Planck’s constant, c is the speed of light, e is electron charge, and λ is incident wavelength. By fitting the available data, the maximum gain achieved as P approaches zero is Gmax ≈ 1.3 × 105 at VD = 5 V with no applied gate voltage. The gain of the ZnO nanoparticle/graphene phototransistor is a function of the applied optical intensity. The number of electron-hole pairs generated is directly related to the optical intensity applied, where with a higher intensity, more hole trap states are filled at the surface eventually reaching a surface saturation state. Once this occurs, the electron-hole pairs generated do not aid in charge transfer into the graphene layer, thus limiting the efficiency of the hybrid device [Here, nit time ,19:(2)G=d device .In addition to responsivity and gain, the wavelength selectivity of the photodetector is critical because it determines the spectral range. The spectral dependence of responsivity for ZnO nanoparticle/graphene devices can be seen in D = 1 V, with no applied gate voltage (VG = 0 V). The graphene-only phototransistors show no photoresponse when illuminated at 365 µW/cm2 over a time scale of several hundred seconds, which can be seen in 2 can also be seen in 2 can be seen in In order to understand the time-varying behavior of the phototransistors, the photocurrent response was measured as a function of time as the devices were exposed to UV illumination and then subsequently turned off. These temporal measurements were conducted at a drain voltage of VBoth the responsivity and the response times of these ZnO nanoparticle/graphene photodetectors can be optimized by adjusting several experimental parameters. It is well known that the nanoparticle layer is strongly dependent on particle packing in its ability to absorb light and efficiently transfer electrons into the underlying graphene layer . Thus, f4 AW−1 with a maximum gain of 1.3 × 105 and superior spectral selectivity below 400 nm, making them ideal solar-blind UV photodetectors. We have demonstrated the use of pulsed laser fragmentation in liquid (PLFL) as a simple, ligand-free alternative to traditional nanoparticle synthesis techniques for the fabrication of ZnO nanoparticles. Hybrid ZnO nanoparticle/graphene phototransistors were demonstrated, exhibiting a responsivity of up to 4 × 10


Employing density functional theory calculations, we obtain the possibility of fine-tuning the bandgap in graphene deposited on the hexagonal boron nitride and graphitic carbon nitride substrates. We found that the graphene sheet located on these substrates possesses the semiconducting gap, and uniform biaxial mechanical deformation could provide its smooth fitting. Moreover, mechanical tension offers the ability to control the Dirac velocity in deposited graphene. We analyze the resonant scattering of charge carriers in states with zero total angular momentum using the effective two-dimensional radial Dirac equation. In particular, the dependence of the critical impurity charge on the uniform deformation of graphene on the boron nitride substrate is shown. It turned out that, under uniform stretching/compression, the critical charge decreases/increases monotonically. The elastic scattering phases of a hole by a supercritical impurity are calculated. It is found that the model of a uniform charge distribution over the small radius sphere gives sharper resonance when compared to the case of the ball of the same radius. Overall, resonant scattering by the impurity with the nearly critical charge is similar to the scattering by the potential with a low-permeable barrier in nonrelativistic quantum theory. Unfortu2V−1s−1 ,9,10, th2V−1s−1 ,12, mech2V−1s−1 ,14, and 2V−1s−1 ,17,18,192V−1s−1  for deta2V−1s−1 . The mec2V−1s−1 ,24,25,262V−1s−1 ,28 proviIn addition, graphene with a gap in the electronic spectrum in the presence of a multiple-charge impurity is of particular interest. Such a system is similar to the relativistic Coulomb problem . In the symmetry . In the symmetry .Since graphene is an almost ideal planar system, the orbital angular momentum may be quantized fractionally ,33. In aues only . Therefoincluded . TherefoIt has long been known that pure Coulomb potential is an excessive idealization in the three-dimensional problem, which . The emlectrons ,49). In n energy . TherefohBN) and graphitic carbon nitride (gC3N4) substrates under the mechanical biaxial strain in the frame of density functional theory, supplemented by the van der Waals dispersion corrections. It was obtained that the weak van der Waals interlayer interactions, alongside the uniform strain of the graphene sheet on the substrate, leads to the considerable bandgap. Moreover, the value of bandgap increases monotonically with an increasing stretch and vice versa. Note that, due to the remarkable mechanical properties, some substrates allow reversible stretching of graphene up to 30% [hBN or gC3N4 substrate has a crucial role since free-standing graphene does not have an energy gap under uniform mechanical stretching. We discuss a method to smoothly change the electronic properties of graphene, which has a gap in the electronic spectrum by mechanical stretching or compression, which includes tuning the critical value In the presented study, we analyze the electronic behavior of monolayer graphene on the hexagonal boron nitride  and its implementation in the program Quantum ESPRESSO ver. 6.5 ,53. We cesh grid  is used.smearing  techniqusmearing  was appl2 and SiC, as well as more complex systems such as Te2Mo, hBN/Ni(111), or C3B/C3N. At the level of theory used, we obtained that, only quasi-2D hexagonal boron nitride (hBN) and graphitic carbon nitride (gC3N4), opened the band gap in graphene. In all other cases, the “Dirac cone” on the electronic band structure conserved its original shape, and graphene remained a gapless semiconductor, or the Fermi level shifted to the conduction band. Thus, graphene began to exhibit a metallic nature.First, we attempted to select such substrates that would facilitate the opening of an energy gap in graphene without imposing additional conditions, such as an external electric field or mechanical stresses. We examined about fifty different substrates, including the common SiO2 and SiC substrates [hBN and gC3N4 substrates. Peculiarities of the interlayer interactions between the graphene and these substrates lead to the “pure gap” that is characteristic of the narrow-gap semiconductor.Nevertheless, some previous studies revealed that the external electric field could effectively tune the energy gap in graphene on the SiObstrates ,62,63 byhBN and gC3N4 substrates by the hexagonal supercells. In the case of the hBN substrate, the supercell contains eight carbon atoms, and, in the case of the gC3N4 substrate, the graphene sheet inside the supercell is represented by 18 carbon atoms VC. Qualitferences ,42 with +e–-loop. Therefore, the impurity charge screening mechanism specified in Reference [. This moduction , when a eference , accordieference , cannot eference . Therefoperiment .All these conclusions remain valid for the gapless radial Dirac equation in the presence of a supercharged impurity the work , which seference ). Howeve ε¯=−ε>0 .(7)δ0. Therefeference .n-th level with the total angular momentum The critical charge Here, At spectrum , and theollowing ,39.(9)Sg theory , and theThe poles of the scattering matrix The solutions give both the positions Near the top of the valence band As in the nonrelativistic theory of scattering, see Chapter 13 in Reference , quasistThe position of the resonance eference ).+e–-pairs, and the width In the review , resonaneference . Howevermentum J . In the mentum J  for the hBN and gC3N4 substrates by optimizing the supercell parameters as well as atomic positions inside the supercell. For considered samples . In the 3.325 Å ). It shotal data ,79. All hBN and gC3N4 substrates  stretching and shearing as well as inhomogeneous deformation opens the energy gap in free-standing graphene [hBN or gC3N4 substrate makes it possible to tune the bandgap in graphene. In this case, stretching leads to an increase of the gap value, and compression leads to its decrease. For example, uniform stretching of the graphene sheet on the hBN substrate results in the gap of 36.8 meV, and, on the gC3N4 substrate, results in the gap of 36.9 meV (see kBT ~26 meV), which indicates the possibility of maintaining the energy gap at normal conditions. Note that, in real straintronic applications, the limits of strain transfer between graphene and substrate should be considered since biaxial deformation is not always completely transferred from the substrate to graphene [hBN and gC3N4 substrates , where eference  and FiguhBN) and graphitic carbon nitride (gC3N4) substrates, the behavior of the electronic properties of deposited graphene is fundamentally different from the free-standing one. In this case, graphene has an energy gap, which makes it possible to classify it as a narrow-gap semiconductor, whereas the uniform stretching increases the value of the gap. These findings can help to overcome the main barrier to using graphene as an element of nano-electronic and straintronic devices. Moreover, uniform stretching affects the Dirac velocity in graphene on the substrate. Dirac velocity is linear with mechanical tension. It increases with uniform compression and decreases with uniform stretching. The Dirac point, near which the dispersion law is similar to the Einstein dependence of the particle energy on its momentum, corresponds, as usual, to the corner of the Brillouin zone for graphene deposited on the hBN substrate, while, for the gC3N4 substrate, it is located in the center of the zone. However, in both cases, the dynamics of charge carriers near the Dirac points is described by the effective two-dimensional radial Dirac equation for states with the total angular momentum In the presented study, we tried to answer the question of how the choice of the substrate and uniform mechanical stresses can affect the electronic properties in graphene, and, in particular, opens the energy gap and contributes to its fine-tuning. It was found that, when choosing the hexagonal boron nitride (The dependences of the critical charges In addition, the resonant scattering of the holes by the Coulomb potential modified at small distances in states with +e–-pairs exist in the supercritical Coulomb problem. However, calculation of the effective dielectric constant of the considered systems is necessary for the direct comparison of experimental data on the spectra of current-voltage characteristics with the theoretical predictions.Based on such experimental data, it would be possible to conclude whether half-integer orbital angular momenta are realized in the two-dimensional considered systems. In addition, the effective two-dimensional radial Dirac equation with the integer total angular momenta


An acoustic plasmon mode in a graphene-dielectric-metal structure has recently been spotlighted as a superior platform for strong light-matter interaction. It originates from the coupling of graphene plasmon with its mirror image and exhibits the largest field confinement in the limit of a sub-nm-thick dielectric. Although recently detected in the far-field regime, optical near-fields of this mode are yet to be observed and characterized. Here, we demonstrate a direct optical probing of the plasmonic fields reflected by the edges of graphene via near-field scattering microscope, revealing a relatively small propagation loss of the mid-infrared acoustic plasmons in our devices that allows for their real-space mapping at ambient conditions even with unprotected, large-area graphene grown by chemical vapor deposition. We show an acoustic plasmon mode that is twice as confined and has 1.4 times higher figure of merit in terms of the normalized propagation length compared to the graphene surface plasmon under similar conditions. We also investigate the behavior of the acoustic graphene plasmons in a periodic array of gold nanoribbons. Our results highlight the promise of acoustic plasmons for graphene-based optoelectronics and sensing applications. Acoustic graphene plasmons are superior to the graphene surface plasmons in field confinement and normalized propagation length, thus promising for applications. Here, the authors report near-field imaging of acoustic plasmons in high-quality CVD graphene, measure the AGP dispersion and propagation loss, and investigate their behavior in a periodic structure. This mode is supported by a structure comprising of a metal, a dielectric spacer, and a graphene layer, where the image charges in the metal effectively “mirror” the charge density oscillations in the doped graphene layer. The acoustic graphene plasmon (AGP) supported by the structure is mostly confined in the dielectric spacer and does not experience cutoff as the spacer thickness decreases8, thus resembling the fundamental plasmonic mode of a narrow metal gap. The AGP excited at the important MIR frequencies10 does not exhibit significant loss and is detectable even when the spacer is reduced to a single atomic layer of hexagonal boron nitride5. Inside such a narrow dielectric spacer, the AGP wavevector can be about two orders of magnitude larger than that of free space light, which grants access to quantum and non-local phenomena in graphene12, and allows for the AGP localization in nanostructures13 with a stunning mode volume confinement factor4 of ~1010. This ultimate capability to compress MIR light outperforms that of other polaritonic species in van der Waals materials9, including graphene surface plasmon (GSP)15, and is similar to the case of image phonon-polaritons in boron nitride16. For that reason, AGP is promising for applications that require strong light–matter interaction such as molecular sensing20, polaritonic dispersion engineering in van der Waals crystals22, and dynamic light manipulation by graphene-based active metasurfaces26.An acoustic plasmon mode supported by a system of two graphene sheets6. However, the near-field optical probing of AGP, which provides detailed microscopic information about the mode, is yet to be demonstrated.The key advantage of the AGP is its confinement within the dielectric spacer, in contrast to the GSP bound to the graphene layer. Therefore, ohmic losses in graphene are expected to hinder the AGP propagation to a lesser extent compared to GSP. On the other hand, the larger AGP wavevector requires an intermediary structure to alleviate the momentum mismatch under the far-field excitation. So far, structures containing an array of metallic elements have been used to couple far-field radiation to the AGP modeIn this work, we employ a scattering-type scanning near-field optical microscope (s-SNOM) based on an atomic force microscope (AFM) for real-space mapping and analysis of MIR AGP. We directly measure the AGP dispersion, evaluate the mode propagation loss, and investigate its behavior in a periodic structure designed for the far-field coupling. Most importantly, our results reveal a relatively low propagation loss of infrared AGP even when unprotected, CVD-grown, large-area graphene is used at ambient conditions, suggesting a practical route to construct large-area graphene-based optoelectronic devices.Ez, penetrates into the free space above the structure, and hence, can couple to the AFM tip of the s-SNOM effectively acting as a z-oriented electric dipole30  for the structure of interest. Figure De calculated for the range of MIR frequencies and spacer thicknesses, assuming EF = –0.5 eV. Considering that the average tip height above the sample is 40–80 nm, which is approximately equal to the tapping amplitude of the tip32, the most favorable experimental conditions are expected in the frequency window of 1000–1300 cm–1 (where Al2O3 absorption stays sufficiently low), while t > 10 nm.In order to estimate the optimal experimental conditions for the near-field AGP probing by s-SNOM, it is instructive to calculate 2O3 films were fabricated using the template-stripping method34, allowing a sub-nm roughness of substrate surface even when the gold film is patterned6 , wet-transferred on top of the alumina-coated gold substrates, and chemically doped (see Methods section for details).Substrates with gold and Al2O3 substrate reveals an abundance of μm-long edges of monocrystalline areas with AGP interference fringes formed due to its reflection from the graphene termination  is proportional to the amplitude of the z-component of the electric field under the tip30, s ∝ |Ez |, thus it can be numerically calculated by full-wave simulations in a quasi-static approximation36 (see Supplementary Information section 39) corresponds to EF = –0.51 eV and carrier mobility μ = 2000 cm2/V s, consistent with a high-quality CVD graphene7. The roughness-mediated scattering of AGP is neglected in the full-wave simulations. Additional simulations support this assumption, considering the measured root-mean-square (RMS) roughness of 0.5 nm at gold and alumina surfaces , the effective index of the AGP is qAGP = kAGP/k0 = 39.06 + 2.92i (55.96 + 4.17i), where kAGP is the propagation constant of the AGP, and k0 = 2π/λ0 is that of free space light. To quantify the dissipation of plasmonic modes, we use the figure of merit (FOM) defined as the ratio of the plasmon propagation length Lp to its wavelength λp: Lp/λp = lp = Re{kp}/(2πIm{kp}), where lp is the normalized plasmon propagation length in optical cycles. It can be noted that the FOM of AGP lAGP = 2.12 (in both samples) is almost twice larger than the reported value for MIR GSP in exfoliated graphene on an SiO2 substrate41lGSP = 1.18. An analytical solution for GSP in the same graphene on a thick Al2O3 substrate provides qGSP = 23.41 + 2.47i (24.43 + 2.62i), and thus, an expected lGSP ≈ 1.5 in both cases. Therefore, our near-field measurements indicate that, while the observed AGP is ×1.7 (×2.3) times more compressed in terms of the wavenumber, its FOM is 1.4 times higher than that of the GSP in the same graphene sheet. The better FOM of AGP has been predicted8 due to the larger compression factor of the plasmonic wavelength compared to the decrease in the propagation distance. However, dispersion analysis also indicates that the AGP is in general less sensitive to the loss in graphene if considered in local limit42  is shown in Fig. EF = –0.46 eV. The near-field data are obtained from a series of measurements over the same sample area, which makes it possible to compare the spectral dependency of the near-field contrast32sref and φref are the amplitude and phase of the near-field signal over the graphene-free area, respectively. Figure –1 to 46 at 1260 cm–1. Furthermore, the spectral dependency of De , which can provide coupling efficiency exceeding 90% when combined with an optical cavityyer Fig. . Due to λAGP does not depend on the geometry of the structure, thus EF in graphene can be fitted using the AGP dispersion obtained from the near-field imaging (see Supplementary Information section w = 240 nm). Dispersion fit to λAGP at different frequencies  is calculated using a simple model of a planar AGP waveguide with an infinite array of nanogaps, treated as partially reflective “mirrors” with complex transmission and reflection coefficients. Then, the dispersion solution is reduced to the eigenvalue problem for a lossy Bloch state in a 1D periodic medium43. The periodicity of the structure does not lead to the opening of a bandgap or flattening of bands at the center or edges of the Brillouin zone, possibly due to the lossy nature of plasmonic modes  and phase φ obtained at ω = 1200 cm–1 in the sample with P = 230 nm and EF = –0.62 eV; λAGP ≈ 228 nm, so that βAGP = 2π/P. The measured and the numerically calculated profiles of s ∝ |Ez | and φ ∝ arg{Ez} across the nanoribbons  of 30 μm thickness was cut into ribbons and placed inside a CVD quartz tube, stretching between the hottest and the coldest zones inside the tube. Then, cycle annealing was introduced with a thermal gradient along the ribbons. The Cu foil was annealed at 1040 °C for two hours in an atmosphere of 40 sccm hydrogen and 1000 sccm argon gases. Then temperature was decreased to 700 °C during 30 min, and then increased up to 1040 °C during the same time. This process was repeated for four cycles in total, after which we opened the chamber to cool naturally.The gold/alumina layers on a Si substrate were prepared with template stripping as described in Ref. 4, H2, Ar) for graphene synthesis. The graphene flake size is controlled by growth conditions such as the ratio between CH4 and H2 concentrations, the total amount of CH4, and the growth time. Here, we purged 5 sccm of CH4, 30 sccm of H2, and 1000 sccm of Ar for a full coverage of Cu by graphene. Then, Cu ribbons were cut and graphene was wet-transferred from Cu onto the prepared samples, and chemically doped by vapors of HNO3 acid by placing the devices over the acid for 4 min at room temperature in a fume hood.For growing the high-quality graphene, we used low-concentration methane (0.1% in argon) in four stages: ramping, annealing, growth, and cooling. First, the temperature was increased up to 1060 °C during one hour and then kept stable for one hour for annealing, which is necessary for removing organic molecules and enlarging the Cu grain size. Then, we used a mix of three gases , was used for near-field imaging. s-SNOM in AFM tapping mode was used to perform surface scans with 5 nm step and tip oscillation amplitude of ≈70 nm.The near-field scans were obtained by commercial s-SNOM (Neaspec GmbH) coupled with a tunable quantum cascade laser , which illuminates the Pt-coated AFM tip . The background-free interferometric signalα = 0.2 nm, having the effective relative dielectric permittivity εr is the background relative permittivity and σ is the optical conductivity of graphene. Dielectric permittivity of gold was taken from Ref. 45, and that of thin film Al2O3 was taken from Ref. 46. See Supplementary Information for details on full-wave simulations.Commercial finite element method software (COMSOL Multiphysics) was used for full-wave simulations. In our 2D simulations, graphene is implemented as a thin film of finite thickness Supplementary InformationPeer Review File


Mandibular condylar osteochondroma (OC) could lead to facial morphologic and functional disturbances, such as facial asymmetry, malocclusion, and temporomandibular joint dysfunction. However, after condylar OC resection, the inaccurate reposition of the neocondyle still needs to be solved. The purpose of this study was to explore the feasibility of the condylar osteotomy and repositioning guide to reposition the neocondyle in the treatment of patients with severe deformity secondary to condylar OC.Three patients with severe deformity secondary to OC of the mandibular condyle were enrolled in this study. With the aid of condylar osteotomy and repositioning guide, condylar OC resection and repositioning were carried out, and the accuracy and stability of these guides were evaluated. All patients healed uneventfully, and no facial nerve injury and condylar ankylosis occurred. Compared with the computerized tomography scans in centric relation before surgery and 3 days after surgery, the results showed that the facial symmetry was greatly improved in all the patients. Also, after the superimposition of the condylar segments before surgery and 3 days after surgery, the postoperative reconstructed condyles had a high degree of similarity to the reconstruction of the virtual surgical planning. Observed from the sagittal and coronal directions, the measurements of condylar positions were very close to those of virtual surgical planning. Moreover, it also showed stable results after a 1-year follow-up.For patients with severe deformity secondary to condylar OC, condylar osteotomy, and repositioning guide was expected to provide a new option for the improvement of facial symmetry and occlusal relationship. Osteochondroma (OC), a kind of osteocartilaginous exostosis, is considered as the common tumor of long bones, comprising approximately 35 to 50% of all benign bone tumors , 2. The In order to improve the aesthetics and function, different treatment methods are selected according to the degree of dental and maxillofacial deformities secondary to condylar OC. At present, condylar OC resection combined with orthognathic surgery, including mandibular sagittal split ramus osteotomies (SSRO), Le Fort I maxillary osteotomies, and genioplasties is the most frequently used treatment methods . For conFor patients with more severe maxillary and mandibular deviation and worse malocclusion, intraoral condylectomy and contralateral SSRO could not be used to place the residual neocondyle into the fossa to restore the occlusal relationship and TMJ function. To correct the deformities and improve the function, bilateral SSRO must be performed intraorally. Unfortunately, during the follow-up process, we found that this technique may cause ischemic absorption or necrosis of the proximal bone segments of the condylectomy side due to excessive periosteum decollement.Considering the problems mentioned above, for patients with severe asymmetric deformities secondary to condylar OC, we chose a preauricular incision to remove the condylar OC and then performed orthognathic surgery to avoid this situation. Different from the conventional preauricular incision, we innovatively applied the condyle osteotomy and repositioning guides to ensure the accurate position of residual neocondyle with respect to the fossa. Also, the preliminary evaluation was made to investigate its accuracy and stability. This approach could provide a new option for the treatment of severe asymmetric deformities secondary to condylar OC.Three patients with condylar OC accompanying dental maxillofacial deformities were enrolled in this preliminary study at the Department of Oral and Craniomaxillofacial Surgery of Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine. Following clinical and radiographic examinations, the patients had no other craniomaxillofacial skeletal syndrome, traumas, or previous orthognathic surgery Fig. . All thePreoperatively, the high-resolution computed tomography (CT) of the craniomaxillofacial skeleton was taken by a GE Medical System LightSpeed CT scanner . All two-dimensional CT images were saved in digital imaging and communications in medicine (DICOM) format and imported into SurgiCase CMF 5.0  for the reconstruction of three-dimensional virtual models of the craniomaxillofacial skeleton. After considering the chief complaint, virtual surgical planning and simulation were created by the surgical team and 3D design engineer according to the clinical physical examinations and three-dimensional malformation analyses. The osteotomies and condylar OC resection were performed virtually by the surgeons so as to achieve an optimal aesthetic appearance. Preoperative virtual condyle osteotomy and repositioning guides were used to ensure accurate positioning of the neocondyle Fig. a–c. The The data of craniomaxillofacial skeleton bone were imported into Unigraphics NX 7.5  for designing of the condylar and other templates. Using the pre-simulated data, skull, maxillary and mandibular stereomodels, condylar osteotomy guide, condylar repositioning guides, and maxillary osteotomy and repositioning guides were manufactured. Condylar guides included condylar osteotomy guide and condylar repositioning guide. The condylar osteotomy guide embraced the neck of the condyle. The position of the condylar osteotomy guide was designed according to the size and position of condylar OC to ensure complete tumor resection. Under the guidance of the osteotomy guide, the osteotomy line was exactly the same as the virtual design. After condylar OC resection, the remaining three holes were placed on the residual bone for the use of a zygomatic-borne condylar repositioning guide. The condylar repositioning guide was divided into two parts: nail hole positioning guide at the root of the zygomatic arch and the zygomatic-borne condylar repositioning guide. The nail hole positioning guide was embraced at the root of the zygomatic arch according to pre-determined virtual design, and the position was unique to ensure the accuracy of the following condylar repositioning. Two holes were also placed on the zygomatic arch for the use of a zygomatic-borne condylar repositioning guide. The zygomatic-borne condylar repositioning guide was fixed on the stationary zygomatic bone. Also, the neocondyle was repositioned using the remaining three holes on the neck of the condyle and two holes on the root of the zygomatic arch. The five holes for fixation screws were carefully placed to avoid damaging the essential tissues and organs while fixing the screws. The zygomatic-borne condylar repositioning guide helped the surgeons to determine the unique optimal site quickly and accurately.The mandible is unstable due to the presence of condylar OC. Hence, the movement of the maxilla was controlled using maxillary osteotomy and repositioning guides to ensure the stability and accuracy of operation. All the guides were made using the three-dimensional printing technique before the operation, and all were tried on the three-dimensional skull, maxillary and mandibular stereomodels to ensure that the guides were placed smoothly Fig. d–f. AfteThe surgery was performed by one team. After adequate exposure of the condylar OC and the neck of the condyle, the articular disc was peeled and perserved intactly without damaging the peripheral anatomic structures. Then, the condylar osteotomy guide was secured to the neck of condyle through designed holes. During the operation, special care was taken to protect the important surrounding tissues, including facial nerve. The osteotomies were performed by referring to the cutting slots to make the condylar OC resection as effectively as in virtual planning Fig. g. After The zygomatic-borne condylar repositioning guide was placed on the bone of the zygomatic arch and the neck of condyle along the holes left by the osteotomy guide and the nail hole positioning guide Fig. i. The fiDue to the instability of condyle, the maxillary movement was determined using 3D printed osteotomy and repositioning guides, after which the surgeon performed BSSRO and repositioned the mandibular segment in the traditional manner to correct the facial asymmetry.A craniomaxillofacial CT scan was taken for each patient at 3 days (T1) and 1 year (T2) postoperatively. The accuracy of the condyle position was assessed by comparing the actual postoperative operation to the virtual planned one (T0). Both postoperative and planned CT data were imported into SurgiCase CMF 5.0, and the deviations were measured by Geomagic Studio 2013 software. Deviations were defined using the following indicators: (1) 3D deviation of the osteotomy, (2) 3D deviation of the condylar segments at 3 days postoperatively. In order to quantitatively compare the accuracy, the following measurements were recorded from the two-dimensional CT images of T0 and T1. For the coronal image, the medial joint space (MJS) and lateral joint space (LJS) were measured from the most prominent medial and lateral condylar points to the glenoid fossa. For the sagittal image, the anterior joint space (AJS), superior joint space (SJS), and posterior joint space (PJS) were measured from the most prominent anterior, posterior, and superior condylar points to the glenoid fossa. The linear measurements of the affected joint spaces in the actual postoperative and virtual planned models were assessed and compared with each other.To observe the stability and accuracy of the condyle repositioning guides, the postoperative CT data (T2) and 3D deviation of the condylar segments were obtained at 1 year postoperatively. The two-dimensional CT images were also acquired to compare the relevant linear indicators mentioned above. The functional and esthetic outcomes were also assessed based on the postoperative dental occlusion, images and clinical examination.The new computer-assisted surgery was performed on three consecutive patients  who required condylar OC resection and orthognathic surgery. The articular discs of the patients in our study were intact with no displacement, rupture, or perforation through observing TMJ MRI before surgery (Additional file After the superimposition of the condylar segments of T0 and T1, the postoperative reconstructed condyles had a high degree of similarity to the reconstruction of the virtual surgical planning Fig. . The 3D To evaluate the stability of condylar position with condylar repositioning guides, the skull and condylar segments were compared with the preoperative virtual ones 1 year after surgery Fig. . The 3D The complications of condylar OC vary with the size of the tumor and its main growth direction , 13. TheCondylectomy or accompanying joint sacrifice and subsequent condylar reconstruction via extraoral incision have been reported in many studies –21. Yet,To achieve the goals mentioned above, individual surgical planning is indispensable. Computed tomography and computer-assisted design could be used to diagnose the lesion, to perform surgical simulation, and to evaluate the accuracy of the technology. With the aid of an endoscope, the condylar OC could be exposed and resected via an intraoral approach , 25. RemIn view of these complications, for patients with severe deformity, the preauricular incision, condylar OC resection, and BSSRO via intraoral incision are necessary. Besides these approaches, maxillary Le Fort I osteotomy or genioplasty or lower border osteotomy might be required in different situations to achieve the goal of correcting deformities and malocclusion . These sIn the present study, we presented a novel condylar osteotomy guide and condylar repositioning guide to solve these problems. In order to overcome the instability of the mandible, maxillary 3D-printed osteotomy and repositioning guides were also simultaneously used to perform orthognathic surgery. Our study demonstrated that this technique could be used to ensure the correct positioning of the neocondyle into fossa after condylar OC resection. The postoperative follow-up results have also shown that the technique could provide clinically acceptable accuracy and stability in repositioning the neocondyle. These guides were designed and made using the CAD/CAM technique based on three-dimensional planning. The condylar osteotomy guide in our surgery could completely guide the condylar OC resection and reduce the additional surgical time and damage caused by repeated grinding. The condylar repositioning guide placed on the predrilled holes of the osteotomy guide could be used for the optimal positioning of the neocondyle with respect to the fossa. The newly designed guides were placed on the fixed zygomatic arch, which eliminated the potential errors caused by the movement of the maxilla or mandible during surgery. Preoperative virtual planning decreased intraoperative changes to ensure the optimal position relationship between neocondyle and fossa and avoid damage that could be caused by anatomical occurrences that were not considered preoperatively. Superimposition of the virtual segments and the actual segments after computer-assisted surgery showed that the mean 3D deviation of the condylar osteotomy guide between T0 and T1 was 0.41 mm; furthermore, the mean 3D deviation of condylar repositioning guide between T0 and T1 was 0.42 mm. The follow-up results demonstrated that the mean 3D deviation of the condylar repositioning guide between T1 and T2 was 0.81 mm. Thus, there is a high degree of similarity between the reconstructed condylar segments and the virtual planned ones. To further evaluate the accuracy and stability of this novel condylar repositioning guide, the distances between the most prominent points to the glenoid fossa in the coronal and sagittal CT images were measured. The results showed that the data of T1 were very close to that of T0 observed from the sagittal and coronal directions. However, compared with the phase of T0 and T1, the condylar position in all dimensions of T2 showed some alterations. However, the facial symmetry and occlusion relationship did not significantly change in any of the patients. It is known that the position and shape of condyle tend to gradually change after surgery . For theThe approach presented in the current study showed certain advantages. First, for patients with severe deformities secondary to condylar OC, the most significant advantage was the use of condylar repositioning guide, which could accurately restore the optimal relationship between neocondyle and fossa. The position and stability of neocondyle were important for maintaining the TMJ function, facial symmetry, and occlusion relationship. Moreover, the design of extraoral incision could avoid ischemic absorption and even necrosis of the proximal bone segment after removing the condylar OC. Another advantage of this computer-assisted surgery was that the condylar osteotomy guide and the repositioning guide used for condyle osteotomies and reposition neocondyle respectively provided a duplication of the preoperative virtual surgical plan and eliminated the need for intraoperative measurement, thus substantially saving time. However, the present study also had some limitations, including the small sample size and short follow-up time, which should be addressed by future studies. Also, the biggest concern related to this technique was extraoral incision resulting in facial nerve injury and scarring. In this study, none of the patients showed any signs of facial nerve injury in the follow-up.Currently, the use of condylar osteotomy and repositioning guide combined with orthognathic surgery in the treatment of asymmetric prognathism caused by condylar OC is still in its infancy. Nevertheless, this surgical technique offers great advantages with no significant complications. The secondary deformities and malocclusion can be corrected by simultaneous orthognathic surgery. Therefore, condylar osteotomy guide and repositioning guide used for condyle OC resection and reposition neocondyle extraorally combined with orthognathic surgery intraorally provide a valuable option for patients with severe jaw deformities secondary to condylar OC.Additional file 1: Figure 1. The evaluation of position and condition through TMJ MRI before surgery. A, B: The TMJ MRI for sagittal view of condylar OC and normal side. C, D: The TMJ MRI for coronal view of condylar OC and normal side.


First isolation of graphene, as a great achievement, opens a new horizon in a broad range of science. Graphene is one of the most promising materials for spintronic fields whose application is limited due to its weak magnetic property. Despite many experimental and theoretical efforts for obtaining ferromagnetic graphene, still, a high degree of magnetization is an unsolved challenge. Even, in most observations, graphene magnetization is reported at extremely low temperatures rather than room temperature. In principle, the magnetic property of graphene is created by manipulation of its electronic structure. Removing or adding bonds of graphene such as creating vacancy defects, doping, adatom, edges, and functionalization can change the electronic structure and the external perturbation, such as external magnetic field, temperature, and strain can either. Recently, single and few-layer graphene have been investigated in the presence of these perturbations, and also the electronic changes have been determined by Raman spectroscopy. Here, we successfully could develop a simple and novel Leidenfrost effect-based method for graphene magnetization at room temperature with the external perturbations which apply simultaneously in the graphene flakes inside the Leidenfrost droplets. Macroscale ferromagnetic graphene particles are produced by this method. Briefly, the graphene is obtained by the liquid-phase exfoliation method in the ethanol solution media and also evaporates on the hot surface as a Leidenfrost droplet in the magnetic fields. Then, the floated graphene flakes circulate inside the droplets. Due to the strain and temperature inside the droplets and external magnetic field (the magnet in heater-stirrer), the electronic structure of graphene is instantly changed. The changes are extremely rapid that the graphene flakes behave as a charged particle and also produce an internal magnetic field during their circulation. The internal magnetic field is measured by sensors. As the main accomplishment of this study, we could develop a simple method for inducing magnetism obtained 0.4 emu/g in the graphene, as magnetization saturation at room temperature, which is higher than the reported values. Another achievement of this work is the detection of the Leidenfrost droplets magnetic field, as an internal one which has obtained for the first time. To investigate magnetic graphene particles, the magnetization process, and the electronic structure of the vibrating sample magnetometer (VSM), magnetic field sensor, and Raman spectroscopy are used, respectively. For example, recent studies indicate that the monolayer graphene is optically transparent and can absorb ~2.3% of the visible light2, and also it is wetting-transparent to substrates such as copper, gold or silicon3. In both cases, transparency is reduced by increasing the number of graphene layers3. Because of the high flexibility of graphene, the folded, wrinkled, and crumpled graphene can be easily created. Consequently, in these structures, the roughness and specific surface area are raised9. Moreover, the results of measuring some properties extremely depend on the measurement conditions, such as temperature, the number of layers, and fabrication method20. Suspended and supported fabrication strategies are two types of developing methods for obtaining the free-standing form of graphene. The suspended form can remarkably enhance its electrical and thermal properties because it can effectively eliminate substrate phonon interaction. Generally, the intrinsic properties of graphene are detected in the suspended form without inconvenience of the substrate. For comparison, the electron mobility of suspended graphene is 200,00012 cm2/Vs and for unsuspended graphene supported by Si/SiO2 substrate is 10,000 cm2/Vs1. Also, the thermal conductivity for suspended monolayer graphene over a trench in Si/SiO2 substrate is 5300 W/mK10 (at room temperature) and is 600 W/mK20 for the supported graphene (near room temperature).Graphene as an ideal material have unique physical properties and extensive usages. The isolation of single-layer graphene in 2004 was a starting point for exploring its structure and properties24. Recently, many methods have been introduced to change the electronic structure and induce magnetization in graphene. For example, unsaturated dangling bond in vacancy defect is responsible for magnetic moment, which is theoretically estimated about 1.12–1.53 μB per atom26. Vacancy defect in graphene and graphite structure is generated by ion  irradiation as well as reduction of graphene oxide31. In addition, adatom such as hydrogen and fluorine can create magnetism in graphene by forming the local magnetic moments37. Recently, low magnetization has been reported for fluorinated graphene (0.2 emu/g at 1.8 K)27, hydrogenated epitaxial one (<30 * 10−7 emu/g)32, and defective one (0.02 emu/g at 2 K)27. Moreover, the zigzag edges in the specific structure of graphene such as graphene nanoribbons have spin-polarized states at the edges which induce magnetism39. Frequently, the graphene oxide is used in the experimental study. The magnetization of ferromagnetic nitrogen-doped graphene created through reduction of graphene oxide in ammonia is 1.66 emu/g 40, and in nitrogen plasma41 is 0.01 emu/g (at room temperature). Using annealing, graphene oxide is reduced in the presence of hydrazine which a magnetization about 0.02 emu/g (at room temperature) was reported by Y. Wang et.al in 200928. The fluorination of the reduced graphene oxide modifies the magnetization up to 2 emu/g at 2 K33. In general, there are limitations in the use of graphene oxide and defective graphene. When graphene is oxidized and reduced, its electrical conductivity is disrupted, and when the defect is created in graphene, in high density of defects, the structural stability become loosed and fragile42. J. Tucek et.al doped graphene by Sulfur and reported its ferromagnetic properties below 62 K (5.5 emu/g) while they observed no ferromagnetic properties at room temperature43. Ultimately, modification of graphene magnetization is followed up by functionalizing with nitrophenyl44. But most of the studies do not achieve suitable ferromagnetic. In general, ferromagnetic graphene is a matter of interest in the spintronic field45. Because the electron structure differs only in the vicinity of the defects, and there are also limitations in the production of these defects, these methods have a low potential for improvement.Among all of these properties, the ferromagnetic property of graphene and graphite is extremely low. The negligible amount of their magnetization relate to defects and impuritiesIn this method, using floated graphene in ethanol droplet during the Leidenfrost effect-based method under the magnetic field at room temperature, the ferromagnetic graphene particles (FGPs) are produced that the magnetization saturation degree is stunningly high in compare to the recent reports. The graphene suspensions are manipulated by Leidenfrost effect. What is important is that the graphene structure is not disrupted by this method.46. Recently, the Leidenfrost droplet has been used to fabricate nanostructures47, create photonic microgranules48, accelerate the chemical reaction49, and used as a chemical reactor51, additionally, many models have been presented for the Leidenfrost droplet54. Despite many studies, dynamics such as vibration, rotation, and internal circulation in the droplet still have not been well understood.When a liquid droplet contact to the hot surface, which the surface temperature is upper than the liquid boiling point, an insulating vapor layer is formed that protects the droplet from boiling rapidly. The droplet floats above its own vapor and become shrinkage during evaporation until the liquid become totally dry. This phenomenon was firstly considered by J.G Leidenfrost in 1756 and known as the Leidenfrost effect55. Adjusting surface tension of solution in the region of 40–50 mN/m is determined as a main parameter for achieving high yield of graphene dispersion56. To adjust surface tension, various liquids and surfactants are used60. In our previous work, the graphene was dispersed in two different surfactants and also their mixture59. In this study, in order to reach the surface tension ~45 mN/m, the ratio of 20:80 ethanol/water was used . In order to control the bouncing of the droplets, the upper plate is drilled. The volume of each hole is about 0.05 ml. As shown in Fig. In recent findings, mechanical control over the electronic structure of graphene is known as “strain engineering”, which affects the magnetic properties of graphene. For more explanation, the pseudo-magnetic field of highly strained nanobubbles of graphene is reported by the N. Levy et.al in 2010. In this study, in addition to strain, other parameters such as temperature and the magnetic fields are applied to graphene flakes by the Leidenfrost effect-based method. Temperature and magnetic field, like strain, can change the electronic structure of graphene flakes and cause magnetic properties in these flakes.61.Raman spectra of single and multilayer graphene indicate useful and prominent features based on the phonon and electronic properties of them. In addition, the changes of lattice vibration and electronic structure in the presence of external perturbations such as magnetic field, strain, and temperature can be determined through the Raman spectroscopy. The three main scattering modes for graphene systems located at ~1340, 1574, and 2707 called D, G  and 2D, respectively. The Raman spectrum of pristine graphite is shown in Fig. 61. Indeed, the frequency of Raman peaks related to temperature62, is raised by isotropic compression and reduced by isotropic tension63. Also, anisotropic stress gives complex features to these peaks (splitting the G mode). In addition, Landau levels can be generated due to the application of an external magnetic field (and in some cases by strain)71, which transition between these levels lead to magneto excitation create72. Resonance, magneto phonon resonances, occurs when the energy between the levels of the Landau and the lattice vibrations of graphene is equal. Other parameters such as number of layers and stacking form can influence Raman spectra. Therefore, we confirm just the change of electronic structure in the graphene by Raman spectra because the graphene flakes inside the droplets feel the non-uniformed perturbation applied in any direction61. The starting point of these changes is observed when the Leidenfrost droplets are placed on the holes. The magnetic field sensor measures the weak magnetic fields for droplets as an internal magnetic field based on the circulation graphene flakes inside the droplets . Here, under the magnetic field, the floated graphene flakes circulate inside the droplet and aggregate during the evaporation. As explained before, the dynamic inside the Leidenfrost droplets is not clear, also, the investigation of the floated graphene inside the droplet is not possible. Therefore, due to these limitations, the probable magnetization process is proposed and interpreted based on the magnetic field measurements. Magnetic field measurement with this procedure detects two sources of magnetic fields. One of them is external, about 420 nT generated by the magnet inside the heater-stirrer, and other is an internal magnetic field in the region of 1–5 nT generated by the circulation of the polarized graphene flakes inside the droplets. Generation of the internal magnetic field contains two steps: (1) the polarization of graphene under the external perturbation when the graphene suspension is injected in the aluminum holes, (2) generation of the internal magnetic field through the circulation of polarized graphene, based on Faraday law.In recent studies, responding of graphene to external factors have been reported such as an alignment of graphene sheets under the external magnetic fieldIn addition, the polarizability of graphene sheets depends on the number of layers and influence the internal magnetic field. To elaborate more, as shown in Fig. Due to high temperature of the heater, measurement of magnetic field faces limitations; therefore, the sensor is located at 30 cm above the heater, as seen in the inset of Fig. 2/gr and 6.2320 m2/g, respectively that are close to each other.Also, high and non-uniform strain is applied on graphene flakes, which creates different shapes and wrinkles on FGPs. Figure H < +10 kOe. Table 24 of graphite investigated by Raman and ICP spectroscopy (Supplementary Table The vibrating sample magnetometer (VSM) was performed for all samples. The (M-H) curve for graphite and graphene shows weak ferromagnetic properties  are purchased from Merck Company. Larger flakes are collected by centrifuge (Hettich EBA20). Set up contains heater stirrer  and two aluminum plates (thickness 2.45 mm). Almost one hundred hole  is drilled in the top plate. The impurities of graphite are measured by ICP spectrometry (ICPS-7000 SHIMADZU). Adjusting surface tension of ethanol solution is performed using Tensiometer (sigma 700 ring mode). Magnetization of FGPs is measured by vibrating sample magnetometer (Kavir IRAN). Magnetic field sensor  is used to detect magnetic fields. Raman spectra are obtained by Raman spectroscopy . The morphologies are investigated by FESEM (TESCAN). UV-Vis (mini 1240) and FTIR (8400S) spectrophotometer analyzes are performed by SHIMADZU instruments. The specific surface area is determined by Micromeritics instrument.Supplementary information.


Here we study ultrathin gold films near the percolation threshold fabricated by electron-beam deposition on monolayer CVD graphene. The advantages of such hybrid graphene/gold substrates for surface-enhanced Raman spectroscopy are discussed in comparison with conventional substrates without the graphene layer. The percolation threshold is determined by independent measurements of the sheet resistance and effective dielectric constant by spectroscopic ellipsometry. The surface morphology of the ultrathin gold films is analyzed by the use of scanning electron microscopy (SEM) and atomic force microscopy (AFM), and the thicknesses of the films in addition to the quartz-crystal mass-thickness sensor are also measured by AFM. We experimentally demonstrate that the maximum SERS signal is observed near and slightly below the percolation threshold. In this case, the region of maximum enhancement of the SERS signal can be determined using the figure of merit (FOM), which is the ratio of the real and imaginary parts of the effective dielectric permittivity of the films. SERS measurements on hybrid graphene/gold substrates with the dye Crystal Violet show an enhancement factor of ~10 Surface-enhanced Raman spectroscopy (SERS) ,2,3,4 isVarious strategies have been suggested to realize a strong and robust FE effect. One of the ways to get strong FE is to create structures with so-called “hot spots” ,18,19 thAs previously shown by near-field and two-photon luminescence microscopy ,25, the 2/Si wafers with and without a single-layer CVD graphene for SERS applications.In this research, we focus on a comprehensive analysis of ultrathin gold films with thicknesses close to the percolation threshold, deposited on SiO2 on the surface (SiO2/Si). The second one was the same silicon wafer, but with a single-layer of CVD graphene, which covered more than 95% of the substrate area (graphene/SiO2/Si). Before metal deposition, the graphene substrates were annealed at 250 °C in a vacuum chamber 10−6 Torr for 1 h to remove residual PMMA and water. The thin gold films for all types of substrate were deposited in one mode by electron beam evaporation in a Nano Master NEE-4000  installation at a high vacuum, with the pressure of residual gases in the chamber being no more than 5 × 10−6 Torr and a deposition rate of 0.5 Å/s, at room temperature (23 °C). Each thickness of the gold film was deposited simultaneously for both types of substrate in one cycle. As a material for deposition, granules of gold produced by Kurt J. Lesker with a purity of 99.999% were used. The quoted “thickness” of the gold films are the average nominal coverage values measured by the quartz oscillator.The ultrathin gold films with thicknesses ranging from 3 to 10 nm with a step size of 1 nm were deposited onto two types of substrates. The first type was a silicon wafer with a thin 90 nm thick layer of SiOThe fabricated films were subsequently imaged by scanning electron microscope (SEM) JEOL JSM-7001F . The thickness of the films and their roughness were measured by an atomic force (AFM) neaSNOM Microscope . Electrical measurements were carried out using a 4-probe station  from Jandel with a collinear geometry of the location of the probes and started with 10 nA, gradually increasing the current to 100 μA.®, J. A. Woollam Co., Lincoln, NE, USA) operating in the wavelength range of 280–3300 nm. The data were collected at multiple angles of incidence from 65° to 75° with a step of 5° and the optical constants of the gold films were obtained by point-by-point fitting of the ellipsometry spectra with 10 nm steps [The dielectric function spectra of the Au thin film were evaluated from data measured using a variable-angle spectroscopic ellipsometer (WVASEnm steps . To be inm steps .2/Si and graphene/SiO2/Si) were covered by an aqueous 10−6 mol/L solution of CV for approximately 1 h and subsequently gently blown dry with compressed air.The experimental setup used for Raman measurements was a confocal scanning Raman microscope Horiba LabRAM HR Evolution . All measurements were carried out using linearly polarized excitation at a wavelength of 632.8 nm, 300 lines/mm diffraction grating, and ×100 objective (N.A. = 0.90), whereas we used unpolarized detection to have a significant signal-to-noise ratio. The spot size was ~0.43 μm. The Raman spectra were recorded with 0.26 mW incident powers and an integration time of 1 s at each point. The statistics were collected from 15 × 15 points maps with a step of 0.8 μm from at least 10 different places of the sample. These scan parameters were selected as a compromise between minimum damage/bleaching of the dye molecules and significant signal-to-noise ratios. Raman dye Crystal Violet (CV) was used for the Raman characterization. Directly before the Raman measurement, the gold films deposited on two substrates  indicates the single layer of graphene ,47, and bstrates a–d. The graphene .2/Si substrate is greater than on SiO2/Si. The data in Routinely, the thickness of the deposited films is estimated by the quartz sensor in an electron beam evaporation installation, but the real thickness can be different. For the gold films, the quoted “thickness” is the average nominal coverage measured by the quartz oscillator. We accurately measured the thickness of the gold films and their roughness by AFM and compared how the obtained values correlate with the detector readings. The results of the AFM measurements are presented in 2/Si substrate can be determined by sheet resistance measurements [2/Si substrate begin to conduct at thicknesses of 8 nm. Therefore, we can assume that the percolation threshold for such films is between 7 and 8 nm. Because graphene has very high electrical conductivity, the definition of the percolation threshold for the graphene/SiO2/Si substrate is more complicated than for previous samples, since this type of sample has conductivity at all thicknesses due to graphene. The sheet resistance of graphene was measured and the value ranged from 700 to 1600 Ω/sq. For films on graphene/SiO2/Si substrate, conductivity is observed over the entire range, but films can be considered percolated when their sheet resistance is much lower than the sheet resistance of graphene. According to this criterion, we can estimate that the percolation threshold is between 5 and 6 nm. Mostly, electrical measurements are enough to uniquely determine the film thickness at which there is a percolation threshold.Although we have shown the difference in thicknesses between those measured by AFM and by the quartz sensor, in the further representation of the experimental data we used the thicknesses given by the quartz sensor. Before the Raman measurements, we found a percolation threshold for deposited films by electrical and optical methods. The percolation threshold for films on the SiOurements ,41. The 2/Si substrate, we used ellipsometry as an additional method to determine the percolation threshold. Ellipsometry measurements give effective optical constants of gold films and can be used to determine the percolation threshold.However, as we cannot uniquely determine these thicknesses for the gold films on the graphene/SiO2/Si substrate starting from 8 nm , which is equal to −ε’/ε” and is presented in 2/Si e and graSi  and ~6.0 × 104 (for the 6 nm gold film deposited on the graphene/SiO2/Si substrate). Thus, the comparison of the hybrid graphene/gold substrates and substrates without graphene shows that, although the level of the SERS signal for the substrate with graphene is almost the same or even slightly lower, the ability to quench of luminescence provides a great advantage over other substrates. There is another interesting feature. If we compare the EF for the film with thickness 7 nm (this thickness is the percolation thresholds for the case of gold on SiO2/Si substrate) on SiO2/Si (EF ~ 1.1 × 105), and graphene/SiO2/Si substrates (EF ~ 2.1 × 104), a significant difference can be observed. Indeed, the presence of only one graphene layer on a SiO2/Si substrate can affect the thickness of the percolation threshold for the gold films and leads to the difference in SERS signal in near one order of magnitude.For the calculation of enhancement, we use the analytical enhancement factor (EF) expression, which quantifies how much more signal can be expected from SERS in comparison with normal Raman for the same experimental parameters . The ave2/Si and graphene/SiO2/Si. We demonstrated that the thickness of the deposited gold films measured by AFM and sensor values can vary significantly. The percolation threshold has been determined by two independent experimental methods as a four-probe measurement of the sheet resistance and spectroscopic ellipsometry. These methods show good agreement with each other for both types of substrate with and without graphene. According to them, the thicknesses of the percolation threshold is between 7 and 8 nm for the films on the SiO2/Si substrate and between 6 and 7 nm on the graphene/SiO2/Si substrate. The maximum SERS signal was obtained for the gold films with thicknesses near the percolation threshold in the case of both types of substrate and amounted to be ~6.0 × 104 (graphene/SiO2/Si) and ~1.1 × 105 (SiO2/Si), respectively. SERS measurements of the dye Crystal Violet demonstrated the ability of graphene to effectively quench luminescence by an average of ~60%. Thus, the substrates with and without graphene show comparable SERS signals. However, the presence of graphene shows the ability to quench luminescence, which provides a great advantage over other types of SERS substrates. Our results demonstrate that the high intensity of the SERS signal is also observed for the films thicknesses adjacent with the percolation threshold, but only for thicknesses slightly below. Additionally, we show a strong correlation between FOM and intensity of SERS spectra. The maximum EF for both types of substrate corresponds to zero slopes of the FOM curves in the infrared region. We propose to use FOM curves as an additional criterion for finding the percolation threshold and the thicknesses of the films that exhibit the maximum EF. We believe that the reported results will be interesting for SERS applications, especially for bio- and molecular sensing.In summary, this article presents a detailed study of semi-continuous gold films near the percolation threshold deposited on two types of substrate—SiO


In this work, we report an available technique for the effective reduction of graphene oxide (GO) and the fabrication of nanostructured zirconia reduced graphene oxide powder via a hydrothermal method. Characterization of the obtained nano-hybrid structure materials was carried out using a scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and Fourier-transform infrared spectroscopy (FTIR). The confirmation that GO was reduced and the uniform distribution of zirconia nanoparticles on graphene oxide sheets during synthesis was obtained due to these techniques. This has presented new opportunities and prospects to use this uncomplicated and inexpensive technique for the development of zirconia/graphene nanocomposite powders. Over the past years carbon-based nanostructured materials such as carbon, CNTs (carbon nanotubes), graphene, CNFs (carbon nanofibers), fullerenes, etc. have accomplished swift evolution and extensive use because of their Van der Waals force and covalent bonding, chemical stability, and high stiffness and strength together with their low weight.The formation of graphene  raised n2 hybridisation that occurs during the formation of carbon atoms is in charge of its stand-out in-plane elastic and mechanical properties, namely Young modulus (0.5–1 TPa [The sp.5–1 TPa ) and ten.5–1 TPa ). Meanwh.5–1 TPa ,11. As a2) have received significant attention from the scientific circles as a potential material for various structural applications, because of their high mechanical characteristics, physical and chemical wear, and thermal stability coupled with corrosion resistance and zero toxicity [2.Recently, zirconia ceramics / 8 mol% yttria stabilized zirconia by colloidal processing subsequent spark plasma sintering and reported that the addition of GO-enriched layer into zirconia laminates is slowing down the probabilities of crack generation and propagation .In the work of Rao et al., the surface modified graphene zirconium oxide was synthesized and characterized as a nanocomposite material. It was utilized as an adsorbent for the removal of chlorophenols from aqueous solution. The removal efficiency was maximum towards 4-chlorophenol at pH 1.0 and decreased with increasing pH .Teymourian et al. reported the synthesis of the high performance zirconium dioxide-reduced graphene oxide composite and its application as a novel architecture for electrochemical sensing and biosensing purposes . Furthermore, Li et al. prepared zirconia/graphene nanosheets composite coatings using a plasma spraying technique. The obtained composite coating demonstrated low friction coefficient and improved wear resistance .Additionally, in recent years the production of nanostructured crystalline particles has gained attention, as they possess enhanced performance abilities over conventional coarser grain materials. In order to prepare nanocrystalline zirconia-based nanocomposites, different methods such as the sol/gel method , vapor pTherefore, in order to produce effectively isolated nanoparticles with a narrower size distribution, the hydrothermal methods are mentioned as having great potential ,45. ThisAll that is required for synthesis is to take a Teflon-lined stainless-steel autoclave and put the materials and precurcors in inside of it.Uniform nucleation processes and very low grain size highlighted are highlighted as the main benefits of this method. Therefore, the purpose of this work was to produce graphene oxide (GO) with the subsequent production of reduced graphene oxide (rGO), which is used as a support to anchor zirconia nanoparticles and leading to their uniform distribution on the surface of rGO. This special nano-hybrid structure provides the opportunity to obtain a uniform composite ceramic/graphene nanopowder that can be used as a raw material to fabricate dense multi-purpose zirconia-based composites reinforced with nanostructure graphene, which combine the desired properties of each component. Thus, the aim of the present work is to obtain a nanocomposite material when there is phase uniformity and good interfacial bonding between the graphene and matrix after sintering.3 and KMnO4 in concentrated H2SO4. Pure graphite powder was steadily added  into a hot concentration of H2SO4 solution that would be cooled in an ice bath. Afterward, KMnO4 was slowly added, in small doses, to keep the reaction temperature below 20 °C. Then the suspension was treated with a hydrogen peroxide (H2O2) solution and washed with HCl and H2O in order to complete the reaction with KMnO4. After filtration and drying, GO sheets were obtained. In the previous works, this process is decribed in further detail [Hummers’ method was used as synthesis technique of graphene oxide (GO) from graphite powder which was oxidized. Briefly, this approach involves Hummer’s reagents with low levels of NaNOr detail ,46,47.−1. The synthesis of reduced graphene oxide-nanozirconia (ZrO2/rGO) composites was carried out by means of a simple hydrothermal process, in which the GO suspension was used as the rGO precursor, N2H4·H2O and ZrOCl2·8H2O acted as the reducing agent and the nZrO2 precursor, respectively. Firstly, 40 mL of GO colloidal suspension was mixed with 20 mL of a ZrOCl2 solution (0.01 M) to obtain a mixed solution of GO and ZrOCl2 with a volume ratio of 2:1. After being sonicated for 30 min, 1 mL hydrazine hydrate was added to the mixture and was poured into a 200 mL stainless steel teflon-lined autoclave, which was sealed and maintained at 180 °C for 18 h, and then naturally cooled at room temperature. For the purpose of removing Cl-ions, the obtained material was washed, centrifuged, and re-dispersed five times in deionized water. Finally, the ZrO2/rGO nanocomposite powders were dried in a FreeZone2.5 freeze-drying system . The collector temperature is continuously set at −50 ± 2 °C.The as-prepared GO was exfoliated ultrasonically for 2 h in order to obtain a stable GO colloidal suspension with a concentration of 2.33 mg∙mL2/GO) composite powder was prepared following the same cycle without the addition of N2H4·H2O.Furthermore, the shell temperature and the chamber pressure were kept at 23 ± 2 °C and 0.02 ± 0.01 mbar, respectively, during the entire process . For comThe microstructure, crystalline structure, and surface morphology characterizations of the as-synthesized samples were evaluated by transmission electron microscopy JEM 3010 . To this end, nanopowders were placed on a TEM grid  .0 and on surface area basis q2 were compiled. A Gaussian normal distribution was fitted to the measured distribution in order to obtain the corresponding geometric mean particle diameters μg(q0) and μg(q2). The cumulative distribution of the particle diameters q0 was fitted with a sigmoid function to obtain the characteristic particle diameter d50. The specific surface area STEM of the zirconia nanoparticles was calculated from μg(q2) using the density of 5.68 g/cm2 and assuming spherical particles [As was described previously, the particle diameter distribution of the nanocrystalline zirconia was determined from TEM micrographs by measuring the diameters of about 800 zirconia nanoparticles in both synthesized nanopowders. From these data, the density distribution of the particle diameters on number basis qarticles . FurtherX-ray diffraction measurements were conducted using an Empyrean diffractometer  ranging from 5° to 70°. The step size was 0.05 with a scan speed of 0.06 /min. The diffractometer used Cu Kα radiation (λ = 1.5405981) working at 40 kV and with an intensity of 30 mA .TM2 Raman Microscope, Thermo FisherScientific, Waltham, MA, USA) with a wavelength of 532 nm and a laser power of 2.0 mW. The laser beam was focused through an optical microscope’s 50× objective lens to a spot size of 2 µm on the studied area . The accumulation time for each Raman spectrum was about 10 s [Raman spectra of studied materials were collected to identify the phase composition. The Raman setup is composed of a laser (DXRout 10 s .2/rGO and ZrO2/GO nanopowders were conducted separately using a Gaussian–Lorentzian function after performing a Shirley background correction.All X-ray photoelectron spectroscopy (XPS) measurements were carried out on a K-Alpha  photoelectron spectrometer equipped with a micro-focused monochromator Al Kα X-ray source. Peak fitting of the C1s and Zr3d spectra for ZrO−1 to 4000 cm−1 . For this purpose, potassium bromide (KBr) pellets (diameter 1.0–1.3 cm) of each powder sample were prepared using a uniaxial press [Fourier transform infrared (FTIR) spectra of the synthesized nanocomposite powders were measured  in the wavenumber range 500 cmal press .g(q0) and μg(q2) of the particle size distribution are 5.1 nm ± 0.8nm and 4.8 nm ± 1.2 nm, respectively, and the d50 diameter is 5.0 nm with an uncertainty of ± 0.8 nm for both ZrO2/GO and for ZrO2/rGO nanopowders. The specific surface area STEM of the nanopowders was calculated from the geometric mean diameter μg(q2) and reached a value of 110.0 m2/g.The nanozirconia crystals have a particularly spherical shape and they are uniformly dispersed. Almost no difference was found in particle size distribution and specific surface area. The geometric mean diameters μ2/rGO nanocomposite powder diffraction peaks in the region up to 20° are not presented as indicated in the case of GO and enables to say that the GO was well reduced during the reaction. In addition, analys of intensities and peaks location appearing in XRD data were compared with the infromation from the International Centre for Diffraction Data (ICDD) for tetragonal (PDF file no.01-083-0113) and monoclinic (PDF file no. 00-024-1165) structures.The most intense apparent peak located at 2θ = 10.8° in the curve of GO, and corresponds to the (001) reflection of GO . The resThe Raman spectra of all the studied materials are shown in −1 is the G band and it is the more intence than the 2D (2720 cm−1) and D (~1350 cm−1) bands  [−1 means that sp2 bonds were broken and, consequently, it indicates that a intended transformation of sp2 hybridization to sp3 was occurred, which shows a rise of the disorder in the sp2-hybridized carbon system [2/rGO  bands a. In the1) bands b–d, a imand sp3) . The morn system . This dim [2/rGO d powders2 nanoparticles onto the surface of the rGO causes additional changes in the characteristics of the vibrations in the material’s lattice. Therefore, the intensity of D bands after the reduction process is higher in comparison to the unreduced ZrO2/GO nanocomposite powders. It was suggested that, besides some defects in reduced graphene oxide, the presence of ZrO2 carbon lattice and consequently its structural changes. However, the elimination of oxygen containing groups leads to the restoration of ordered graphitic stacking in rGO and causes the appearance of 2D peak [It should be noted that after the graphite’s chemical oxidation process, the presence of peaks attributed to 2D band were not observed in GO b. It can 2D peak ,54,55.−1 is a second-order peak and denoted by S3.Another band, known as conjunction between D and G intensities and appears near 2900 cm−1, 269 cm−1, 407 cm−1 and 616 cm−1 were interpreted as tetragonal zirconia phases [S3/2D intesity ratio is related to the diminution in defects and involves a lower quantity of oxygen in graphene . Raman sa phases ,58.To investigate the chemical changes between reduced and not reduced graphene oxide powder XPS measurements have been carried out .2/GO  peak, C-C (sp3 C) peak and C=O (the carbonyl groups), respectively [For further interpretation and to better understand the mechanism of formation and the chemical states of elements, comprehensive analysis of C and Zr XPS spectra were carried out. The detailed C1s spectra of not-reduced and chemically reduced powders are shown in ectively ,60. Moreectively ,62,63, wectively .2/rGO powder had an additional peak at 285.9 eV ) and bending (δ (−OH)) vibrations of coordinated or absorbed water molecules retained on the ZrO2 suface [−1 indicates the existence of OH deformations in the C–OH groups [−1 [2 stretching mode demonstrates the broad absorption peaks around 2930 cm−1 [Obviously, the clear expresed absorption that appeared at peaks at 3420, 1635 and 1558 cm2 suface . The peaH groups . The Zr-oups [−1 ,74,75. M930 cm−1 .−1, while the O–H bending vibration of water molecules and the C=C skeleton stretching mode was assigned at 1626 cm-1 [−1. The peak located at 1227 cm−1 belongs to the C–O–C stretching vibration. The absorption bands at 1121 and 1072 cm−1 corespond to the epoxy C-O and the alkoxy C-O stretching vibration, respectively [−1 was recognized to stretching of the aromatic C=C mode, while the band at 1234 cm−1 corresponds to the epoxy C−O and C=O, indicates that GO was reduced incompletely [−1 was assigend to C=O stretching frequency belonging to the ZrO2/rGO nanocomposite was shifted to 1746 cm-1 because the relationship between the C=O group and Zr and its relative intensity was also decreased [2 solution provides the interaction between the oxygen-containing groups of GO and Zr(IV) and the appearance of two vibrational bands located at 1460 and 1395 cm−1, which were assigned to the creation of either a monodentate or bidentate complex [−1 can be assigned to the C=C stretching mode and Zr–O vibration, respectively [2 nanoparticles and rGO nanosheets occured, i.e., ceramic nanoparticles were directly attached to the surface of the rGO nanosheets.The C=O stretching vibration of –COOH and C=O was observed at 1735 cm626 cm-1 . The O–Hectively ,81,82,83mpletely ,85,86. Oecreased . The hyd complex ,79,80,812 nanoparticles were seen  plane surface. We believe that this synthesis route will allow for the effective production of uniform ceramic/graphene nanopowders that can be used as a building block to fabricate zirconia matrix composites reinforced with a homogenous nanostructure graphene after sintering.In the present work, a nanostructured ZrO


Arabidopsis thaliana is negatively regulated by ABI4 and plays a key role in water uptake by seeds, ensuring that imbibed seeds proceed to germination.PTR2 in Arabidopsis thaliana, the physiological role of the tonoplast-localized, seed embryo abundant PTR2 is unknown. In the present study, a molecular physiological analysis of PTR2 was conducted using ptr2 mutants and PTR2CO complementation lines. Compared with the wild type, the ptr2 mutant showed ca. 6 h delay in testa rupture and consequently endosperm rupture because of 17% lower water content and 10% higher free abscisic acid (ABA) content. Constitutive overexpression of the PTR2 gene under the control of the Cauliflower mosaic virus (CaMV) 35S promoter in ptr2 mutants rescued the mutant phenotypes. After cold stratification, a transient increase in ABA INSENSITIVE4 (ABI4) transcript levels during induction of testa rupture was followed by a similar increase in PTR2 transcript levels, which peaked prior to endosperm rupture. The PTR2 promoter region containing multiple CCAC motifs was recognized by ABI4 in electrophoretic mobility shift assays, and PTR2 expression was repressed by 67% in ABI4 overexpression lines compared with the wild type, suggesting that PTR2 is an immediate downstream target of ABI4. Taken together, the results suggest that ABI4-dependent temporal regulation of PTR2 expression may influence water status during seed germination to promote the post-germinative growth of imbibed seeds.Peptide transporters (PTRs) transport nitrogen-containing substrates in a proton-dependent manner. Among the six PTRs in The online version of this article (10.1007/s11103-020-00967-3) contains supplementary material, which is available to authorized users. Seeds comprise the seed coat, endosperm, and embryo. Germination of dormant seeds commences with the uptake of water and terminates with the emergence of the radicle through the seed coat Bewley . During CYP707A , gibberellic acid (GA), ethylene, and auxin, and the antagonism between GAs (which stimulate seed germination) and ABA (which establishes and maintains seed dormancy) is the primary factor that regulates seed germination , ABA2 (encoding xanthoxin dehydrogenase), and ABA3 (encoding molybdenum cofactor sulfurase) genes, which in turn suppresses germination   and a wide range of nitrogen (N)-containing substrates, including nitrate, amino acids, and di-and tri-peptides , which is formed from vacuoles during seed maturation as a result of protein deposition and water displacement , seven ptr mutants (ptr1–6) including ptr2-1 and ptr2-2 alleles, two PTR2 complementation lines (PTR2CO2 and PTR2CO5), abi4 mutant, and ABI4 overexpressor (ABI4OE) were used in this study. Seeds from seed batches grown and harvested at the same time and stored for less than 2 weeks were treated with 70% ethanol for 1 min and then sterilized with 0.8% sodium hypochlorite. After washing five times with sterilized distilled water (SDW), seeds were imbibed in water in the dark at 4 °C for 2 days to break seed dormancy and then transferred to continuous light (30 µmol m−2 s−1) at 22 °C. Seeds were sown in SDW supplemented with 2% Glc, 1 µM ABA, 50 µM GA, ABA inhibitors (20 µM nordihydroguaiaretic and 10 µM diniconazole), 20 mM glycine di-peptide (Gly-Gly), 20 mM leucine di-peptide (Leu-Leu), or 10 ∝g ml−1 of a transcription inhibitor, Cordycepin. The osmotic potential of SDW was adjusted to − 0.23, − 0.39, − 0.68, or − 0.91 Mpa with polyethylene glycol 4000 .PTR genes were identified at The Arabidopsis Information Resource (TAIR) database (https://www.arabidopsis.org/index.jsp). Seeds of ptr1-1 (SLAK_131530), ptr2-1 (SALK_400_D08), ptr2-2 (SAL_65_B10), ptr3-2 (SALK_097591), ptr4-1 (SALK_062626), ptr5-2 (SALK_116120), and ptr6-1 (SALK_149283) were obtained from the Arabidopsis Biological Resource Center (ABRC) at Ohio State University, OH, USA. To map the T-DNA insertion sites, genomic DNA was isolated from the leaves of mutant plants using lysis buffer . Reverse transcription PCR (RT-PCR) was carried out using the SALK LBa1, LBba1, and LB1 primers and PTR2 gene-specific primers (Supplementary Table S1). The resulting PCR products were sequenced and compared with the genomic sequence of each gene to map the T-DNA insertion site .T-DNA insertion mutant lines of all six Arabidopsis PTR2 was amplified by PCR using sequence-specific primers (Supplementary Table S1). The PCR product was digested with XbaI and BamHI restriction endonucleases and cloned into the pJJ461 vector. The construct was confirmed by sequencing and transformed into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method. Transformation of ptr2 mutants was conducted using the floral dip method. PTR2CO lines with a single-copy of the hygromycin resistance (HygR) gene and PTR2 were identified by RT-PCR .The coding sequence of g for 10 min. Then, 600 µl of the supernatant (embryo extract) was transferred to an Eppendorf tube, followed by the additional of an equal volume (600 µl) of chloroform. The mixture was mixed thoroughly by shaking for 10 min at room temperature. After the addition of 600 µl of buffered phenol (pH 8.0), the organic phase was transferred to two new 2.0 ml Eppendorf tubes (300 µl each). Then, five volumes of methanol containing 0.1 M ammonium acetate were added to each tube, and the mixture was incubated at − 20 °C for 1 h to precipitate the proteins. Proteins were separated by centrifugation at 15,000×g for 10 min and washed first with 100% cold acetone and then with 80% cold acetone. The protein pellet was air dried and resuspended in SDS sample buffer . Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) using 15% resolving and 5% stacking polyacrylamide gels.Seed proteins was extracted using the chloroform-assisted phenol extraction (CAPE) method. Briefly, seeds (0.1 g fresh weight) were homogenized using a cold pestle and mortar in 1.0 ml buffer . The homogenate was centrifuged at 12,000×AP2M (at5g46630) gene  and treated with DNaseI (Takara). After NucleoSpin RNA Cleanup (Macherey-Nagel), cDNA was synthesized from 500 ng of total RNA using the iScript cDNA Synthesis Kit (Bio-Rad) and amplified by qRT-PCR on the CFX96 Real Time System (Bio-Rad) using the iQ™ SYBR Green Supermix (Bio-Rad), according to the manufacturer’s instructions. Reactions were performed in triplicate in a 10 µl volume, containing 5 µl SYBR Green Master Mix, 0.5 µl of each primer (10 pmol), 4 µl of 20-fold diluted cDNA, and 0.5 µl nuclease-free water, using the following conditions: 95 °C for 15 min, followed by 40 cycles of amplification at 94 °C for 10 s and 62 °C for 30 s, and lastly a final extension at 72 °C for 30 s. Fluorescence was measured at the end of each extension step. Amplification was followed by melting curve analysis, with continual fluorescence data acquisition during the 65 °C to 95 °C transition. A negative control containing water instead of cDNA template was included in each run. Raw data were analyzed with the CFX Manager software, and gene expression was normalized to the expression of −1) (Money Imbibed or germinating seeds (20 mg) were frozen in liquid N and ground using a pestle and mortar. The ground powder was immersed in 1 ml of 80% methanol and stored at 4 °C for 1 h. The extracts were filtered with C18 (Sep-Pak Vac) cartridges  to remove pigments and other polar materials. The filtered solution was dried and concentrated using a rotary vacuum concentrator and suspended in Tris-buffered saline (TBS). The level of ABA was determined using the enzyme-linked immunosorbent assay (ELISA) Kit (Agdia). To measure the seed water content, ca. 330 seeds immersed in SDW were collected at a given time, and external liquid was removed using a silica-based membrane column by spinning at 12,000 rpm for 5 min. Then, dry seed weight was subtracted from the fresh seed weight to determine the amount of water absorbed by the seeds. External osmotic potential of varying concentrations of PEG4000 was calculated from the estimated osmolality (mol kg1) Money .Escherichia coli and purified using a metal-affinity resin (Qiagen)  and competitor DNA fragments, including specific unlabeled fragment and ABI5 promoter fragment , were used with the purified GST-ABI4 protein in EMSAs .Glutathione S-transferase (GST)-tagged DNA-binding domain of ABI4 (GST-ABI4) was expressed in PTR2 and CaMV 35S promoters were cloned and fused to the β-glucuronidase (GUS) reporter gene in pCAMBIA3301 using primers listed in Supplementary Table S1. The resulting constructs were transiently transformed into the WT, abi4, and ABI4OE lines. Four-day-old seedlings grown in darkness were vacuum-infiltrated with Agrobacterium twice for 1 min each ; 4-methylubelliferone (Sigma-Aldrich) was used for calibration. Protein content was determined using bovine serum albumin as the standard 6, and 0.5 mM K4Fe(CN)6, and subsequently incubated in a staining solution containing 1 mM 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (Duchefa)  were analyzed using the two-tailed Student’s t test, after normality assessment. Means with p values less than 0.05 were considered statistically significant.Data . Thus, PTR2 activity appears to be regulated at the post-transcriptional level during early seed germination and then at the transcriptional level during late seed germination.Publicly available microarray data show that transcript levels of α- and β-subunits of the legumin-type 12S globulins , suggesting a transcriptional regulation of both these genes during seed germination. However, transcripts of CYP707A3, CYP707A4, GTs, and BGs showed minimal or no changes (data not shown).We also quantified the water and ABA contents of germinating seeds, as aquaporin-independent uptake of water  and was almost complete at 36 HAS  or amplified by that of ABA and Glc , and was fully rescued by the overexpression of PTR2 under the control of the CaMV 35S promoter (35SCaMVp::PTR2) in the complementation lines PTR2CO2 and PTR2CO5 .To identify the physiological role of PTR2 in the seed germination process, ptr2-1 seeds exhibiting the delayed TR phenotype. Rapid water uptake during phase I was not affected by the PTR2 mutation. However, water uptake during phase II and phase III was lowered by 17% and 8%, respectively  and the GST-ABI4 fusion protein. The recombinant ABI4 protein containing only the DNA-binding domain bound directly to all PTR2 promoter fragments, like ABI5 (a positive control), but this binding was inhibited by unlabeled, cold fragments  and transformed into hypocotyls of WT, abi4, and ABI4OE. The expression of PTR2-GUS was lowered by 10% in the ABI4OE background, consistent with the results of the EMSA, thus confirming that ABI4 represses PTR2 transcription by directly binding to the CCAC motifs in PTR2 promoter , which is shaped by ABI4, suggests that PTR2 is involved in water uptake rather than in N mobilization from the endosperm to the growing axis. Our results are consistent with those of Song et al. , thus ruling out the role of PTR2 in the mobilization of seed storage proteins during seed maturation.Di- and tri-peptide sources available for the tonoplast-localized PTR2 seem different between the first half and second half of the water uptake phase II. Time-course analysis of water and storage protein contents of seeds clearly revealed that the inhibition of water uptake observed in the PTR2 belongs to NRT1/PTR family (NPF) proteins. In addition to nitrate and di- and tri-peptides, some NPFs are involved in the uptake of other substrates, such as nitrite, chloride, glucosinolates, auxin, ABA, GA, and jasmonates Electronic supplementary material 2 (DOCX 22 kb)Below is the link to the electronic supplementary material.