The real capacity of graphene and the lithium-storage process in graphite are two currently perplexing problems in the field of lithium ion batteries. Here we demonstrate a three-dimensional bilayer graphene foam with few defects and a predominant Bernal stacking configuration, and systematically investigate its lithium-storage capacity, process, kinetics, and resistances. We clarify that lithium atoms can be stored only in the graphene interlayer and propose the first ever planar lithium-intercalation model for graphenic carbons. Corroborated by theoretical calculations, various physiochemical characterizations of the staged lithium bilayer graphene products further reveal the regular lithium-intercalation phenomena and thus fully illustrate this elementary lithium storage pattern of two-dimension. These findings not only make the commercial graphite the first electrode with clear lithium-storage process, but also guide the development of graphene materials in lithium ion batteries. The mechanism of lithium storage in graphenic carbon remains a fundamental question to be addressed. Here the authors employ suitable bilayer graphene foam to investigate various physiochemical phenomena of lithium intercalation and propose a storage model. Before its commercial application in 19902, intensive studies had been performed to determine its related Li-storage process/mechanism4. Examples include the classical pleated-layer model by Daumas and Hérold in 1969, and the earlier Rüdorff model in 19657. Recently, in the wake of the discovery and development of graphene  and the numerous reports of enhanced energy-storage performances of graphene-modified electrode materials, many researchers believe that the capacity of LIB can be significantly enhanced through simply replacing the traditional graphite anode by this ultimate carbon material that has metal-level conductivity, large surface area (2630 m2 g−1 in theory), and possibly two exposed sides to adsorb Li atoms (namely forming the expected Li2C6 stoichiometry with a doubled capacity of 744 mAh g−1)10. However, despite the considerable investment in money and time, this target has not been achieved by using pure graphene materials. Even the Li-storage mechanism (or the storage locations) on graphene is still up for debate both experimentally and theoretically12. For instance, based on density functional theory (DFT) calculations14 and the different electrochemical behavior of monolayer graphene supported by Cu foil from those of multilayer graphenes/graphite for LIBs15, it has been proposed that Li atoms cannot be adsorbed onto pristine monolayer graphene, instead they only intercalate into the graphene interlayer or the interspace between graphene and substrate through edge planes or high-order defects . The understanding and resolution of this problem are rather essential for the development of graphene materials in LIBs.Despite its limited capacity (maximum of 372 mAh gn) into account, the considerable difference between the deduced theoretical capacities assuming the above opposite viewpoints . However, basic requirements for graphene in LIBs, such as a sufficient monolithic mass and absence of extrinsic interference from the as-applied substrates  to address this issue.Taking the number of graphene sheet layers  route17 Fig. , we have17 Fig. . Such a 17  was measured to be about 0.49, in good agreement with the computational and experimental values of I{0110}/I{1210} ≈ 0.4–0.5 for the Bernal (AB) stacking bilayer graphene23. By distinguishing the height, width, Lorentzian fits, intensity, and chemical-shift features of the 2D and G bands, Raman spectroscopy can provide more definitive identification for graphene materials with diverse numbers of layers regardless of the macroscopic appearance27, as displayed in Fig. 8 C6-ring units with an area of 0.052 nm2) were randomly collected from different locations on the surface and cross-section of the as-prepared samples (before and after removing the Ni substrate). In this way, the bilayer-dominated region in our target material was estimated to be over 90%25. Moreover, the specialized Lorentzian fitting analyses of the 2D bands in Fig. 26, together with the matched FWHM and ID2/IG values 27, can help confirm the predominant Bernal stacking configuration of this CVD-grown bilayer graphene foam. This is also in line with subsequent X-ray diffraction (XRD) results showing an interplanar spacing d002 of ~3.35 Å ). The weak D band from the Ni-free bilayer graphene indicates the mere existence of some intrinsic structural defects originating from the edge planes, grain boundaries, vacancies, or geometrical curvatures of the graphene sheets17. These defective sites on graphene are essential in coordinating the 3D nanoporosity17 as well as suppressing the aforementioned restacking effect29. Combining the preparation mechanism and the above characterization results, we presume that such bilayer graphene foam can help elucidate the Li-storage mechanism in graphene-based electrodes.As shown in Fig. 6 in ethylene carbonate/dimethyl carbonate in R2032-type coin cells, the common half-cell configuration2 was adopted for the electrochemical characterizations of the freestanding bilayer graphene foam. For graphite-based LIBs, it is well known that (1) the insertion/deinsertion potential of Li+ ions is always below 0.3 V (vs. Li+/Li)31, a range that contributes to the overwhelming capacity of graphite (close to 372 mAh g−1), and that (2) the inevitable solid-electrolyte interphase (SEI) is usually generated at above 0.5 V during the initial charging–discharging cycles34. Both the highly reversible galvanostatic charge–discharge (GCD) measurements  that are close to the theoretical value of LiC12 at 8040 C mol−1  to gain deep insight into the Li-intercalation behavior in bilayer graphene. Clearly, there are seven intrinsic and quasi-reversible redox peaks in each CV profile . Compared to those reported for graphite36, other than the emergence of one new pair of redox peaks , especially considering the staged distribution densities of Li atoms on the X–Y plane  between the CV peak-current response (i) and the sweep rate (ν), the pseudocapacitive intercalation for Li+ ions, namely the surface-controlled behavior with b ≈ 1.0 at the initial C5–C3 and unique C1 stages, tended to become slow solid-state diffusion (like the case in a battery with b ≈ 0.5) for the PI generation , further verified this rate-determining step. These values and their trends are also similar to those of natural graphite electrodes (DLi+ ≈ 10−9–10−7 cm2 s−1)45. Obviously, it is the planar distribution density of Li atoms, not the number of graphene layers, that intrinsically determines the intercalation kinetics of Li-graphene/graphite products, in line with the above-mentioned 2D model , all the characterization results are statistical and thus can reflect the macroscopic features and general phenomena. Firstly, the highly reversible in situ Raman spectra9 for this graphene electrode , ex situ Raman spectroscopy, transmission electron microscopy (TEM), and electron energy loss spectroscopy (EELS) techniques  by the outer graphene sheets in the CRC-stacking configuration31. Furthermore, according to the simultaneous XRD patterns of the typical bilayer and multilayer electrode samples  to Li atom (radius: 1.52 Å))28, the d002 value  seemed to increase slightly in the foregoing P5−P3 phases of ARB stacking (<0.02 Å), only relying on a small population of Li atoms. However, after the unstable P2 phase48 with a theoretically transitional stacking configuration from ARB to ARA28 28. These inequalities likely result from the varied electrostatic repulsions between neighboring graphene layers bonding with the interbedded Li atoms of different volume concentrations  and the following ex situ XPS results also favor these observations and discussions. That is to say, Li intercalation into the interlayers of various graphenic carbons not only follows similar mechanisms in the 2D plane  and Li-free graphene sheets at the highly lithiated PI or P1 phases  and 0.40 (1.17) for A–B (A–A) stacking C6C6 and C6LiC6, respectively). In particular, for the P3 phase with a large enough but still unsaturated Li concentration, the plentiful vacancies would make the initial movements of its interlayered Li atoms rather disordered , when the peaks at 52–51 eV were ascribed to metallic Li species (like in the ARB mode)50. Thus, from the point of view of chemical composition change, the combined experimental and theoretical results further reveal and verify the phase-wise stacking configurations suggested by the XRD and SAED characterizations performed at different analytical ranges. This is seen in: the maintained ARB structure at P5  and P4  modes at P3 (C14LiC14) with a large enough Li concentration  with ionic Li 1s species of high BE 47 and a simultaneous nearly single C 1s species. In brief, upon the Li intercalation, the holistic ionic character of Li atoms is enhanced along with the stacking configuration transformation, while the metallic character decreases .In view of the roughly equal concentrations (at 50 ± 3 at.%) and the common source of their O components  t 28 Fig. , and (3) 28 Fig.  accordin 28 Fig. , the obs 28 Fig. , and the 28 Fig. . All theits Fig. .Fig. 5ScD graphenic carbon material. It is found that there is no fundamental difference between bilayer or fewlayer graphenes and graphite electrodes in the Li-storage manner and kinetics behavior, and the whole Li-storage process goes through four-time fractal-way Li intercalations to achieve the saturated composition of LiC6, with the final step the rate-controlling step. This study not only clarifies the Li-storage mechanism for commercial graphite anodes, but also highlights the potential of defect-free graphene in LIBs to guide its development in the energy-storage field.In summary, by developing a high-quality freestanding bilayer graphene foam and systematically studying various aspects of Li storage in it , we have demonstrated that Li atoms can only intercalate into graphene interlayer and proposed a planar Li-storage model for 230Mn70 foil in 2.0 M (NH4)2SO4 solution for 10 h, was put at the center of a quartz tube (φ30 × φ27 × 1000 mm) of the CVD furnace. The initial flow of Ar was 500 sccm until that the temperature reached the low temperature Tl shown in Supplementary Fig. Th, the flow of Ar and H2 was gradually increased to be 2500 and 100 sccm, respectively. After the above reduction pre-treatment, benzene  was introduced with the gas flow of Ar and H2 for graphene growth. For bilayer graphene, the initially total pressure and the partial pressure at the tube’s front end should be controlled at 5–6 and 0.5–0.7 Mpa, respectively. The minimum Tl value was determined to be 700 °C, below which a large amount of defects would generate on the graphene sheets. The furnace was immediately opened to let the inner quartz tube rapidly cool to room temperature with a fan  for the target material.Nanoporous Ni foam, by directly dealloying the Ni2O < 0.5 p.p.m., O2 < 0.5 p.p.m., MBraun, Unilab) with LiPF6 solution of 1 M in ethylene carbonate (EC)/dimethyl carbonate (DMC) (EC:DMC = 1:1 vol/vol) as the electrolyte, the freestanding graphene foams, the graphene@np-Ni composites, monolayer graphene on plate Cu foil, bilayer graphene on plate Ni foil directly as the working electrodes, fresh Li foil as the counter electrode, and Whatman glass fiber as the separator. No binder was adopted for the coin-cell assembly. For the graphite electrode supported by Rany Ni current collector or not, polytetrafluoroethene (with the mass ratio to graphite powder at 1:9) dissolved in ethanol was used as the binder. Both GCD and CV measurements were carried out at room temperature on an electrochemical workstation and a battery-measurement equipment. The alternating-current electrochemical impedance spectrum with varied open circuit voltages were recorded simultaneously at a frequency range from 100 kHz to 0.1 Hz and an amplitude of 5 mV. ZView software was applied to simulate the Nyquist plots in view of the reported equivalent electric circuit proposed for graphite electrode42.Coin 2032-type test cells were assembled in a high-purity argon-filled glove box (HKα radiation and nickel filter (λ = 0.15406 nm), and the operating voltage and current were 40 kV and 30 mA, respectively. A micro-Raman spectrometer (Renishaw InVia RM 1000) with laser wavelength of 514.5 nm (excitation energy: 2.41 eV) was used for Raman measurements. The laser power was set at 2.0 mW to avoid possible damage by laser irradiation. The spectrum acquisition time was 150 s. The surface species of the samples were determined by XPS  with an Al Kα (mono) anode at 150 W in a vacuum of 10–7 Pa. Both the Raman and XPS spectra were recorded from micron-sized regions.The microstructure was characterized by both of a field-emission SEM  and a field-emission TEM  equipped with two aberration correctors (CEOS GmbH) for the image- and probe-forming objective lens systems. The SAED patterns of each sample were recorded from areas of 30–100 nm in diameter. High-resolution EELS spectra of the samples were collected using a Gatan Enfina spectrometer, and energy dispersion was 0.5 eV per pixel. The XRD patterns were collected on X-ray diffractometer (SmartLab) using Cu To prepare lithiated graphene samples for the ex situ TEM and XPS measurements, in case of oxidation, each coin cell loading the target Li-graphene product Fig.  was disa51 was applied to generate projected electrostatic potential of the Li and C atoms. The applied accelerating voltage was set at 200 kV. Standard deviation of the random Gaussian distribution accounting for atom vibration was 0.085 Å, and phonon configuration number was 300. Considering the large area selected for the SAED, the periodic boundary condition was set in order to acquire intense Bragg peaks. Different rectangle supercells were constructed to meet the rectangle dimensional of simulated image size (1024 × 1024). Meanwhile, CaRine Crystallography 3.1 software was applied to simulate the diffraction patterns of various structure models to obtain the information of lattice spacings.The multislice method was adopted to simulate the diffraction spot brightness of various graphene-based structures. The scattering factors in ref. −1. In the static calculations of total energy, a 10 × 10 × 1 Monkhorst–Pack k grid was employed and the electron wavefunctions were expanded by using a plane wave basis set with a cutoff energy of 400.0 eV.First-principles calculations were performed by using the Vienna ab initio simulation package, based on the spin-polarized DFT. The electron-ion interactions were presented by the frozen core all-electron projector augmented wave pseudopotentials, and generalized gradient approximation of the electron exchange-correlation functional was adopted. The atomic layer distance was fully optimized until the Hellmann–Feynman force was <0.01 eV Å14 sheet and LiC6 sheet with normal in-plane lattice parameters  were constructed in the first step. Then, we restricted the Z-axis relaxation and merely allowed atoms to relax in the X–Y plane  to obtain the optimized structure achievable in theory. Finally, the above CaRine Crystallography 3.1 software was applied to simulate the in-plane lattice parameters of the new structure (namely the optimized unit cell) used for comparison with those experimental SAED data.On the one hand, to confirm the in-plane lattice-expansion phenomena of lithiated bilayer graphene induced by electron beam irradiation Fig.  and 18, X–Y plane, we fixed the interlayer spacing , restricted the in-plane relaxation, and merely allowed individual atoms to relax along the Z-axis direction in the binding-energy calculations  was calculated asEn−1)Ligraphene+ and n Li atoms interlaminated between two graphene atomic layers. μLi is the average Li chemical potential calculated as the unit cell merely contains isolated Li atoms. As for the BE of different equivalent C sites in different stacking configurations with Li interlaminated, its value was calculated asV). Similarly, μC is the average C chemical potential calculated as the unit cell merely contains the specific isolated C atoms.On the other hand, considering different configured Li sites relative to carbon lattice in the ons Fig. . The litSupplementary InformationPeer Review File


Graphene, a two-dimensional nanomaterial, has shown tremendous promising applications in a broad range of fields. Mass production of defect-free graphene is a prerequisite for its applications. In this work, by using a needle valve, we propose a simple hydrodynamic-assisted exfoliation method to produce high-quality few-layer graphene flakes. The prepared graphene flakes, with an average layer of 5 (~ 71% less than five layers) and a Raman D/G intensity ratio as low as 0.1, are free of defects and oxidation. The average thickness and length of the few-layer graphene flakes are 2.3 nm (~ 90% < 4 nm) and 1.9 μm (~ 50% in the range of 1–7 μm), respectively. In a lab-scale trial, the concentration of graphene can reach 0.40 g/ml under mild operating conditions , and the corresponding production rate is 0.40 g/h. The hydrodynamic-assisted exfoliation by needle valve potentially offers a simple and efficient method for large-scale production of high-quality graphene. Graphene, a single layer of graphite, has been attracting growing attention since its discovery in 2004 . Owning To date, many methods such as micromechanical cleavage , chemicaID/IG, a measure of defect content) and low aspect ratios. For example, Liang et al. [ID/IG value of 0.38 for jet cavitation exfoliated graphene, while the length was unknown. The graphene produced by kitchen blender  and high-pressure homogenizer  were also featured with high ID/IG values and low aspect ratios [ID/IG and length in the range of 0.14–0.78 and 0.02–1.41 μm [Recently, as an alternative pathway, fluid dynamics-assisted liquid phase exfoliation, has been proposed to produce graphene with low defect content on large-scale , 23–33. g et al.  reportedt ratios , 31. Dif–1.41 μm –33, respN-methyl pyrrolidone aqueous solution as solvent [In this work, a simple method based on hydrodynamic mechanism was proposed for scalable production of high-quality few-layer graphene flakes. A simple needle valve was used as exfoliation device. The exfoliation process was exemplified using 80 wt%  solvent , 35. QuaN-methyl pyrrolidone (NMP) (purity 99.5%) and graphite powder  were purchased from Aladdin Industrial Corporation in Shanghai (China). Deionized water was purified by a laboratory water purification system .A schematic view of the needle valve used for exfoliation is shown in Fig. The morphology and size of graphene were analyzed by SEM . Samples for SEM were coated with gold in an argon atmosphere. TEM was performed by a Tecnai G2 F30 S-Twin and operated at 300 kV. The samples were prepared by dropping the graphene dispersion onto holey carbon grids. AFM images were captured in tapping mode using a Bruker Dimension Icon. A newly cleaved mica was used as substrate for AFM analysis. Raman spectroscopy was conducted with a Lab RAM HR800 (λ = 532 nm) at room temperature. XPS was employed to detect the oxidation defect of graphene using an ESCALAB 250Xi analyzer. UV-Vis absorption was performed to measure graphene concentration by a Lambda 35 spectrophotometer (PerkinElmer) at a wavelength of 660 nm.Figure To identify the formation of mono-layer or few-layer graphene, TEM analysis was performed to visually observe the number of graphene layers. Figure To further identify the thickness and length of graphene, AFM analysis was performed by using mica wafer as the substrate. Shown in Fig. −1), G band (~ 1580 cm−1), and 2D band (~ 2700 cm−1) were observed for these two graphitic materials. For graphene, the 2D band was in the shape of symmetric peak. The full width at half maximum (FWHM) of G band was 13 cm−1, well matching with the previous reports for thin graphene flakes (12–14 cm−1) [ID/IG) for the prepared graphene was 0.10, lower than that of ultrasonication exfoliated graphene (0.29) [Raman spectroscopy was performed to detect the defect content of graphene. Figure 14 cm−1) . Moreovee (0.29) , shear fe (0.29) , 26, ande (0.29) , 32, furThe oxides content of the prepared graphene flakes was investigated by XPS. As shown in Fig. P), number of cycles (N), and initial concentration of bulk graphite (Ci), on the concentration of few-layer graphene dispersion were investigated.To maximize the productivity of few-layer graphene, the effects of operating parameters, that is, working pressure , the concentration obviously increased with increasing working pressure from 1 to 20 MPa, and a concentration as high as 0.40 mg/ml was reached at 20 MPa. However, further increasing the working pressure to 30 MPa, no significant increase in the concentration was observed. Such results may be explained by the following reasons. In hydrodynamic-assisted exfoliation process, increasing in the working pressure results in an increase in the collapse intensity of cavity, due to which there is an increase in the magnitude of the stress derived from cavitation and turbulent, thereby favoring the delamination of graphite. The concentration did not change appreciably above 20 MPa, probably due to the agglomeration and re-stacking of graphene flakes caused by the rise of temperature under higher working pressure [As shown in Fig. pressure .Fig. 8EfP = 20 MPa, Ci = 10 mg/ml). As expected, the concentration was increased with increasing number of cycles. With 16 cycles, the concentration reached a maximum value of 0.4 mg/ml. However, further increasing the number of cycles, the concentration was basically unchanged. In the exploitation process by needle valve, passing the suspension through the valve more than once could result in the fragmentation of graphite, which was favorable for exploitation due to the fact that smaller graphite pieces are easier to delaminate into graphene than larger ones [Figure ger ones . Howeverger ones . TherefoP = 20 MPa, N = 16), the concentration increased from 0.146 to 0.40 mg/ml with increasing the initial concentration of graphite from 2 to 10 mg/ml. In liquid phase exfoliation process, particle-particle collisions are beneficial to the delamination of graphite. A higher initial concentration of graphite leads to the reinforcement of particle collision, thereby contributes to the self-exfoliation of graphene [Initial concentration of bulk graphite also had a significant influence on graphene concentration . As showgraphene . When thgraphene  and Araographene , indicatID/IG of the few-layer graphene produced by different fluid dynamics methods. The graphene produced by needle valve had a concentration as high as 0.40 mg/ml, which was higher than most of the reported values. In a lab-scale trial, the production rate was calculated to be 0.40 g/h. The concentrations reported by Varrla et al. [ID/IG as low as 0.1 (lower than the reported ratios). Therefore, it can be concluded that the hydrodynamic-assisted exfoliation by needle valve was an efficient approach to produce few-layer graphene flakes with high quality.Table a et al.  and Araoa et al.  could reWe suggest the superiority of the hydrodynamic-assisted exfoliation be ascribed to the exfoliation mechanism. Considering the structure of the needle valve, the flowing fluid dynamics effects are responsible for the delamination of graphite: First, when the suspension containing graphite passes through the narrow gap of the valve, the total pressure of the liquid falls sharply below its vapor pressure. As a result, a turbulent jet that causes huge hydrodynamic stress is formed at the outlet of the valve and generating a large volume of cavitation bubbles. The bubbles then grow from micro gas nucleuses and subsequently collapse intensely. With the collapse of bubbles, intensive microjets and shock waves that surround the graphite are generated, thus resulting in the delamination . Second,ID/IG ratio as low as 0.1 were free of defects and oxidation. In a lab-scale trial, the concentration of few-layer graphene could reach 0.40 mg/ml under mild operating conditions , and the corresponding production rate was 0.40 g/h. Hydrodynamic-assisted exfoliation by needle valve was potentially an efficient method for scalable production of high-quality graphene.In summary, we have demonstrated the production of high-quality few-layer graphene using a simple hydrodynamic-assisted exfoliation method. The results indicated that ~ 71% of the prepared graphene flakes were less than five layers, while the average thickness and length of the flakes were 2.3 nm (~ 90% < 4 nm) and 1.9 μm (~ 50% in the range of 1–7 μm), respectively. The flakes with


The geroscience hypothesis holds that targeting fundamental mechanisms of aging has the potential to prevent or reduce severity of multiple age-related diseases. Cellular senescence is a key mechanism that may be driving disease in human aging, including Idiopathic pulmonary fibrosis (IPF), a progressive, ultimately fatal, senescence-associated disease. Importantly, cellular senescence may be targeted therapeutically. Senolytic agents are drugs that selectively induce senescent cell apoptosis by transiently disabling anti-apoptotic pathways. Selective ablation of senescent cells using the senolytic drug combination dasatinib plus quercetin (DQ) alleviates IPF-related dysfunction in bleomycin-administered IPF mouse model. We conducted the first-in-human trial of senolytics in IPF patients, and our data indicate that short-term, intermittent administration of DQ may alleviate physical dysfunction that accompanies IPF in human aging, including clinically-meaningfully improvements in mobility (p<0.05). This geroscience-guided clinical feasibility study supports evaluation of senolytics in larger randomized, controlled trials of cellular senescence-associated age-related diseases.


In contrast, the accumulation of quinochalcones (hydroxysafflor yellow A and carthamin), kaempferol glycosylated derivatives (kaempferol-3-O-β-rutinoside and kaempferol-3-O-β-d-glucoside), apigenin, and luteolin in CtACO1 overexpression lines were decreased. Conclusion: This study confirmed the feasibility of applying the floral dipping method to safflower and showed a novel regulatory effect of CtACO1 in the flavonoid biosynthetic pathway. It provides hypothetical and practical groundwork for further research on regulating the overall metabolic flux of flavonoids in safflower, particularly hydroxysafflor yellow A and other quinochalcones, by using appropriate genetic engineering strategies.Background: Flavonoids with various structures play a vital role in plant acclimatization to varying environments as well as in plant growth, development, and reproduction. Exogenous applications of ethylene and 1-aminocyclopropane carboxylic acid (ACC), could affect the accumulation of flavonoids. Very few attempts have been made to investigate the effect of 1-aminocyclopropane carboxylic acid oxidase (ACO), a unique enzyme that catalyzes ACC to ethylene, on genes and metabolites in the flavonoid biosynthetic pathway. In this study, two ACOs in safflower (CtACOs) were cloned, and then transgenic safflower with overexpressed CtACO1 was generated through the Agrobacterium-mediated floral dipping method. Results: CtACO1 and CtACO2 were both characterized by the 2-oxoglutarate binding domain RxS and the ferrous iron binding site HxDxnH as ACOs from other plants. However, the transcript levels of CtACO1 in flowers at stages I, II, III, and IV were all higher than those of CtACO2. At the cellular level, by using electroporation transformation, CtACO1 was found to be localized at the cytomembrane in onion epidermal cells. CtACO1 overexpression had varying effects on genes involved in the ethylene and flavonoid biosynthetic pathways. The metabolites analysis showed that CtACO1 overexpression lines had a higher accumulation of quercetin and its glycosylated derivatives (quercetin 3-β- Solanum lycopersicum [Malus domestica [Glycine max [Gossypium hirsutum [ACO1 and ACO2) gene expression has also been reported to prolong the shelf life of Eksotika papaya fruit [Flavonoids originating from phenylalanine and acetate metabolism are extensively scattered across most plants in nature . Becausepersicum , Malus domestica , Glycinecine max , and Goshirsutum . A previhirsutum . RNA intya fruit . These sCsF3′5′H resulted in the production of new delphinidin derivatives in the corollas of transgenic tobacco plants, and increased the content of cyanidin derivatives, and produced deeper and redder flowers, in transgenic plants [Carthamus tinctorius L.) is a versatile oilseed crop with several desirable features. Apart from being cultivated as an oilseed crop, it has also emerged as a medicinal plant that has been used to treat a broad range of ailments in traditional Chinese medicine, such as coronary heart disease, cerebrovascular diseases, gynecologic diseases, and hypertension [O-β-rutinoside, and kaempferol-3-O-β-d-glucoside, which are known to be the predominant active contents though which safflower displays its potency. Nevertheless, no previous studies have explored the modification of flavonoids in safflower through transgenic approaches.In addition, advances in molecular biotechnology have produced another way to modify content accumulation in plants. Specifically, the transgenic approach has provided novel possibilities for overproducing or silencing enzymes involved in the metabolic pathway, leading to the preferential accumulation or absence of some compounds in a target organ or tissue . For exac plants . Overexpc plants . Safflowrtension ,17. Thusrtension . Howeverrtension . As a flThe present work first describes the isolation and characterization of CtACOs from safflower. The open reading frame of CtACO1 is then subcloned into an overexpression vector for transforming safflower through the Agrobacterium-mediated floral dipping method and for determining the subcellular localization of CtACO1. Simultaneously, the transcriptome expression patterns of selected genes involved in the ethylene and flavonoid metabolic pathways in transgenic safflower are analyzed. Moreover, the accumulation of flavonoids in different organs is quantified. Overexpression of CtACO1 in transgenic safflower is found to lead to widespread changes in flavonoids, supporting the role of CtACO in regulating flavonoid metabolism and suggesting a metabolic engineering strategy for improving the accumulation of flavonoids in safflower.Flower transcriptome sequencing was carried out to study the functional genes in safflower. The expressed sequence tags (ESTs) were annotated by BLASTx and BLASTn in the Nr and Nt databases (unpublished). Two genes were annotated as ACO enzymes in safflower flower. Their missing 5′ fragment region and 3′ region were cloned through RACE, and the entire assembled sequences were labeled as CtACO1 and CtACO2, respectively. The cDNA of CtACO1 and CtACO2 both contained a 1098 bp open reading frame encoding a 365 amino acid with a predicted molecular weight of 40.8 kDa and a pI of 5.35. CtACO1 and CtACO2 were subjected to phylogenetic analysis with the ACOs of other plants . The seqTo detect the expression patterns of CtACO1 and CtACO2 in flowers at different flowering times, flowers at stages I, II, III, and IV were collected . As showBy using the WoLF PSORT prediction program for the subcellular localization of the CtACO1 protein, we predicted that the CtACO1 protein may be localized in the cytoplasm or nucleus. To examine this prediction, the recombinant of CtACO1 and pCAMBIA-1380-CaMV35S-MCS-EGFP-NOS (PMT-39) was introduced into onion epidermal cells through Agrobacterium strain LBA4404 mediation. The empty PMT-39 vector was also introduced into onion epidermal cells as a control. As shown in To explore the in vivo function of CtACO1, transgenic safflower that overexpressed CtACO1 under the CaMV 35S promoter was generated. Based on screening by genomic DNA PCR , 14 of 1As shown in p < 0.05 were selected as important differential metabolites. Finally, 114 metabolites in the flower (F) part, 192 metabolites in the bracteole (BP) part, 245 metabolites in the leaf (L) part, and 113 metabolites in the stem (J) part, with significant differences between the transgenic and the control group, were identified.UHPLC-Q-TOF/MS was undertaken to analyze the four organs of safflower in eight groups; Twelve target metabolites were identified by comparison with standards. The quantities of these metabolites were determined by applying LC-MS. As shown in The changes of transcript abundance and compounds in the CtACO-overexpressing flower. The increased (with red up arrow) and decreased compounds (with black down arrow) are shown in the diagram. The upregulated genes are marked with a red up arrow and the downregulated genes are marked with a black down arrow.Flavonoids derived from phenylalanine and acetate metabolism are ubiquitously scattered across the plant kingdom and constitute various classes of metabolites. In the process of plant growth and development, flavonoids act as UV filters, signal molecules to the environment, visual and olfactory attractors to pollinators and herbivores, and protectors against light intensity, temperature, and biotic and abiotic stresses ,22,23. TIn this study, two CtACOs from safflower were first cloned and identified. Similar to ACOs from other plants, CtACO1 and CtACO2 were characterized by the conserved 2-oxoglutarate and Fe (II)-dependent oxygenase superfamily domain. Bioinformation analysis suggested a physiologic function of CtACO1 and CtACO2 in the ethylene biosynthesis pathway. The transcript levels of CtACO1 in flower stages I, II, III, and IV were all higher than those of CtACO2. Regarding the amino acid level, CtACO1 was highly similar to CtACO2, with 99% identity. However, four amino acids were different; valine in position 21 of CtACO1 mutated into phenylalanine of CtACO2, serine in 208 into asparagine, arginine in 220 into cysteine, and asparagine in 362 into cysteine asparagine, which might lead to their dissimilar expression patterns at different flowering times.At the cellular level, a previous study on tomato suspension cells showed that the location of ACO was a cytomembrane localized protein with cell fractionation technique . HoweverO-β-rutinoside and kaempferol-3-O-β-d-glucoside) displaying lower accumulation in the CtACO1-overexpressing flower. In contrast, the detected quercetin and its glycosylated derivatives (quercetin 3-β-d-glucoside and rutin) were increased in the flower. A previous report has shown that the different transcript expression ratios of the F3’H/CHS gene could led to disparate quercetin/kaempferol metabolite accumulation in tissue [d-glucoside and rutin). This report also showed that the ethylene-induced increases in flavonol accumulation, as measured by DPBA fluorescence, were most apparent in the elongation zone and thus well- positioned to contribute to root elongation and gravitropism. Additionally, the accumulation of apigenin and luteolin in different tissues affected by CtACO1 overexpression, especially their antipodal accumulation pattern in the leaf, was evidently dissimilar, which further enriched the potential effect of ethylene on apigenin derivatives.The use of transgenic technology to alter plant characteristics has been shown to be a feasible way to quickly establish plants to improve the production of bioenergy crops . Regardin tissue . In our Quinochalcones are considered to be the characteristic and major active constituents of safflower. Our previous study showed that the small heat shock protein from safflower might be involved in the formation of hydroxysafflor yellow A . Thus faIn sum, the floral dipping method was applied to generate transgenic safflower with overexpressed CtACO1. Meanwhile, the effect of CtACO1 overexpression on the transcript and metabolite levels in safflower was confirmed, which indicated varying effects between genes and metabolites. Taken together, the results of this study validated a novel regulatory effect of CtACO1 in the flavonoid biosynthetic pathway, thus providing hypothetical and practical groundwork for further work on regulating the overall metabolic flux of flavonoids in safflower, particularly hydroxysafflor yellow A and other quinochalcones, by applying appropriate genetic engineering strategies.Discovery of CtACO from the transcription profiling database. To investigate the functional genes in safflower, flower transcriptome sequencing was done. The expressed sequence tags (ESTs) were annotated by BLASTx and BLASTn in the Nr and Nt databases (unpublished). Two genes were annotated as ACOs in safflower flower.Carthamus tinctorius L. by Professor Meili Guo, was planted in the greenhouse at the School of Pharmacy in the Second Military Medical University . The voucher specimen was SMMU141226 and was deposited at the Medicinal Plant Herbarium of the Department of Pharmacognosy, School of Pharmacy, Second Military Medical University.A safflower line (No. 0025), identified as The safflower line No. 0025 was grown in the greenhouse of the Second Military Medical University . The plants were maintained at a mean temperature of 25 °C over a photoperiod of 16 h.To decrease the gene copy variation at the transcription level, normalized cDNA libraries of safflower flowers at different developmental stages were constructed. Then, safflower transcriptome sequencing was undertaken to detect more genes related to the biosynthetic pathway of the active components. The genes were annotated by BLASTx and BLASTn search homologs in the Nr and Nt databases. Two ACOs were selected from the annotated genes.Carthamus tinctorius L., 5′- and 3′-RACE experiments were carried out by using the SMART-RACE cDNA amplification kit . Gene-specific primers denoted as GSP-1/-2 . The CtACO1 and CtACO2 protein sequences were aligned with the protein sequences of ACO genes from other plants through the National Center for Biotechnology Information (NCBI: http://www.ncbi.nlm.nih.gov/). To identify the conserved motifs of the CtACO1 and CtACO2 proteins, their deduced amino acid sequences were aligned with ACOs from other plants using DNAMAN 8.0 software . Phylogenetic trees were constructed with the MEGA 5.10 program using the neighbor-joining (NJ) method and 1000 bootstrap replicates [The respective sequences of the CtACO1 and CtACO2 mRNA have been submitted to GenBank under accession numbers KU291182 and KU291190. The theoretical isoelectric points and mass values for the proteins were predicted using the ExPASyProtParam tool (plicates .http://www.genscript.com/psort/wolf_psort.html). To further identify the outcomes of the prediction, the recombinant of CtACO1 and pCAMBIA-1380-CaMV35S-MCS-EGFP-NOS  vector was constructed with specific primers  agarose gel and by detecting the A260/280 ratio. RNA quantification was done on a Biomate 3S spectrophotometer . Subsequently, first-strand cDNA was synthesized using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit . cDNAs were diluted five times and then mixed with TransStart Green qPCR SuperMix  and 0.4 uM of each primer for amplification. −∆∆Ct method [Regarding the analysis of CtACO1 and CtACO2 expression in the flower at different flowering times, flowers at stage I , stage II , stage III , and stage IV  were collected. For the qPCR analysis of transgenic safflower that overexpressed CtACO1, 3-day-old flowers of transgenic and control plants, as well as tissues of the leaf, stem, and bracteal leaf, were harvested in the same period. Total RNA was extracted from plants using TransZol reagents  according to the manufacturer’s instructions. The RNA integrity was examined by running on 1.0% . Kaempferol-3-O-β-rutinoside, kaempferol-3-O-β-d-glucoside, and carthamin were synthesized in our laboratory. All other chemicals were of analytical grade, with purities higher than 98%.Chemicals and reagents: Chromatography grade methanol and acetonitrile were purchased from Merck . High liquid chromatography grade formic acid was purchased from Fluka . Ultrapure water was prepared using a Milli-Q water purification system . The following standard compounds were obtained from Aladdin: rutin, kaempferol, quercetin, apigenin, quercetin 3-β-Before the analysis, the whole dried sample was ground to powder by using a disintegrator. Next, 1000 mL of 60% methanol was added to the powder (5 mg), which was then soaked overnight. After treatment by ultrasound for 40 min and passing through micropore filters, the clear supernatant (150 μL) was transferred into a sampling vial for UHPLC–MS analysis.A metabolomics approach that uses ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) was developed to perform four different parts of safflower—F, BP, J, and L—metabolic profiling analysis and search for differential metabolites in the transgenic group and control group. On this basis, the present research explored the metabolic effect and regulation of ACO overexpression on the flavonoid metabolism of safflower.m/z. The reference ions were 121.0509 and 922.0098 in positive mode, and 119.03632 and 966.000725 in negative mode.UHPLC-Q-TOF/MS analysis was carried out on an Agilent 6538 Accurate Mass Quadrupole Time-of-Flight mass spectrometer . Chromatographic separations were done at 25 °C on an XBridge TM BEH C18 column . The mobile phase consisted of 0.1% formic acid (A) and ACN modified with 0.1% formic acid (B); the post time for equilibrating the system was 6 min, and the flow rate was set to 0.4 mL/min. The optimized UPLC elution conditions were: 0–2 min, 5% B; 2–4 min, 5–20% B; 4–6 min, 20–21% B; 6–9 min, 21–26% B; 9–10 min, 26–40% B; 10–15 min, 40–80% B; 15–17 min, 80–95%; and 17–19 min, 95% B. An electrospray ionization source (ESI) was operated in both positive and negative modes. The optimized conditions were as follows: capillary voltage, 4 kV in positive mode and 3.5 kV in negative mode; drying gas flow, 11 L/min; gas temperature, 350 °C; nebulizer pressure, 45 psig; fragmentor voltage, 120 V; and skimmer voltage, 60 V. The mass spectrum was collected in centroid mode, ranging from 50 to 1100 m/z data, and normalized ion intensities, was imported into the SIMCA-P program  for multivariate statistical analysis to screen the differential metabolites.All data were acquired using the Agilent MassHunter Workstation B.04.00 analysis software. The MS data were determined, extracted, and integrated with the Agilent Profinder B.06.00 software. All data were weight-normalized using Excel. The three-dimensional data matrix, including the retention times, After analyzing and fitting the samples from four different parts of the transgenic and control groups using metabolomics, liquid chromatography tandem mass spectrometry was used to detect the accurate concentrations of target metabolites in the four parts of these groups.The column was kept at a temperature of 25 °C, and the flow rate was 0.4 mL/min, with an injection volume of 4 μL. The UHPLC binary solvent system consisted of mobile phases A (0.1% formic acid) and B (ACN modified with 0.1% formic acid). The gradient elution UHPLC conditions were: 0–2 min, 5% B; 2–2.5 min, 5–15% B; 2.5–7.5 min, 15% B; 7.5–8 min, 15–20% B; 8–10 min, 20–21% B; 10–18 min, 21–95% B; and 18–20 min, 95% B; the system was equilibrated with a post time of 5 min. The 12 components were measured well in the negative mode. Thus, the negative mode was used to analyze the differences. The mass spectrometry method was the same as previously described.O-β-rutinoside, 1.67 mg/mL; kaempferol-3-O-β-glucoside, 1.04 mg/mL; apigenin, 1.06 mg/mL; HSYA, 1.3 mg/mL; isoquercitrin, 1.23 mg/mL; luteolin, 1.05 mg/mL; d-phenylalanine, 3.72 mg/mL; and carthamin, 0.266mg/mL. The same volume of standard solutions was mixed and titrates ratio diluted. l-para-chlorophenylalanine was added as an internal standard, with a final concentration of 10 μg/mL.The stock solutions of the standards were resolved with 60% methanol. The concentrations of the standards were as follows: naringenin, 1.36 mg/mL; rutin, 1.32 mg/mL; quercetin, 1.04 mg/mL; kaempferol, 1.11 mg/mL; kaempferol-3-The standard curve of 12 objective compounds was drawn. The accuracy and limit of quantification were evaluated.t test was used to determine the statistical significance.The internal standard was added to the samples at the same concentration. The above-mentioned chromatography and mass spectrometry methods were used to detect metabolites in the samples from four parts of the transgenic and control groups. Agilent Profinder B.06.00 software  was applied to complete the peak extraction and integration. Students


The understanding of the transfer of charge carriers during the photocatalytic processes remains unclear because of their complexity. In this study, the characteristics of the deposited WO3/graphene layered materials were investigated by Raman spectroscopy, UV–vis spectroscopy, and SEM. According to the results, p-graphene exhibits and enhances the characteristics of the WO3/graphene film. The photocatalytic activities of WO3/graphene layered materials were assessed by the photocatalytic degradation of oxytetracycline antibiotics as irradiated by UV light. Here, a higher current of cyclic voltammetry and a higher resistance of impedance spectra were obtained with the as-grown WO3/graphene directly synthesized on Cu foils under UV light using an electrochemical method, which was different from traditional WO3 catalysts. Thus, it is urgent to explore the underlying mechanism in depth. In this study, a large layered material WO3/graphene was fabricated on a Si substrate using a modified CVD method, and a WO3/graphene device was developed by depositing a gold electrode material and compared with a WO3 device. Due to photo-induced doping effects, the current-voltage test suggested that the photo-resistance is larger than dark-resistance, and the photo-current is less than the dark current based on WO3/graphene layered materials, which are significantly different from the characteristics of the WO3 layered material. A new pathway was developed here to analyze the transfer properties of carriers in the photocatalytic process.Graphene enhanced WO For this end, photocatalytic water splitting generates hydrogen and oxygen from water, which plays an increasingly important role as the clean energy . In thisehaviors , 5. Thuscatalyst –12.3/GR, numerous studies have been conducted to reveal the underlying mechanism that graphene improves WO3 characteristics associated with photo-generated charge transfer, and several well-established explanations have been made. For instance, Wu et al. considered that the graphene can serve as an electron–acceptor material and reduce the recombination of photo-excited electron–hole pairs, thereby increasing photoconversion efficiency [3 nanorods can provide another possible electron route between WO3 and coupled rGO nanosheets, thereby exhibiting excellent visible-light catalytic activity for hydrogen production and clarifying the Z-scheme catalytic mechanism [Given the superior photocatalytic performance of the WOficiency . Furtherechanism –17.2 surface [Besides, a few experiments were performed to explain the mechanisms of oxide semiconductor materials and graphene hybrid nanocomposite , 19. Pan surface . Recentl surface , 22. Mor surface .3/GR were deposited, of which the characteristics were investigated under the Raman spectroscopy, UV–vis spectroscopy and SEM. All the results show that p-graphene emerges and improves the characteristics of the WO3/GR film. The photocatalytic activities of the layered materials were assessed by the photocatalytic degradation of oxytetracycline antibiotics under UV light irradiation. The characteristics of cyclic voltammetry and electrochemical impedance spectra of the as-grown WO3/GR directly fabricated on Cu foils under UV light using electrochemical behavior were obtained here and compared with traditional WO3 catalysts. To explore the charge transfer mechanisms associated with photo-induced doping, the stacks of large area layered materials WO3/GR were designed on the Si substrate using a modified CVD approach, and WO3/GR and WO3 devices were developed by depositing an electrode material of gold foil for comparison. The characteristics of WO3/GR were analyzed and compared with those of WO3 due to photo-induced doping effects using the current–voltage test. Charge transport behaviors of p-graphene can be modified to improve photocatalytic ability. Furthermore, graphene was used as the photogenerated electron acceptor and effectively suppressed the charge recombination in the WO3/GR layered materials.In this study, the layered materials WO3/GR thin flake transistor: first, large-area graphene films of the order of centimeters were formed on copper substrates by chemical vapor deposition using methane. Graphene films were removed from the Cu foils to SiO2/Si substrate by etching in an aqueous solution of iron nitrate. The WO3 thin film was formed from 50 nm WO3 powder on a clean Si wafer with a 275-nm SiO2, graphene top layer [3 device without graphene was prepared under the same conditions.Characterization of WOop layer . During λ = 532 nm). All spectra were recorded at low power levels to avoid laser-induced modification or ablation of the samples.The band-gaps of the fabricated films were obtained by measuring absorbance using an UV–vis instrument . The morphology and microstructure of the nanostructured films were assessed with a JEOL JSM-7600F field emission scanning electron microscopy (FE-SEM). Raman measurements were performed in a Witec system in a backscattering configuration. The excitation was achieved by visible laser light  solution in a typical activity test. The suspension was left in the dark for 1 h to reach the adsorption equilibrium, and the photocatalytic reaction was initiated under UV light for 160 min. The light source was a 250-W mercury lamp. By measuring the changes in the UV–vis absorption spectrum as a function of irradiation time, this study monitored antibiotic degradation.3/GR/Cu foil and WO3/Cu foil served as the working electrode, Pt foil as a counter electrode, and a saturated Ag/AgCl as a reference electrode. All the potentials were calibrated by a reversible hydrogen electrode (RHE). Linear sweep voltammetry with a scan rate of ~ 0.1 V s−1, from + 0.20 to − 0.20 V vs. RHE was performed in 0.5 M H2SO4. The Nyquist plots were obtained at the frequencies ranged from 100 kHz to 0.1 Hz at the overpotential of 40 mV. To extract the series and charge-transfer resistance, the impedance data fitted to a simplified Randles circuit.All the electrochemical measurements were performed in a three-electrode system for CHI 604E electrochemical workstation (CH Instruments), in which WOAll the electronic and optoelectronic characterization were performed in a probe station in a vacuum and at ambient temperature. The photocurrent was recorded by the Agilent 1500 A semiconductor analyzer. The light excitation was achieved by the 253 nm lamp used for the UV excitation.3/GR and WO3 films by CVD is shown in Fig. 3/GR thin films. It is found that the WO3/GR thin film materials are uniform and smooth here. Moreover, from inspection, small crack gaps about 100 nm in size were found on the surface of WO3/GR. Figure 3/GR surface. Obviously, both W and O are uniformly distributed over the surface with a higher percentage. Since graphene is grown below WO3, element C can be found at the position of crack gaps with a low percentage [The deposition process of WOrcentage .Fig. 1Sc3/GR, as well as pure WO3. In general, monolayer graphene has two peaks at nearly 1348 cm−1 and 1586 cm−1, suggesting that the intensity ratio of IG/ID peak is about 2 of a Raman spectrum. Similar peaks at D-band (round 1370 cm−1) and G-band (round 1599 cm−1) were observed in the WO3/GR composite. According to the spectra in Fig. G/ID ratio decreased from 2 for the graphene to 1.2 for the WO3/GR composite. Thus, the smaller the IG/ID peak intensity ratio of a Raman spectrum, the higher the defects and disorders of the graphitized structures will be in the WO3/GR composite due to the high temperature of nearly 400 °C. Due to the stretching mode O–W–O in the sample of WO3/GR composite, Raman vibrations centered at 815 cm− 1, the characteristic of pure WO3 was detected, which was constantly narrowed in the sample of WO3/GR composite. It is noteworthy that the G-band of WO3/GR had risen from 1584 to 1599 cm−1 compared with graphene. This G-band up-shift was the general evidence of chemical doping of carbon materials. The trend here is consistent with previous studies with the p-type doping of the graphene, leading to upshift of the G-band. According to the Raman G-band shift, charge transfer between the graphene and the WO3 in the WO3/GR composite was demonstrated [−1 for pristine (undoped) graphene and round at 2700 cm−1 for p-doped graphene, respectively [Figure nstrated , 27. Theectively .Fig. 2a 3/GR composite were extracted into intensity mapping, and Fig. 3/GR composites obtained from the G-band of the graphene. The “bright” regions with high intensity illustrate the presence of the graphene, and it can be confirmed that p-doped graphene and defects exist in the layered materials due to the local high bright regions. Also, the “dark” regions are related to the WO3 information, which present the large area distribution of the graphene in the layered materials [The Raman data of WOaterials .3, UV–vis absorption spectra were recorded as shown in Fig. A × (hν-Eg)n/2 was used, where α, ν, Eg, and A are the absorption coefficient, the light frequency, the band gap, and a constant, respectively [1/2-hν curves of as-prepared samples are shown in Fig. 3/GR in the visible light region was more sensitive than that of pure WO3. The mixture of graphene onto the WO3 improved the absorption capacity to the light. Compared with pure WO3, the band gap of WO3/GR was narrowed from 3.88 to 3.68 eV  as shown in Fig. 3, WO3/GR led to a high degradation of oxytetracycline. The kinetics of oxytetracycline degradation under UV light can be obtained by pseudo-first-order reaction, where C0 and C are initial and concentration at given degradation time t and k is the rate constant, respectively. The diagram of ln(C/C0) was plotted as a function of t ) compared with Ret (75(Z′/Ω)) in the dark. Note that under the UV light, WO3/GR/Cu electrode shows a relatively obvious semicircle arc (Ret = 42(Z′/Ω)), indicating a higher electron transfer resistance behavior than that of Ret (38(Z′/Ω)) in the dark. The increase in the value of electron transfer resistance (Ret) due to photo-induced doping effects improved Fermi energy level of graphene on electrode surface under UV light. These results also demonstrated that the graphene can improve the electron transfer rate between the electrode and WO3, which is consistent with the CV results.The interfacial characteristics of the modified electrode, which were of huge significance to the electrical conductivity, and the electrocatalytic properties of the modified electrode were analyzed here by EIS. The electron-transfer kinetics and diffusion characteristics can be concluded from the shape of the electrochemical impedance spectrum. The semicircular portion, Ret, obtained at higher frequencies represents an electron transfer-limited process, and the linear portion at lower frequencies was attributed to the limited mass transfer of the as-prepared sample ion , 33. Fig3/GR layered materials can be surveyed under UV light, as shown in Fig. 3/GR composite and the reference device with pure WO3 were measured in the dark and under UV light at 253 nm with the intensity of 0.3 mW/cm2 as shown in Fig. 3/GR composite device was nearly 106 times higher than that of the reference device from pure WO3. Note that the photocurrent was less than the dark current of the WO3/GR composite, which is significantly different from the reference device from pure WO3. The typical I–V characteristics of the device were similar to I–T characteristics , the current of the device was decided by RWG + RG. Therefore, the conductivity properties had been significantly improved in the presence of graphene.Figure ance RWG . The dev3/GR hybrid composites and diagram of the photo-induced doping mechanism are shown in Fig. 3/GR heterostructure device without light illumination is consistent with the previous result of a stable p-type doped graphene transistor, in which electrons were transferred from the graphene thin film to WO3. Initially, graphene was hole-doped in the dark, and an electric field appeared from graphene to silicon. As shown in Fig. 3 were excited to the conduction band to create electron–hole pairs [3 were excited by photons to the conduction band. The ionized defects were positively charged and localized in the WO3. These excited electrons in both cases can be mobile, move towards, and then enter the graphene. It was suggested that significant photo-induced electron transfer occurred from WO3 to graphene at the WO3/GR device [Schematics of the band structure of the WOle pairs –39. On tR device .3/GR heterojunction emerged. Subsequently, the experimental data shows a decrease in conductivity with the increase in Fermi energy, EF of graphene, thereby leading to a slow decrease in the UV photocurrent. This is well consistent with the theoretical model [3 when the WO3/GR device is exposed to light. Photo-induced doping effects were also reported by some authors. Tiberj et al. reported that the charge carrier density of graphene can be finely and reversibly tuned between the hole and electron doping due to photo-induced doping, which was significantly affected by the substrate cleaning method [The excited electrons entered the graphene, and the positively charged defects led to modulation doping in the graphene. Under this modulation doping in the graphene, WOal model . It is tg method . Ju et ag method .3/GR, as the primary photosensitive particles, has more photogenerated holes than the pure WO3 surface under UV light. The more active sites of the WO3/GR surface pores, the more efficient the improvement of photosensitivity [3 nanorod nanocomposites, which improved the visible-light photocatalytic performance compared with bare WO3 nanorods [3 composites mixed with different amounts of graphene . Moreover, the sensor based on 0.1 wt% GR–WO3 composite exhibits good selectivity and high response in comparison with those of pure WO3 [3, decreasing the amount of the active sites. Subsequently, the proper proportion of WO3 and graphene can gain the best experimental effect. Akhavan et al. also analyzed the characteristics of TiO2/GO (graphene oxides) sheets at different irradiation times [Under the light-induced doping effect, the surface of WOsitivity . In genenanorods , 46. Thenanorods , 48. Chupure WO3 , 50. It on times . They foon times . Accordi3/GR directly synthesized on Cu foils under UV light through electrochemical behavior were obtained, which was also different from traditional WO3 catalysts. The characteristics of WO3/graphene layered materials were investigated under the Raman spectroscopy, UV–vis spectroscopy, and SEM. All results show that p-graphene emerges and enhances the characteristics of the WO3/GR film. The stacks of large-area WO3/GR layered materials were designed on the Si substrate using a modified CVD approach, and WO3/GR and WO3 films were fabricated on an electrode material of gold foil for comparison. Due to photo-induced doping effects, the current–voltage test suggested that the photo-resistance was larger than dark-resistance, and photocurrent was less than dark current based on WO3/GR layered materials, which were different from the characteristics of WO3 layered materials. Besides, charge transport behaviors of p-graphene could be modified to improve photocatalytic ability. Graphene serves as the photogenerated electrons acceptor and effectively suppresses the charge recombination in the WO3/GR layered materials. This study is considered a significant advance towards unraveling photocatalytic dynamics processes based on graphene and oxide semiconductor. Hopefully, these results can motivate scientists to explore high efficient catalysts for related applications.In this study, the photocatalytic activities of the layered materials were assessed by the photocatalytic degradation of oxytetracycline antibiotics under UV light. A higher current of cyclic voltammetry and large resistance of impedance spectra with the as-grown WO


Significant progress has been made on the development of electrolyte-gated graphene field effect transistor (EGGFET) biosensors over the last decade, yet they are still in the stage of proof-of-concept. In this work, we studied the electrolyte matrix effects, including its composition, pH and ionic strength, and demonstrate that variations in electrolyte matrices have a significant impact on the Fermi level of the graphene channel and the sensitivity of the EGGFET biosensors. This is attributed to the polarization-induced interaction between the electrolyte and the graphene at the interface which can lead to considerable modulation of the Fermi level of the graphene channel. As a result, the response of the EGGFET biosensors is susceptible to the matrix effect which might lead to high uncertainty or even false results. Then, an EGGFET immunoassay is presented which aims to allow good regulation of the matrix effect. The multichannel design allows in-situ calibration with negative control, as well as statistical validation of the measurement results. Its performance is demonstrated by the detection of human immunoglobulin G (IgG) from serum. The detection range is estimated to be around 2–50 nM with a coefficient of variation (CV) of less than 20% and the recovery rate for IgG detection is around 85–95%. Compared with traditional immunoassay techniques, the EGGFET immunoassay is label-free and ready to be integrated with microfluidics sensor platforms, suggesting its great prospect for point-of-care applications. Field effect transistor (FET) biosensors are operated by measuring the conductance changes of a channel induced by the binding of target molecules to it. Compared with other biosensing techniques, FET biosensors are particularly favored for their potential in portable and point-of-care biomedical applications due to their high sensitivity, simple configuration and cost-effective mass producibility ,2. Over Significant progress has been made in the development of electrolyte-gated graphene field effect transistor (EGGFET) biosensors over the last decade . VariousDespite the reports of outstanding performances, most results have been based on experiments using simplified samples in the laboratory setting ,2. In EGIn this work, we studied the impact of the variance in electrolyte matrices on EGGFET biosensors—known as the matrix effect—by varying the composition, ionic strength and pH of the electrolytes. The underlying mechanisms were discussed with regarding to the strong polarization-induced interaction between the electrolytes and the graphene and its impact on the Fermi level of the graphene channel. The influence of the matrix effect on the gate potential and the sensitivity of the EGGFET biosensors were also studied. For the regulation of the matrix effect, we present an immunoassay based on the EGGFET immunosensors. Its performance was demonstrated by the successful detection of human immunoglobulin G (IgG) from serum in spike-and-recovery experiments.A prototype of the EGGFET immunoassay chip and an enlarged view of the EGGFET immunosensor configuration are shown in 3)3  for 3 h to etch the copper. The PMMA/graphene film was lifted by a glass slide  and deionized (DI) water for 5 min, successively) and allowed to dry for 1 h at room temperature. The sample was then heated at 75 °C for 30 min to improve the contact between graphene and the substrate. The PMMA was removed by acetone and rinsed with IPA and DI water, respectively. The sample was annealed at 250 °C in a nitrogen atmosphere with a flow rate of 1000 sccm to remove the PMMA residues. To remove the post-annealing residues, the graphene was cleaned using an electrolytic method at −5 V vs. an Ag/AgCl reference electrode in 0.5 M sulfuric acid for 30 min as we reported previously [Poly(methyl methacrylate)  was spin-coated on CVD graphene  at 2000 rpm for 30 s and then cured at 120 °C for 1 min. The sample was treated by oxygen plasma asher at 30 W for 90 s to remove the backside graphene, and then was placed to be floating on iron nitrate  followed by etching in gold etchant  and nickel etchant  for 10 s, successively. A second photolithography process was applied to create a shielding photoresist layer on the graphene channel, and then the graphene channel was patterned by oxygen plasma etching (100 W for 90 s with oxygen flow at 49 sccm). The shielding photoresist layer was finally removed using acetone followed by rinsing with IPA and DI water.3) and 1 M ammonia  solution were injected into the chamber. First, an oxidative pre-treatment at +0.95 V was applied for 30 s. For better results, the sample was placed in a vacuum for 30 min to remove the dissolved oxygen and the microscopic gas bubbles on the electrode surface. Then the electroplating was driven at −0.5 mA for 300 s, resulting in a Ag layer of around 5 μm in thickness. A quantity of 0.1 M HCl solution was injected into the chamber after rinsing with DI water, and the chloridization was driven at +0.2 mA for 60 s. After removing the electroplating chamber, the sample was rinsed with DI water.A three-electrode cell with a standard Ag/AgCl reference electrode, a gold wire coil counter electrode, was used for the electroplating of Ag/AgCl on the as-fabricated gold electrode. The schematic and experimental setup are shown in The sample delivery channel was made of polydimethylsiloxane (PDMS) using the soft lithography technique. Due to the vulnerability of graphene, conventional oxygen plasma cannot be used for the activation of the glass surface for bonding with PDMS. As an alternative, the EGGFET chip was immersed in 0.1 M NaOH solution for 30 s and then rinsed with DI water with a layer of DI water left on the EGGFET chip afterwards. The PDMS sample delivery channel was activated with oxygen plasma and then applied onto the EGGFET chip. The alignment of the sample delivery channel and the EGGFET chip was accomplished under a microscope. The interfacial water layer serves as the lubricant and prevents the immediate bonding of glass and PDMS, while preserving their bonding capability. The aligned sample was then placed in a 60 °C oven for 3 h for bonding.N-hydroxysuccinimide ester  was injected into the channels and kept for 2 h. PBASE can be adsorbed on graphene through a π–π interaction without damaging its electrical properties, and it is widely used for the functionalization of graphene and carbon nanotubes [N-hydroxysuccinimide (NHS) ester in PBASE and the amine group on the 5′ end of the IgG aptamer. The remaining unconjugated sites were blocked by bovine serum albumin  after rinsing with 1× PBS. X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy were performed to study the functionalization of the graphene surface. The transfer curves of the EGGFET biosensors were measured after each step of functionalization. Details are provided in the A schematic diagram in anotubes . A 5′-amThe electrical measurements were conducted using Keithley 4200 SCS  and a Micromanipulator probe station. The operation parameters, including the gate voltage scan rate, gate voltage setting and drain-source voltage, were optimized to minimize the hysteresis-induced deviation and maximize the transconductance . The datThe operation of the EGGFET biosensors is based on the modulation of the Fermi level in the graphene channel by electrostatic gating upon specific adsorption of the charged biomolecules. Graphene is a zero-bandgap semiconductor with its conduction band and valance band meeting at the Dirac points with linear energy dispersion . At low n of IgG b.In EGGFET biosensors, the graphene channels are directly exposed to the electrolytes, which makes it important to learn if and how electrolytes would affect the operation of the EGGFET biosensors. The electrolyte matrices for bioanalysis are highly variant and complex; to better understand the underlying mechanisms of the matrix effects, we chose several simple electrolytes and studied the matrix effect as a function of their composition, pH and ionic strength. In this study, we focus on the matrix effect on the + shows a stronger electrostatic gating effect than K+ [+ ions are more strongly absorbed onto graphene than Na+ ions [+ and Na+ have one positive charge, the stronger adsorption of K+, which leads to a higher surface charge density on graphene, will introduce stronger n-doping in the graphene channel due to the electrostatic gating effect. The stronger n-doping effect of K+ compared with Na+ is further verified by the measurement of the 3 and Na2SO4 , respectively, suggesting that the We measured the transfer curves of a set of EGGFET biosensors in different electrolytes using a standard Ag/AgCl reference electrode as the gate and the rves see . As show than K+ . Our resNa+ ions . Because3O+ and OH− on graphene, as discussed above, the dipole–π interaction might be a more important contributing factor [3O+ to p-doping OH− causes the continuous positive shift of + and K+ surpass Cl− in capability to dope graphene and reveals the significant impact of the ions in the electrolytes on the transfer characteristics of EGGFET biosensors. The minimum conductivity and the transconductance (the slope of the transfer curve) of EGGFET biosensors are not sensitive to the matrix effect (see More significantly negative shift in trolytes a. We furg factor . Studiesg factor . Due to  reports . Becausefect see .− using the Nernst Equation (2):−. We measured the For the application in the EGGFET biosensors, a standard Ag/AgCl reference electrode is preferred due to its well-defined potential. However, standard Ag/AgCl reference electrodes are difficult to miniaturize and integrate with microfluidics devices. The Ag/AgCl pseudo-reference electrode is an alternative because its fabrication is compatible with microfabrication techniques . In EGGF3. The results indicate that the Debye lengths are highly dependent on the ionic strength of the electrolytes, resulting in a strong impact on the sensitivity of the EGGFET biosensors.In electrolytes, the electrostatic effect of charges is screened by the attraction of the opposite charges and the specific orientation of the dipoles around them. Debye length, which is dependent on the ionic strength of the electrolyte, is normally used to characterize the distance of the electrostatic effects’ persistence. The sensitivity of the FET biosensors is significantly limited by the Debye screening effect . Figure − is low, as discussed in As shown in As discussed above, matrix effects including the composition, ionic strength and pH of the electrolytes can significantly impact the operation and response of a EGGFET biosensor. These matrix effects must be carefully regulated during applications of EGGFET biosensors, otherwise high uncertainty or even false results might be obtained. Inspired by the well-developed enzyme-linked immunosorbent assay (ELISA), a novel EGGFET biosensor immunoassay a was devWe first measured the response of one EGGFET immunosensor to IgG. It is worth noting that 0.01× PBS was used as the detection buffer because it provides relatively high sensitivity and low uncertainty, as discussed earlier, and is shown in The EGGFET biosensor was also used for real-time measurement by monitoring the drain current  and immunoglobulin M (IgM) and IgG were added successively into the channel and the drain current was monitored. No significant drain current change was observed upon the additions of IgA and IgM, which indicates that there is a good selectivity of the EGGFET immunosensor to IgG a,b.To allow the regulation of the matrix effect and obtain reliable test results, we developed a standard operation protocol for the EGGFET immunoassay, as shown in The operation protocol of the EGGFET immunoassay is described as follows: (1) The seven immunosensor sets were filled with detection buffer after rinsing with washing buffer three times; (2) the transfer curves of the EGGFET immunosensors were measured and the initial A spike-and-recovery test was used for examining the performance of the EGGFET immunoassay. The standards were prepared by dissolving human IgG  in 1× PBS to specific concentrations; goat serum (Sigma Aldrich) was used as the blank sample for the negative control. The samples to be measured were prepared by spiking goat serum with human IgG with specific concentrations. Following the protocol mentioned above, the initial and modulated  20% see .The reliability of the EGGFET immunoassay was further verified by a linearity-of-dilution assessment. As shown in In summary, the EGGFET immunoassay exhibits high reliability due to (1) the high uniformity in the electrical properties  theIn this paper, matrix effects of electrolytes were investigated for electrolyte-gated graphene biosensors; the results show that the composition, pH, and ionic strength of the electrolyte used have considerable impact on the characteristics and performance of EGGFET due to polarization-induced interaction at the interface between electrolyte and graphene. The study on the effect of the matrix on the EGGFET biosensors provides a more in-depth understanding of the characteristics, optimization, and application of the EGGFET biosensors. An EGGFET immunoassay was shown to allow good regulation of the matrix effects and statistical validation of the measured results in human IgG detection, suggesting its great potential for point-of-care applications.


Understanding and manipulation of surface impedance in graphene hybrid structure is a significant issue for applications of graphene-based optoelectronics devices. In order to achieve this purpose in the terahertz region, analytical expressions for the impedances of metasurface were derived, which allows us to easily understand the relationship between physical dimensions and impedance. Simulation results show an excellent agreement with the analytical predictions. In addition, we focus on the synthetic impedance when square patch and graphene sheet joined together, discuss the influence of the size of metasurface as well as chemical potentiality as for graphene on the synthetic impedance. Based on these results, a number of absorbers as well as optical devices can be designed that utilize impedance metasurfaces. In recent years, new artificial impedance metasurfaces, exhibiting anomalous electromagnetic properties, were proposed and investigated in the previous literatures –6. MeanwIn the meantime, several analytical models for calculating the equivalent impedance of metasurfaces or graphene sheet have been employed to explain the physical mechanism , 15–20. μc, and the impedance of the composite structure. Here, the surface impedance of metasurfaces/grapheme hybrid structure is discussed by calculating its real and imaginary components. To the best of our knowledge, there is almost no literature reported this mechanism comprehensively.In this paper, a 3D artificial absorber was utilized to analyze and predict the impedance of metasurfaces/graphene hybrid structures, which takes into account the relationship between metasurfaces and graphene. For fast calculation of the surface impedance of metasurfaces, the analytical formulas were firstly developed. These simple and precise analytical formulas can allow a complete elucidation and basic requirement about impedance design. Then, the impedances of the graphene sheets are calculated. Finally, we focus on the relationship between the size of the metasurface, chemical potential h. For a small size square patch in comparison with the wavelength (period of array D ≪ λ) and patches are separated by a narrow slots (width of slot D − w ≪ D), the present model is valid. According to the transmission line theory, an equivalent circuit model of the absorptive structure can be constructed , at normal incidence the equivalent impedance of the patch is represented by Zm, and can be expressed as follows [In the case of a lossy pure resistive sheet impedance  follows , 18:4\doD/w is the geometric element. The effective relative permittivity can be approximated asWhere α for an electrically dense array of ideally conducting patches can be written asFurthermore, the grid parameter μ0, ε0, and k0 are the permeability, permittivity, and the wave number, respectively. Furthermore, it is worth to point out that relation . In order to extract the patch impedance Zm, according to the relationship between the simulated input impedance Zin and the surface impedance of the grounded dielectric slab Zgd, the impedance of the metasurface patch can be expressed as follows:According to the equation , we can Zgd = jZd tan(kdh), Where Analytical results are verified by comparison with the simulated ones based on the extracted reflection coefficient, as shown in Fig. Zm and validate the effectiveness of the formula 10). FiguZmg is equal to the parallel combination of the square patch impedance Zm and the graphene sheet impedance Zg, i.e., Zmg = Zm ∥ Zg. However, through our simulation and calculation, it is found that this relationship is not valid. In order to verify the authenticity, we simulated a metasurface-graphene absorber unit shown in Fig. Zmg at different chemical potentials with w = 19 μm.In the case of a square patch on a graphene sheet, the surface impedance for this hybrid structure should be determined. In the prior literatures , 30–37, μc from 0 to 0.8 ev. However, the analytical and simulated results show the same trend that the impedance will tend to be stable with increasing frequency. The reason is that the impedances of the graphene sheet and square patch become smaller when the frequency increases. It is noteworthy that, compared the impedance of metasurface-graphene film at 0 ev with the other results, the impedance Zmg is quite different. This is due to the values of the graphene sheet impedance at 0 ev is quite different from the higher chemical potential (seen in Fig. From Fig. Zmg is not strictly equal to the parallel combination of Zm and Zg. However, second, there exists a certain relationship between them. In order to demonstrate these conclusions, we first simulate the structure of the absorber shown in Fig. μc = 0.4 ev is displayed in Fig. Zmg can be obtained. Figure Zmg with different patch sizes. According to Fig. w increases from 17 μm to 19.5 μm. However, the opposite trend is found when the frequency is higher than 0.31 THz. On the other hand, Fig. Thus, we can make the following conclusions from the calculated and simulated impedance in Fig. w = 17, 18, and 19 μm with the chemical potential μc = 0.4ev. The color represents the intensity of the field. Obviously, as the size increases, the magnitude of surface current decreases. Taking into account Eq. To further explore the physical origins of surface resistance as a function of patch size, the surface current distributions of the metasurface-graphene film in normal incidence are investigated at 3 THz. Figure J. The qualitative agreement between the simulated and the theoretical results can be clearly observed. To quantificationally analyze this physical phenomenon, the integral value of the surface current distribution on the metasurface-graphene film is calculated by using the HFSS Fields Calculator, and the values are 1.10e-6, 1.07e-6, and 1.04e-6 A, respectively. These results are consistent with Fig. From relation , we can In summary, for metasurface-graphene thin film in THz frequency, the fundamental and effective surface impedances were investigated. Analytical formulas were derived and verified for calculating the impedance of a square patch. As for the metasurface-graphene hybrid structure, the simulated results based on the extracted reflection coefficient were compared with the analytical results obtained from the parallel combination of the square patch and the graphene sheet impedances. Additional analysis was performed on discussing the effect of patch size on effective impedance. Furthermore, the relationships between patch size and film impedance were qualitatively and quantificationally explained by plotting and integrated the surface current. This analysis method can be extended to study the impedance problem with two other different conductive layers. In addition, extensive numerical simulation as well as analytically optimize composite layers for specially applied to antenna and absorber can be avoided by our analysis that made in this work.


Besides, the morphology, structure and graphitization of the resulting carbon nanostructures were investigated. On the other hand, the mechanisms of CNS growth and the synthesis of graphene sheets on the CNS surface were studied. Finally, the mechanical and thermal properties of the CNS/PP, CNS-FLG/PP, and CNS-MLG/PP composites were analyzed by applying tensile test and thermogravimetric analysis (TGA), respectively.This paper focuses on the synthesis and mechanism of carbon nanospheres (CNS) coated with few- and multi-layered graphene . The graphitic carbon encapsulates the core/shell structure of the Ni/NiO nanoparticles via the chemical vapor deposition (CVD) method. The application of the resulting CNS and hybrids of CNS-FLG and CNS-MLG as reinforcement nanofillers in a polypropylene (PP) matrix were studied from the aspects of mechanical and thermal characteristics. In this research, to synthesize carbon nanostructures, nickel nitrate hexahydrate (Ni(NO The discovery of new carbon forms has triggered a great interest in the carbon material science community ,2. AmongAmong the methods employed to synthesize CNS and G, such as the arc plasma technique ,10, hydrGrowing graphene as a two-dimensional structure on other nanomaterials including carbon nanospheres, carbon nanofibers and carbon nanotubes makes hybrid nanomaterials with remarkable properties, such as thermal and mechanical properties, which have the potential to be used in many fields of science and engineering ,24,25,26On the other hand, based on the number of graphene layers, graphene sheets have different types including single-layered (one layer), double-layered (two layers), few-layered (three to five layers) and multi-layered (six to 10 layers), each of which have different properties ,28,29. STo date and to the best of the authors’ knowledge, there is no study reporting the synthesis of carbon nanosphere-few layer graphene (CNS-FLG) and carbon nanosphere-multi layer graphene (CNS-MLG) hybrids using the CVD method and studying its growth mechanisms. In some existing research, authors have mixed CNS and G sheets to achieve CNS-G, but in this research, the CVD technique was employed to synthesize CNS, followed by few-layered and multi-layered graphene on its surface to produce CNS-FLG and CNS-MLG. Besides, the mechanisms to synthesize few- and multi-layered graphene on the grown CNS were studied. Finally, the nanomaterials were used as nanofillers in a polymer matrix to produce a nanocomposite with improved mechanical properties and thermal stability ,31. FurtIn order to characterize the produced nanomaterial by its surface morphology, structure, components, number of graphene layers, size and graphitization, scanning electron microscopy (SEM), energy dispersive X-ray (EDX), transmission electron microscopy (TEM) and Raman spectroscopy were used.The CVD reactor  was used to synthesize carbon nanomaterials including carbon nanosphere, and few layered and multi layered graphene. In order to obtain different morphologies and sizes of the nanomaterials, CVD parameters such as reaction temperature and time should be changed. Therefore in this case, the temperature was set to range from 950 to 1050 °C to synthesize CNS and G, respectively. Besides, the number of graphene layers was changed from few layers to multi layers by changing the time from 30 to 50 min.3)2·6H2O solution for 2 h. Then it was dried at 100 °C under airflow for 1 h. After that, the boat was placed in the middle of the CVD reactor and the system was set. Then, the furnace was turned on to increase the temperature and at this time, N2 gas was released into the CVD chamber to remove O2 inside the reactor. When the temperature reached about 300 °C, the surface of the boat was calcinated and the nitrate compound was removed and the Ni/NiO particles remained. When the temperature reached 950 °C, the CNS fabrication started for 30 min by the use of acetylene (carbon source), with a flow rate of 50 standard cubic centimeters per minute (sccm) under flowing H2/N2  at atmospheric pressure (1 bar). After that, at the end of reaction time, the acetylene flow was stopped and the temperature was increased to 1050 °C under flowing C2H2/H2/N2  to synthesize graphene sheets. To synthesize few layered and multi layered graphene, different time durations (30 to 50 min) were used. In order to analyze the structural characteristics and graphitization of the resultant CNS, CNS-FLG, CNS-MLG, Raman spectroscopy  was used. Raman spectrometer with a green Argon laser excitation source having a wavelength of 532 nm to observe the spectrum of the unstressed specimen.Initially, quartz substrate (the boat of the CVD reactor) was immersed in Ni , energy dispersive X-ray (EDX) and transmission electron microscope (TEM) .−2 pressure by the use of a HSINCHU hot press machine  and then cooled. Finally, based on the guideline in ASTM D638 standard, the specimen and cut into dog bone shape [The last part of the experimental work was to prepare the polymer composite. In this section, Thermo Haake Poly Drive R600/610  as mixer was used to melt and blend the polypropylene (PP) at 180 °C with a 55 rpm rotor speed for 5 min. After that, the nanofillers (5 wt %) were added into the mixer and was blended again for 15 min . Then, tne shape .In order to perform mechanical tests, an Instron Universal Testing Machine  was used with a crosshead speed of 5 mm/min to determine and measure the strength modulus and tensile stress of the PP, CNS/PP, CNS-FLG/PP and CNS-MLG/PP composites . FurtherThe agglomeration of produced CNS coated with the grown few- and multi-layered G formed by CVD was demonstrated by electron microscopy (SEM and TEM) images. The synthesized CNS had a spherical shape, which made it a porous structure a. Beside−1 corresponded to the Eg and A1g modes, respectively [−1 corresponded to the D peak (breathing modes of the sp2 atom due to defective structure), G peak  and 2D peak  of the carbonic nanomaterials, respectively [Raman spectroscopy is a powerful yet relatively simple method to characterize the thickness and crystalline quality of graphene layers . Figure ectively , while tectively .2 cluster size and number of graphene layers of the carbonic structures, the intensity ratio of the D peak and G peak (ID/IG) and the intensity ratio of the 2D peak and G peak (I2D/IG) were calculated [D/IG decreased, the graphitization increased. As well, by increasing the I2D/IG ratio, the number of the graphene layers decreased.In order to estimate the degree of graphitization, the splculated ,38. It iID/IG ratio was about 0.87 and the I2D/IG ratio was 0.52 for CNS, the ID/IG ratio was about 0.63 and the I2D/IG ratio was 0.94 for CNS-FLG, and the ID/IG ratio was about 0.72 and the I2D/IG ratio was 0.81 for CNS-MLG. Therefore, it can be concluded that the few growing layers of graphene sheets on the carbon nanosphere surface led to the increment of the graphitization of the hybrid.The Raman spectra obtained for CNS and CNS coated with few-layered and multi-layered G sheets are revealed in 2H2/H2/N2 flow is revealed in The SEM/EDX image of the carbon nanospheres at 950 °C under C2/N2 flow and without C2H2 flow were composed of C, Ni and O with an atomic ratio of approximately 66:30:4. This result states that the amount of Ni particles increased while the O atoms decreased in comparison with carbon nanospheres at 950 °C. Hence, by increasing the temperature to 1050 °C under H2/N2 flow, more Ni salt was converted to NiO and more NiO was converted into Ni particles. EDX spectroscopy and elemental imaging in On the other hand, based on the SEM/EDX images in 2H2 flow into the CVD reactor, the graphene sheets were grown on the Ni particles placed around and on top of the CNS surface.After that, by inserting the CAccording to the results for characterization, the schematics for the formation of the different carbon nanomaterials including CNS, CNS-FLG and CNS-MLG are depicted in 3)2·6H2O particles. When the temperature is increased to about 300 °C and the H2/N2 gases are inserted into the CVD reactor, the decomposing process of Ni(NO3)2·6H2O particles starts as follows [ follows .Water separation:Decomposition:Oxide decomposition to NiO:2/N2 to Ni:Reduction with H2H2 molecules started to break up to form C atoms  composites including CNS/PP, CNS-FLG/PP and CNS-MLG/PP are depicted in Therefore, regarding Indeed, the multi-layered graphene covered the agglomerate of CNS refer c and alsMoreover, Raman spectroscopy was employed to further analyze the interaction between the different nanofillers and polymer matrix, which should be reflected in the change in peak shift or peak width . Based oBased on the results achieved from the mechanical test, it was found that the nanofillers improved the mechanical properties see . BesidesAccording to the thermogravimetric analysis (TGA) results, it was found that the existence of nanofillers in the polymer matrix led to improved thermal resistance of the polymer nanocomposite due to the high heat absorption capacity of the carbon nanomaterials see . Once th2/N2 gas flow, more Ni/NiO evaporated around the produced CNS, which led to the synthesis of graphene sheets on the CNS surface. Finally, the resultant nanomaterials including CNS, CNS-FLG and CNS-MLG were used as reinforcing nanofillers in the polymer matrix to improve the mechanical and thermal properties of the polypropylene matrix. According to the obtained outputs, it is concluded that CNS-FLG had more a significant role in improving the properties of the polymer composite such as tensile strength, strain and thermal resistance.The most important contribution of this research is the synthesis of the few- and multi-layered graphene on the CNS, as well as the synthesis of the CNS encapsulating the Ni/NiO particles using the chemical vapor deposition technique. The reaction commenced by depositing carbon atoms on the Ni particle surface. Also, CNS was grown at 950 °C for 30 min and FLG and MLG were synthesized on CNS surfaces at 1050 °C for 30 and 50 min, respectively. Moreover, we studied the mechanism for hybrid growth and developed a model for growing a carbon nanosphere–graphene hybrid, which encapsulated the Ni nanoparticles. Regarding the results, firstly, CNS was grown on Ni/NiO nanoparticles at 950 °C. Thereafter, by increasing the temperature under H