After that, the films are perforated with smaller, nanoscale holes. Experimental studies indicate a dependency of low-frequency 1/f noise on the underlying SiNx geometry. The contribution of the suspended graphene area to capacitance which affects the noise level in the high frequency range reveals that the graphene free-standing film area influences noise level over a wide frequency region. In addition, the low-frequency noise demonstrates a weak dependency on salt concentration, in deviation from Hooge’s relation. These findings and attendant analysis provide a systematic understanding of the noise characteristics and can serve as a guide to designing free-standing monolayer graphene nanopore devices.Graphene-based nanopore devices have shown tantalizing potential in single molecule detection for their monoatomic membrane thickness which is roughly equal to the gap between nucleobases. However, high noise level hampers applications of graphene nanopore sensors, especially at low frequencies. In this article, we report on a study of the contribution of suspended graphene area to noise level in full frequency band. Monolayer graphene films are transferred onto SiN Difx) , aluminux) [2O3) , and sil2O3) [2) , graphen2) [2O3/graphene stack structure [x when applying a bias voltage on both sides of the membrane, which produces sub-nanometer scale sensitivity. Therefore, the original expectation of single-nucleobase resolution will be sacrificed as the result of any thickness increase during the modification process.Indeed, the reduction of noise in graphene nanopore sensor is the most intractable problem. In allusion to that, several studies have been implemented, such as utilizing multiple layers of graphene ,8, depos2)  or polyi2)  over gratructure . Howevertructure  demonstrx layer beneath the graphene had been put forward to cope with these issues. The underlying SiNx membrane is more than a supporter to offer mechanical stabilities, but also mitigates pin-hole effect as well. Schneider [x films supported by silicon substrates, from which they found it was apparently feasible to reduce the noise level of graphene by reducing the suspended area; although this work was simply verified by comparing individual current traces, it indicated an underlying reason of mechanical fluctuation contributing to the high noise level.Under the premise of holding the line on single-atomic thickness, one simple and effective method of depositing SiNchneider  and his chneider  fabricatf is frequency, and β is a fitting parameter , which means that the fluctuation of current and surface charge contribute to the 1/f noise below this thickness [Noise characteristic polynomial is beneficial for the improvement of experimental program to minimize noise level as much as possible and optimize the signal-to-noise ratio in single molecule translocation experiment. Noise in a solid-state nanopore consists of four components and can be described by the noise characteristics:ectively . The noiectively . At low-ively [f , while tively [f . The effively [f . The empcarriers , while Bcarriers . The 1/fhickness .f noise significantly. In addition, resistance and capacitance of the membrane are corroborated to be associative with suspended area of graphene as well, which are in vital connection with noise power magnitude in the low and high frequency ranges separately. Moreover, deviating from Hooge’s relation, the 1/f noise shows a weaker dependency on electrolyte concentration, and surface charges are found to impact on pore conductance fluctuation in graphene nanopores.Here, we discuss the influence of free-standing area of graphene membrane on low-frequency noise level. We carried out a systematic study from which we concluded that the mechanical fluctuation of monolayer graphene membrane contributes to the low-frequency 1/x pore. To investigate the dependency of noise power on mechanical membrane fluctuation, we fabricated 8 graphene nanopores with similar diameters over a series of larger silicon nitride pores. The free-standing film area of graphene film, named suspended area, is equivalent to the size of SiNx. f noise, high-frequency dielectric noise and thermal noise, respectively.f noise displayed in After Fourier transform, the noise power spectra of 8 graphene nanopores at varied KCl concentrations and bias voltages are obtained. The noise power spectral density of a graphene nanopore (No. 5) at 100 mM KCl is shown in tionship :(2)SII2=f noise, the low-frequency noises  of graphene membrane diminishes drastically and its equivalent capacitor can be omitted. The equivalent circuit should be represented by However, the theoretical model is quite distinct when there is not much difference between the two apertures of graphene and SiNx lies in the range of 5–320 nm.From experimental observation, the inversion frequency that divides the full band into a low-frequency range and the high-frequency range is found to be variable. A typical inversion frequency at 1 M KCl solution and 150 mV bias voltage ranges from 300 to 20 kHz while the aperture of underlying SiNI-V) characteristic of single-layer graphene nanopore with diameter of 1.2 nm at varied KCl concentration. The calculated slopes of the solid lines fitting to the dots represent the conductance of individual nanopores. The estimated conductance of 8 fabricated graphene nanopores lies in the range of 4–39 nS. The conductance and surface charge density as functions of electrolyte concentration are shown in x-based device. It can be obviously observed in x nanopore. Additionally, the current fluctuates much more dramatically than the current of the SiNx nanopore, which exhibits a significant low-frequency variation. As a matter of fact, the disparity of noise levels in different materials appears more prominent in the noise power spectrum. From f noise of graphene is more than two orders of magnitude higher than that of SiNx. Meanwhile, current noise in high-frequency seems disparate as well, even though the substrate materials are identical. Normally, a demarcation frequency separates the full spectrum band into low-frequency and high-frequency regions. A previous work has proposed that it lies at about 1 kHz, below which the 1/f noise would govern the noise power of the whole system [f noise ceases to dominate beyond 10 kHz. Calculated 1/f noise power after normalization is nearly equal, which further proves the applied voltage’s irrelevancy with 1/f noise power.Graphene is the most widely used 2D material which shows great prospect of future success in realization of single-base spatial resolution in DNA sequencing. However, as shown in e system . In thisf regime shows a strong dependency on electrolyte concentration SD=4kConductance of all graphene chips range from 4 to 39 nS, which, compared to previously reported studies , attestsx chips  as substrates of graphene membranes. The 20 nm-thick low-stress SiNx membrane is freestanding over a silicon flake with a 20 × 20 μm window at its center. After N2 plasma cleaning for 2 min, holes of diameters ranging from 5 to 320 nm were drilled and measured through the SiNx membrane with focused ion beam  [As the schematic of the devices illustrated in MA, USA) ,24.2O2:H2O = 1:1:10 to remove the copper. Subsequently, the PMMA film with graphene on it was covered onto the fabricated SiNx chips. Finally, the chips were drowned in acetone to wash the PMMA off.The graphene we used is produced with chemical vapor deposition method on copper sheet and a layer of polymethyl methacrylate (PMMA) was spin-coated on it. After 20 min heating, the PMMA film formed and then was immersed into acid solution consisting of HCl:Hx reported [The graphene nanopores were drilled by dielectric breakdown . In advareported , and thef noise contributes in a finite frequency region and the limiting frequency, as we have observed, varies from 300 Hz to 20 kHz, perhaps even higher depending on values of the bias voltage, solution concentration, suspended area and diameter of graphene nanopore.Power spectral densities were calculated by taking the Fourier transformation of every open current in diverse applied voltage. Then the power spectral density was divided by the squared value of the mean current that was worked out through Gaussian fitting, which revealed the normalization of noise power density. Under general conditions, the low-frequency 1/f noise should scale with resistance according to Hooge’s relation with a power law of f noise on charge carrier density In summary, we have provided an elaborate description and detailed analysis about the impact caused by the suspended area of monolayer graphene film to noise characteristic in a nanopore system. In the low-frequency region, noise with a dependency on the reciprocal frequency does originate from mechanical membrane fluctuation following a relation of x pore geometry. The fit of noise versus membrane capacitance (At high frequencies, noise power is related to nanopore capacitance dominated by its suspended area as well. Capacitances of 8 fabricated monolayer graphene nanopores are measured by utilizing membrane test and the results suggest a dependency on underlying SiNIn addition, we also discuss ionic behavior of monolayer graphene nanopore devices in this article and show that measured conductance deviates dramatically from linear fit of complete bulk conductance at low salt concentration. It indicates that surface charges and their fluctuation contribute to conductance and its uncertainty significantly.


Escherichia coli (E. coli) detection. Here, the G-FETs for bacterial detection are modeled and simulated with COMSOL Multiphysics to understand the operation of the biosensors. The motion of E. coli cells in electrolyte and the surface charge of graphene induced by E. coli are systematically investigated. The comparison between the simulation and experimental data proves the sensing probe size to be a key parameter affecting the surface charge of graphene induced by bacteria. Finally, the relationship among the change in source-drain current (∆dsI), graphene-bacteria distance and bacterial concentration is established. The shorter graphene-bacteria distance and higher bacterial concentration give rise to better sensing performance (larger ∆dsI) of the G-FETs biosensors. The simulation here could serve as a guideline for the design and optimization of G-FET biosensors for various applications.Foodborne illness is correlated with the existence of infectious pathogens such as bacteria in food and drinking water. Probe-modified graphene field effect transistors (G-FETs) have been shown to be suitable for  The extraordinary electrical properties of graphene, characterized by its high carrier mobility and conductivity, have driven exploration of applications in nanoelectronic devices ,2, incluqC) when DNA hybridization occurred on the graphene surface, with results yielding more than 97% accuracy [dsI) was simulated when the graphene biosensor was exposed to the complementary DNA [There have been a few reports on modeling and simulation of FET-based biosensors. The Incremental Support Vector Regression (ISVR) algorithm was employed to study the aptamer-modified G-FETs for interferon-gamma (IFN-γ) detection, in which the change of Dirac voltage and the shift of transfer characteristics were mathematically modelled and simulated . Recentlaccuracy . Anothertary DNA .Escherichia coli (E. coli) detection by using antibody [E. coli concentration-dependent electrical response. More importantly, the biosensors exhibited rapid detection of E. coli, with a response time of about 120 s. Here, the motion of E. coli cells in solution and the surface charge of graphene induced by E. coli are modeled and simulated with COMSOL Multiphysics to interpret the operation of graphene biosensors. The size of the sensing probe proves to be the important factor affecting the efficiency of the induced charges on the graphene surface; the smaller-sized sensing probe affords more efficient induced charges on the graphene surface. Additionally, the relationship between the bacterial concentration and the electrical current of the graphene biosensor is established in this work, showing that higher bacterial concentration gives rise to larger electrical response , and the saturation is correlated with the active sensing size of graphene and the bacterial size. The simulation of the G-FET biosensors here could help to understand their operation and pave the way to designing and optimizing new biosensors with better sensing performance.Previously, we have demonstrated G-FETs for antibody  or aptamantibody  sensing The general schematic of the sensing probe-modified G-FET biosensor is shown in E. coli, the detection time was about 2 min. We assume that when a droplet of E. coli solution is added to the PDMS chamber, E. coli cells move in the solution. When the cells are close to the graphene surface, they would be sensed by the biosensor. The process from the addition of E. coli solution to the stability of the detection signal could take 2 min. Generally, the motion of E. coli in the electrolyte is affected by different forces, which include gravitational force (gF), Brownian force (bF), drag force from the liquid (dF), the binding force created by the sensing probe (pF) and the swimming force created by E. coli (sF) as shown in When the G-FET biosensor was exposed to different concentrations of E. coli cell as particle) is described byAccording to Newton’s second law , the forpV, pρ, ρ and g are the volume of the particle, the density of the particle and fluid, and the acceleration due to gravity, respectively.Considering buoyancy, the gravitational force can be expressed as:Bκ is the Boltzmann constant, µ is the viscosity of the liquid, T is the absolute temperature, pr is the radius of the particle, and Δt is the magnitude of the characteristic time step. The parameter ζ is a Gaussian random number with zero mean and unit variance.Random collisions of molecules of the fluid with the suspended particles cause a random movement called Brownian motion. The Brownian force can be estimated as :(3)Fb=ζ1µ is the viscosity of the fluid, and su and pu are the velocities of the fluid and the particle, respectively. The apparent diameter of the composite particle cd can be estimated based on the size of the E. coli cell.For a particle suspended in a fluid flowing under conditions of low Reynolds number, the drag force is estimated from Stokes’ law and the relative velocity :(4)Fd=3μE. coli is mainly based on electrostatic attraction. Considering the sensing probe and E. coli as two charged particles, the binding force between them can be expressed asek, q1, q2 and r are the Coulomb’s constant (8.99 × 109 N·m2/C2), the signed magnitudes of the sensing probe and E. coli, and the distance between the sensing probe and E. coli.The interaction between a sensing probe such as an antibody (or aptamer) and E. coli can swim in the solution, which will generate a swim force for its motion. The swim force (sF) is given bypm is the mass of E. coli, and pu is the velocity of E. coli.In addition to the forces discussed above, other forces acting on a particle in a liquid also exist, such as particle-particle interaction forces, van der Waals attraction force, thermophoretic force, lift force and magnetic forces between particles . DependiE. coli imposes an external electric field with direction towards the electrolyte. The external electric field shifts the Fermi level of graphene downwards. Therefore, when the negatively charged E. coli is added and captured by the sensing probe (antibody or aptamer), the hole carrier density of the graphene increases, as shown in dsI in the operating region (p-region) of the transfer characteristics are observed from the experimental results [Since graphene is electrically grounded, the negatively charged  results ,13.E(r) is the electric field, and ρ(r) is the total electric charge density on the surface, and ε is the free permittivity of space. Using the relationBecause of the long-range nature of Coulombic interactions , electrorε, is defined as the ratio of the permittivity in the dielectric, ε, to the permittivity in vacuum, ε0:The dielectric constant or relative permittivity, Finally, the differential form of Gauss’s law for a dielectric can be written asE. coli cells.Equations (1) and (11) were solved here by the Finite Element Method (FEM)-based commercial software COMSOL Multiphysics to visualize the time-dependent bacterial motion in the electrolyte and the charge distribution on the surface of graphene induced by charged E. coli solution. The radius of the droplet is about 1 mm. The smaller green particles are E. coli cells as shown in E. coli cells is approximately 0.5 µm. The duration of particle motion in solution is set at 120 s, which is the same as the real-time electrical current monitoring experiment. Adding E. coli solution at different concentrations allows the number of E. coli cells close to graphene surface to be simulated and counted.The input parameters for the simulation were taken from the experiment in . As show2, 103, 104, 105 and 106. The distribution of the particles at different concentrations is shown in 3 and 105 CFU/mL concentrations may be due to the interactions between bacterial particles. Similarly, when 103 CFU/mL bacterial particles was simulated, the number of particles on the graphene surface increased slightly in the first 20 s followed by a much more significant increase from 20 to 120 s. However, when higher concentrations of bacterial particles  were simulated, most particles reached the graphene surface within a shorter time (~20 s), and a saturation behavior could be observed for all three higher concentrations. Detailed information can be found in the supporting information . Theoretically, when a particle containing −3 × 10−16 C charges is located on a plate, positive charges of 3 × 10−16 C will be induced on the surface of the plate. In our simulation, the induced charges exhibit a distance-dependent decrease as shown in E. coli particle decreases when the graphene-E. coli particle distance increases, which indicates that the efficiency of charge inducement will decrease when large-sized sensing probes are employed as the sensing component. The hole carriers induced by a single E. coli particle is calculated byN is the carrier number; Q is the surface charge; e is the elementary charge (1.6 × 10−19 C). As shown in E. coli particle distance increases. As the heights of the linker-antibody complex and pyrene-tagged DNA aptamer are 5 nm and 3.6 nm, the hole carriers induced by single E. coli are 1635 and 1725, respectively. We used these two values in the following calculation.We simulated the surface charge induced by negatively charged E. coli cell, we calculated the source-drain current change (∆dsI) when the graphene device was exposed to various concentrations of E. coli. As discussed above, the source-current modulation is expressed as a function of the change in the carrier density (∆N) in the graphene channel, which is proportional to the number of targets attached on the graphene surface [w is the width of the graphene channel; l is the length of the graphene channel; e is elementary charge (1.602 × 10−19 C); µ is the carrier mobility; dsV is the source-drain voltage.Based on the simulation results of bacterial motion and the hole carriers induced by single  surface :(13)ΔIdsE. coli induces more holes in the graphene channel. Based on Equation (13), the net change in source-drain current (∆dsI) caused by E. coli was evaluated at the given gV (0.04 V) and dsV (0.05 V). The carrier mobility (1030.99 cm2/V·s) for the source-drain current calculation was obtained from our previous experimental results [6 CFU/mL) reveals  reached the graphene surface by 120 s. This behavior can be explained as follows. The bacterial motion in solution is affected by the gravitational force, Brownian force, drag force and swimming force in the first 30 s. When the E. coli particles are close to the graphene surface, the binding force between the sensing probe and bacteria could dominate the motion of E. coli, causing the number of bacteria on the graphene surface to increase fast. Accordingly, the ∆dsI increases gradually, which is consistent with the change of the number of bacteria on the graphene surface. The simulated ∆dsI is smaller than the experimental result as shown in dsI in the first several seconds in the experiment.As shown in E. coli , a slow increase in the number of bacterial particles on the graphene surface is seen in the first 8 s, followed by a much faster increase until 30 s, affected by the binding force. The rate is slower again from 30 to 90 s with saturation happening afterwards until 120 s. Accordingly, the calculated ∆dsI is consistent with the change in the number of bacteria on the graphene surface. Similarly, a negative peak is observed in the first several seconds, and the simulated ∆dsI is smaller than the experimental value, as shown in dsI could reach saturation rapidly with the addition of high concentration of bacterial particles  to the electrolyte.As shown in 2 CFU/mL), indicated by the slope value in the first rise period. The larger value from the experiment is attributed to more particles reaching the graphene surface in the experiment. At high concentration , the slopes in different rise periods from the experiment and simulation are at the same level, except for a slow increase of the ∆dsI within a very short rise period. For the first rise period (0–30 s) and the second period (31–90 s), the slopes from the simulation and experiment are of the same order. The above analysis on the slope suggests that the simulation fits the experiment well. Additionally, a 2-stage response can be found in both low- and high-concentration cases.We then compare the slope of the simulated and experimental current vs. time curve as shown in E. coli concentration-dependent ∆dsI and compare the simulation and experiment results. dsI of both the antibody-modified and aptamer-modified G-FETs for different concentrations of E. coli solution. According to the simulation results, the number of E. coli cells reaching the graphene surface is about 10,000 CFU/mL with the addition of 11,950 CFU/mL E. coli. When the graphene channel is completely occupied by E. coli and all these E. coli cells could induce holes in the graphene channel, only about 10,000 E. coli cells could attach to the surface of graphene, even when adding a higher concentration of E. coli solution. When higher concentration of E. coli (105 and 106 CFU/mL) is added, the number of E. coli reaching the graphene surface is still 10,000 CFU/mL, implying the attainment of saturation. As shown in dsI obtained from the experiment  is slightly smaller than that of the simulation result (black line). Some antibody molecules may lose their bio-activities, resulting in a smaller electrical response. The saturation of the source-drain current for the aptamer-modified G-FET happens with the addition of 11,950 CFU/mL E. coli (black line in n = 5) is similar as in the simulation results. However, the value of ∆dsI is larger in the simulation because of the use of E. coli K12 as the target in the simulation, in contrast to E. coli 8739 as the target in the aptamer-related experiment. We assumed that E. coli 8739 might carry more surface charge than E. coli K12. To date, there is no report on the surface charge of E. coli 8739. Therefore, we calculated the value of the positive charge induced by single E. coli 8739 cell. When the simulation results were consistent with those of the experiment, the value might be about 2062. By using Equation (12), the surface charge of E. coli 8739 was evaluated as ~−3.59 × 10−16 C. Based on the experiment and the simulation results, the surface charge properties of the target could be evaluated.Next, we discuss the E. coli concentration. Smaller graphene-bacteria distances  and higher bacterial concentrations give larger source-drain current change. Overall, this study provides a comprehensive understanding of the target motion in the electrolyte and surface charge induced by the target (E. coli). The established relationship between the source-drain current and graphene-bacterial distance here could help to design new graphene biosensors with excellent sensing performance. Since G-FETs are a material-specific subset of bio field effect transistors (BioFETs) with silicon and other materials serving as channels [Although there are some gaps between the experiment results and the simulation, the simulation results still give us a deeper understanding of the operation of G-FET biosensors designed for high performance and provide a reference for evaluating the sensing performance. channels ,24, the E. coli particle and its motion at different times, functionalization with differently sized sensing probes and surface charge induced by adding E. coli cells at different concentrations were carefully investigated. The graphene-bacteria distance, defined by the size of the sensing probe, is found to play a key role in improving the sensing performance of the biosensors since it leads to more efficient induced surface charge and a resultant larger electrical response. More importantly, the relationship between the source-drain current, the graphene-bacteria distance, and the concentration of the bacterial particles was established. Smaller graphene-bacteria distances and higher bacterial concentrations yield larger changes of source-drain current of the biosensor. This relationship can help to predict the sensing performance of the G-FET biosensor. This study can serve as a guideline for the design and optimization of G-FET biosensors in the future.We have modeled and simulated the operation of G-FET biosensors, including the bacterial motion in the electrolyte and the surface charge induced by the charged bacterial particles. The charged


The synthesis of large-size graphene materials is still a central focus of research into additional potential applications in various areas. In this study, large-size graphene platelets were successfully produced by pulsed discharge of loose graphite strips with a dimension of 2 mm × 0.5 mm × 80 mm in distilled water. The graphite strips were made by pressing and cutting well-oriented expanded graphite paper. The recovered samples were characterized by various techniques, including TEM, SEM, optical microscopy (OM), atomic force microscopy (AFM), XRD and Raman spectroscopy. Highly crystalline graphene platelets with a lateral dimension of 100–200 μm were identified. The high yield of recovered graphene platelets is in a range of 90–95%. The results also indicate that increasing charging voltage improves the yield of graphene platelets and decreases the number of graphitic layers in produced graphene platelets. The formation mechanism of graphene platelets was discussed. This study provides a one-step cost-effective route to prepare highly crystalline graphene platelets with a sub-millimeter lateral size. Graphene, firstly synthesized by Noveselov et al.  in 2004,2SO4, P2O5 and K2S2O8, exfoliated by stirring with addition of KMnO4, and then reduced using hydrazine monohydrate to form large-size graphene. Through CVD method, the wafer-scale graphene was also synthesized [4 or C2H4 gases at high temperatures (800–1077 °C). Bae et al. [3-intercalated graphite mixed with 30% H2O2 in a reactive bottle for 2 h to exfoliate the graphitic layers to form graphene in a size of 40 × 60 μm2 with scalable production. However, till now, a great challenge has remained in realizing large-quantity and high-quality production of large-size graphene materials for further industrialization.Various methods have been reported to synthesize graphene materials ,34,35,36thesized  on diffee et al.  synthesie et al.  employed2 and TiN nanopowders using the pulsed discharge of Ti wire in air and liquid nitrogen, respectively. Tanaka et al. [Pulsed wire discharge  employs a high-density current to heat a thin conductive wire, leading to rapid melt and vaporization of the wire at high temperature and pressure . Subsequa et al.  have reca et al.  synthesia et al. ,47. In aa et al. ,49, carba et al. ,51, and a et al. . HoweverIn our previous study, a graphite stick was connected to electrodes for pulsed discharge to prepare mono-layer and few-layer graphene in distilled water . ThroughTo recover the as-prepared samples, a cylindrical stainless steel explosion chamber a with an2 F20 S-Twin). Raman spectra of the raw graphite strip and recovered samples were recorded on a LabRAM Aramis Raman spectrometer with a He-Ne laser at an excitation wavelength of 633 nm. The corresponding XRD patterns were recorded on an X-ray diffractometer (XRD) (Rigaku Ultima IV multipurpose XRD system) with Cu Kα radiation (λ = 0.15406 nm) at a step size of 0.05° (2θ) and a step scan time of 1.0 s. The sample suspensions were dropped on a Si plate and dried at room temperature for optical microscopic, SEM and Raman spectroscopic analysis. The sample suspensions were also dropped and dried on a copper mesh grid for TEM analysis. The atomic force microscopy (AFM) analysis was taken by MFP-3D Infinity AFM.The morphology and microstructure of as-prepared samples were examined by optical microscopy , and analyzed by field emission scanning electron microscopy  and high resolution transmission electron microscopy , G band (1581 cm−1), D’ band . The 2D band has been widely used to distinguish mono-layer graphene and few-layer graphene from graphite [2D/IG indicates less graphitic layers [D/IG value is used to characterize the disorder degree of the graphitic layers [D/ID’ reveals the disorder type of graphene [graphite ,61. A hic layers ,56 in prc layers . The ID/graphene .2D/IG values of the recovered samples were 0.70–1.03, much larger than that of the raw graphite strip (0.29), indicating that the recovered samples were few-layer graphene [2D/IG value increased from 0.70 to 0.91 and 1.03 accordingly, indicating that increasing the charging voltage can decrease the number of graphitic layers of produced graphene. Additionally, the 2D band position also revealed the thickness of the graphene materials [−1 to 2662 cm−1 was observed, revealing that the graphitic layers in FLGPs are less when the charging voltage is higher. The results of the influence of charging voltage on graphene layers is in good agreement with the TEM results. In addition, all the ID/IG values of recovered samples are in a range of 0.15–0.20 similar to that of the raw graphite strip (0.17), suggesting that the disorder degree of the recovered samples and the raw graphite strip are nearly the same. Thus, the pulsed discharge process in our experiments can hardly generate structural defects. Furthermore, the above ID/IG values are also smaller than those of graphene produced by arc discharge method (ca. 1.0) [D/ID’ values of the recovered samples were in the range of 2.88–3.69, close to 3.5, suggesting that the main disorder in the raw material and formed FLGPs is boundary-like disorder [D/ID’ is close to 7 [The above intensity ratio values of the raw graphite strip and the recovered samples are listed in graphene ,56. Moreaterials . In Figuca. 1.0) , oxidatica. 1.0) ,66,67, aca. 1.0) , showingdisorder . It alsoose to 7 . Raman rThe yields of produced FLGPs under different conditions are listed in Based on the above characterization results, we propose the mechanism regarding the formation of FLGPs using pulsed discharge  and the density of the graphite stick (1.9 g/cm3) in Ref 35, implying a loose structure and high porosity of graphite strip. During pulsed discharge, under the sharp increase of temperature and pressure, the gas molecules in this loose material expand rapidly to exfoliate graphitic layers. Thus, both good orientation and the gas in the loose structure are conducive to the exfoliation under high temperature and high internal pressure, leading to a higher exfoliation efficiency of the graphite strip and a higher yield (90–95%) of FLGPs.However, note that the graphite strip in these experiments is a well-oriented graphite material, in which the lateral dimension of graphitic layers is in sub-millimeter scale, rather than the graphite stick consisting of micro graphite particles with randomly-distributed orientation. Since the expansion direction of graphitic layers is approximately along the z-axis of graphite crystallites and vertical to the surface of graphite strips, the expansion of well-oriented graphite crystallites can be rarely inhibited by the expansion of adjacent crystallites. Consequently, the exfoliation efficiency of graphite strips is high. Moreover, the density of the graphite strip is approximately 1.4 g/cmThe energy input during pulsed discharge is controlled to only cause the rapid thermal expansion effect to exfoliate graphitic layers without destroying the atomic structure of the graphitic layers, therefore the high crystallinity and large dimension of the original graphite crystallites in the graphite strips results in the high crystallinity and large lateral dimension (100–200 μm) of the exfoliated FLGPs after pulsed discharge. In addition, a higher charging voltage increases the energy input during pulsed discharge, resulting in a higher temperature and internal pressure. Consequently, when the charging voltage is increased, the exfoliation efficiency is also increased to generate a slightly higher yield of FLGPs with less graphitic layers.This study demonstrates an efficient, cost-effective and environmentally friendly route to produce highly crystalline large-size FLGPs in distilled water at room temperature.The highly crystalline FLGPs with a lateral dimension of 100–200 μm were produced successfully with a high yield of 90–95%. The mechanism of graphene production is supposed to be the exfoliation of graphitic layers from a graphite strip under the action of joule heating induced by pulsed discharge. Two factors, charging voltage and the loose structure of the graphite strip, are critical to the exfoliation of graphitic layers and the exfoliation efficiency. As the increase of charging voltage from 20 to 40 kV, the yield of FLGPs is slightly increased with less graphitic layers owing to the higher exfoliation efficiency induced by higher internal pressure.Additionally, the main cost of this experiment is the graphite strips cut from graphite paper with a cost of $22.8 per kilogram , implying the great economical advantage of the synthetic route compared with the price of commercial graphene , etc.). Moreover, the experiments were carried out using graphite strips in distilled water without any other chemical agents, indicating that this route is environmentally friendly. Considering the high yield of this route and the large lateral dimension of produced graphene, this route could have a high potential in the areas of electronics, solar energy devices, and mechanical structures.


One of critical bottlenecks on some important classes of applications, such as printed electronics, conductive coatings, and composite fillers, is the lack of industrial-scale methods to produce high-quality graphene in the form of liquid suspensions, inks, or dispersions. Since 2008, when liquid-phase exfoliation (LPE) of graphene via sonication was initiated, huge progress has been made in the past decade. This review highlights the latest progress on the successful preparation of graphene in various media, including organic solvents, ionic liquids, water/polymer or surfactant solutions, and some other green dispersants. The techniques of LPE, namely sonication, high-shear mixing, and microfluidization are reviewed subsequently. Moreover, several typical devices of high-shear mixing and exfoliation mechanisms are introduced in detail. Finally, we give perspectives on future research directions for the development of green exfoliation media and efficient techniques for producing high-quality graphene. This systematic exploratory study of LPE will potentially pave the way for the scalable production of graphene, which can be also applied to produce other 2D layered materials, such as BN, MoS Graphene is a new type of two-dimensional 2D) carbon nanomaterial, which exists as a single layer of carbon atoms that are tightly arranged in a honeycomb lattice. It is the basic constructional element for all other dimensions of graphitic materials  1]. It . It 1]. D carbon One of the critical bottlenecks on some important classes of applications, such as printed electronics, conductive coatings, and composite fillers, is the lack of industrial-scale methods to produce high-quality graphene in the form of liquid suspensions, inks, or dispersions ,10. At pSince 2008, when LPE of graphite through the sonication of graphite powder in N-methylpyrrolidone (NMP) was first proposed by Coleman et al. , there hIn this paper, we will review the latest progress on the preparation of graphene by liquid-phase exfoliation in organic solvents, ionic liquids, water/polymer or surfactant solutions, and some other green dispersants. Though several reviews ,29,30,31Traditionally, LPE includes mainly two different exfoliation techniques of graphite : cavitatSonication is an effective exfoliation method and has the potential to produce monolayer or few-layer graphene at relatively high concentration. Typically, the power of sonication is used to induce physical or chemical changes in some systems through the generation of cavitation bubbles . As the There are two types of sonication, namely bath sonication (BS) and tip sonication (TS). They have been employed simultaneously or individually to prepare monolayer or few-layer graphene sheets by exfoliating graphene . Sonicat−1 and 4 wt % monolayers by using drastically longer sonication times (460 h) . Th. Th60]. 2 [−1 were obtained in the aqueous solution of 75% acetone. However, micro-jet cavitation results in the absence of severe functionalization of graphene and the sizes of graphene sheets cannot be controlled. Similarly, the technique of jet cavitation was proposed by Shen et al. ,69 to ex2 . Some en2  by means2. The yield of graphene nanosheets of less than three layers thickness reached 88%. The mechanism of exfoliation graphene by jet cavitation with or without supercritical CO2 is shown in 2, there is one more mechanism, i.e., the intercalation and collision of supercritical CO2 molecules has a positive effect on the relaxation of Van Der Waals force among the graphite layers [Recently, Shang et al.  presentee layers .For LPE, one of the most important factors controlling the overall yield of exfoliation graphene is the appropriate solvents. The van der Waals interaction between the graphene layers held within a π–π stacking distance of 0.34 nm should be effectively overcome by the ideal solvent .−2 [Previous studies have shown the interfacial tension plays a crucial role when a solid surface is immersed in a liquid medium . The ran−2  because 6F6, C6F5CF3, C6F5CN and C5F5N, has been reported. The exfoliation capability of each solvent in ascending order was as follows: C6F5CF3≈C5F5N<C6F6<C6F5CN. Hence, C6F5CN provided the highest yield and concentration of no oxidization of graphene dispersion . Moreover, Qian et al. [In addition to the above common organic solvents, a series of perfluorinated aromatic molecules , includin et al.  reportedn et al. .−1 [Although the LPE has been rapidly developed in the past decade, many severe problems still exist. One of the most serious challenge is the lover concentration of graphene dispersions obtained in the pure organic solvents, generally, less than 0.01 mg mL−1 . Besides−1 , which i−1 , such as−1 .+ and OH- increasing the interlayer spacing of graphite. Du et al. [3C6H5O7, Na2C10H14N2O8, Na2C4H4O6, and KNaC4H4O6)   These orhthalene , serving−1. Coleman et al. [−1 by optimizing the parameters of the dispersion procedures (−1) in 1-propanol (23.4 mJ m−2), a volatile solvent, which could be removed just by air drying without heating. This means that the surface tension is not always a valid criterion for predicting the dispersibility of graphene in solvents, which should be characterized by the Hildebrand or Hansen solubility parameters [Some researchers try to exfoliate graphene in low boiling points solvent in order to overcome the limitations of using high boiling solvents. Zhang et al.  demonstrn et al.  demonstrocedures . Howeverrameters .Ionic liquids, an organic molten salt, having a melting point below 100 °C ,89, usua−1) graphene dispersion can be produced via sonication in 1-butyl-3-methyl-imidazolium bis (trifluoro-methane-sulfonyl) imide, an ionic liquid. Ionic liquids that containing non-aromatic cations gave similar results. Nuvoli et al. [−1, without any chemical modification. Ager et al. [Wang et al.  demonstri et al.  found 1-r et al.  demonstrHigh concentration of graphene dispersion can be obtained in ionic liquids, but ionic liquids are expensive and difficult to remove, which limits its wide application. In addition, the viscosity of the ionic liquids is large, which will have a certain effect on the exfoliation of graphene.−2, can be reduced by the addition of suitable surfactants so that water/surfactant system has potential to apply to the exfoliation and stabilization of graphene (this review focuses only on water/surfactant system considering the limitations of organic solvents). Actually, surfactants in aqueous solutions are more environmentally friendly while producing comparable concentrations of graphene to pure solvent exfoliation. The exfoliation of graphene in organic solvents or ionic liquids is facile, involving only two phase dispersions ,27, name−1, which was similar to that of nanotube dispersion in the surfactant aqueous solution [−1. Coleman et al. [−1.The exfoliation of graphite in surfactant solutions is one of the most promising methods to produce high-quality graphene . A rangesolution ,96. Greesolution  shown thn et al.  disperse−1 by increasing the total sonication energy/volume and bath sonication energy/volume (−1) in the aqueous solution of sodium taurodeoxycholate (STC), an anionic surfactant, via only 24 h tip sonication. A novel anionic surfactant (sulfonated used engine oil (SUEO)), which was prepared from used engine oil, was employed to exfoliate graphene sheets under sonication. The exfoliating and dispersing efficiencies of SUEO, estimated by the concentration of graphene dispersions (0.477 mg mL−1), were higher than that of traditional surfactants, such as SDS, SDBS, CTAB, and PVP.In order to improve the concentration of graphene dispersion, long-time sonication (up to 400 h) was utilized in SC aqueous solution, the final concentration is up to 0.3 mg/mL . Multi-sy/volume . Sun et ® P123 provided the highest concentration of graphene dispersions (up to 1 mg mL−1). LPE in non-ionic surfactant systems, providing better stability and higher concentrations of graphene nanosheets, performed better than that of in ionic surfactants. However, surfactants were added only once at the beginning of sonication or high-shear mixing, the concentration of exfoliated graphene was too low to be used for a potential industrially scalable process. Fortunately, Notley et al. [−1) by the continuous addition of Pluronic®108, a nonionic surfactant, this method could be also used with other surfactants  without oxidation in the aqueous phase via sonication. Sodic carboxymethylcellulose (CMC-Na) and DNA showed the similar behavior. The dispersions of PVP-stabilized graphene obtained by tip sonication was stable and expediently re-dispersible in series of organic solvents  [−1 in vinyl pyrrolidone (VP). Sun et al. [−1) can be prepared in low boiling point alcohols, ethanol and isopropanol, with the addition of polymer P20 via bath sonication. Phiri et al. [−1 by 120 min high-shear mixing in PVP aqueous solution, which means that the efficiency of high-shear mixing is several times than that of sonication exfoliation [Bourlinos et al.  first reethanol) , among wn et al.  demonstri et al.  found tholiation . Modifieoliation .Besides, it has been recently shown that graphite can be exfoliated in pure water or organic solvent by adding a various of conventional polymers, such as polystyrene(PS) , ethyl cThe use of polymers in the LPE process is no doubt better than that of pure organic solvents. However, the majority of graphene prepared by polymer-assisted LPE cannot be extracted from graphene–polymer composites on account of the strong interactions between graphene and polymer. In general, polymers or surfactants as stabilizers are challenging to be removed, which results in the poor performance of the graphene prepared by polymer- or surfactant-assisted LPE. The extensive applications of graphene are limited by the lack of cost-effective massive production methods . Therefo2, MoSe2 [−1  with the addition of ammonia solution, which released the gaseous ammonia playing a pivotal role in the graphene exfoliation. This preparation method was simple and did not involve any organic solvents, surfactants or polymers. In order to optimize the exfoliation strategy, the starting graphite concentration and sonication time (ts) were investigated, CG = 20.28 ts1/2 , the results accorded with that presented by Coleman’s research group (CG∝ts1/2) [−1 at 1000 rpm, while it reduced to 13.80 μg mL−1 at 3000 rpm.Shen et al.  reportedG∝ts1/2) . In gene−1. Similarly, pulping black liquor, a main source of papermaking pollution, contains about 65% alkali lignin. The treatment technology of black liquor is complex, expensive, and may bring secondary pollution. Fortunately, Ding et al. [2 and BN, could be efficiently exfoliated by high-shear mixing in pulping liquor. This green, environmentally friendly method not only can solve the pollution of water liquor, but also give high-concentration dispersions of 2D layered materials  , despite the probable functionalization of graphene by oxygen groups in black tea.Lignin, a low-cost by-product of the pulp and paper industry, has been utilized directly or with chemically modified as dispersant agent . Recent g et al.  found laraphene) . These sgraphene  [121,1223-graphite intercalation compounds were treated with dodecylamine (DA) at 90 °C to product amine-treated graphene (G-90), and DA could be completely removed through the subsequent heating treatment; secondly, G-90 was heated at 900 °C in air to product graphene (G-900); Finally, G-900 was further heated at 2000 °C under nitrogen atmosphere to obtain well-expanded graphene (G-2000). Though the exfoliation efficiency of GICs is higher than the direct exfoliation of flake graphite, this requires a special intercalation process.In addition to the direct exfoliation of flake graphite above, another prevalent method of graphite exfoliation is the conversion of graphite into graphite intercalation compounds (GICs). The intercalation can extend the graphite so as to reduce the adhesive forces among the graphene layers. Therefore, GICs can be exfoliated easily by sonication or high-shear mixing in the appropriate solvents . For exaThis review concentrates on the devices that can offer high-shear force to mildly exfoliate graphene, such as the Taylor–Couette flow reactor, rotor-stator mixer, rotating-blade mixer, and microfluidizer . Meanwhi−1 after 100min of mixing time  and the yield of high-quality  few-layer (90% of flakes less than five layers) graphene can achieve up to 5% by optimizing the parameters of vortex flow process based on computational fluid dynamics.Taylor-Couette flow reactor , a vorteTaylor vortex flow can generate the high local shear rate and high wall shear stress in each vortex cell at the same time, which is potentially responsible for the efficient exfoliation of the starting graphite flakes .D/IG = 0.18) graphene by Silverson model L5M mixer  using the method scales as PR∝V1.6 for the surfactant and PR∝V1.1 for NMP, which means the scalable production can be achieved just by increasing the volume of liquid and surfactants were more promising for scalable production.Paton et al.  first re5M mixer , a rotor−1. The shear exfoliation of graphite was carried out in PVP and SC aqueous solution using an available high-shear colloidal mixer, which was equipped with high-shear generator system of three-stage rotor-stator , means that production rate declines with mixing time. This problem may be bound up with the details of the interaction of liquid and rotor-stator [Though the exfoliation of graphene using rotor-stator mixer can achieve scale-up by just increasing the volume of liquid, the graphene concentration, C∝tr-stator .D/IG < 0.12, free of basal-plane defects or oxidation) graphene by using a kitchen blender, a rotating-blade mixer, the concentration reached up to 0.22 mg mL−1 and the yield was 7.3% after 8 h of high-speed mixing. Similarly, Coleman et al. [−1, which was much higher than those generally reached via shear or sonication exfoliation, owing to the strongest negative charge of BSA in all the proteins used. For the shear exfoliation using the rotating-blade mixer, the concentration of graphene dispersion increases linearly with time so that it is 10 times higher than those reached using rotor-stator mixer. More importantly, the concentration declines tardily with volume, which results in the production rate increasing with volume [To explore a new equipment to offer high shear, Shen et al.  first ren et al.  utilizedn et al.  reportedh volume .For exfoliation mechanism of rotating-blade mixer, it involves multifarious fluid dynamics events, such as turbulence , shear,It is now well-acknowledged that one of critical bottlenecks on some important classes of applications—such as printed electronics, conductive coatings, and composite fillers—is the lack of industrial-scale methods to produce high-quality graphene in the form of liquid suspensions, inks, or dispersions . LPE is 2, WS2, etc. However, it is still an enormous challenge to achieve fundamental goals of commercial availability of massive high-quality graphene with cost-efficient and environmentally friendly LPE. There are multitudinous conundrums to be further studied and overcome, some of which and our perspectives are discussed as follows:This review has highlighted the latest progress on the successful preparation of graphene in various media, including organic solvents, ionic liquids, water/polymer or surfactant solutions, and some other green dispersants. The techniques of LPE, namely, sonication, high-shear mixing and microfluidization have also been reviewed subsequently. Moreover, several typical devices of high-shear mixing and exfoliation mechanisms were also introduced in detail. This systematically exploratory study of LPE will pave the way for the scalable production of graphene, which can be applied to produce other 2D layered materials, such as BN, MoSThe yield of LPE is still too low to meet the macroscopical requirement of industrial application of graphene, and the production of single-layer, large-size graphene is a huge challenge as well. The amount of available solvents that are propitious to LPE of graphene is limited. In addition, most solvents that can be used to disperse graphite are relatively expensive and toxic. Polymers or surfactants as dispersants are challenging to be removed, which results in the poor performance of the graphene prepared by polymer- or surfactant-assisted LPE. Sonication or shear exfoliation generally result in the reduction of the size of graphene nanosheets. The devices of high shear mixing are utilized directly from other fields of science, there is no equipment wholly designed for LPE. Graphene nanosheets prepared by LPE tend to aggregate easily and irreversibly in most solvents due to van der Waals forces and high surface energy, so separating graphene from the dispersion is another important issue . More imThough the specified centrifugation strategy  can sepa


Second, the intensity of this pronounced PIA effect can be optimized by adjusting the coupling distance. Third, the resonance frequency can be easily tunable by tuning the graphene Fermi level. This system may have potential applications in dynamically optical switching and biochemical sensing.The constructive interference of bright and dark plasmonic modes results in plasmon-induced absorption (PIA) effect. Here, we theoretically investigate PIA effect, which is realized by the constructive interference between a Fabry-Perot (F-P) resonance mode and a graphene quasi-guided mode. Numerical simulation reveals at least three advantages of our structure over previous ones. First, the extinction ratio can reach ~ 99.999%, resulting in the ultra-high figure of merit* (FOM*) as high as 10The online version of this article (10.1186/s11671-019-3121-9) contains supplementary material, which is available to authorized users. Plasmonics has attracted wide attention due to its extraordinary properties –15 and hR-T, where R and T are the reflectance and transmittance, respectively. It is simply 1-R in our system since the transmittance here is 0. As a result, an ultra-high FOM* as high as 106 in the graphene/metal system can be achieved and the resonance frequency can be dynamically tunable by adjusting the gate voltage of graphene. These prominent properties can be applied in biochemical sensing and dynamically optical switching.However, traditional devices based on the PIA effect of the metallic structure are hard or impossible to obtain tunability, which seriously restricts its applications. Graphene , 30, kno2O3 isolated layer on top of a grooved silver. The thickness of the Al2O3 is g. The system is illuminated by a normal-incident plane wave of transverse magnetic (TM) polarization. The other structural parameters are expressed as follows: d is the depth of the silver groove; w is the width of the silver groove; P is the period of the unit cell. In the mid-infrared region, intraband scattering dominates in highly doped graphene, and its conductivity takes on a Drude-like form σg = ie2EF/[πħ2(ω+iτ-1)]. The electron relaxation time is expressed as τ = μEF/eυF2, where υF = c/300 is the Fermi velocity, EF is the Fermi energy and μ = 10 m2/Vs is the DC mobility of graphene [2O3 are from ref. [ε0 = 1).The schematic of our structure is shown in Fig. graphene , 31, 32.rom ref.  and ref.rom ref. . The perw = 100 nm, P = 250 nm, d = 2000 nm, and the result is shown in Fig. Q factor ~ 0.8, which is due to an F-P resonance induced by the SSP excited by incident light [EF = 0.3 eV, as shown in Fig. EF = 0.3 eV and different unit cell P and is shown in Fig. f = 32.84 THz can be observed for P = 250 nm with a Q factor ~ 304. The high Q resonance with a narrow resonance band can serve as the subradiant (dark) mode in our PIA system. The reflectance dip is due to the resonance of plasmonic quasi-guided mode in graphene with the normal incidence [mth order phase-matching condition [We simulated the reflection spectrum of the silver groove with nt light . This rencidence  since thondition , 37Fig.kx = k0sinθ, k0 = 2π/λ is the wavevector in free space, θ is the angle between the incident light and y-direction, neff is the effective refractive index of TM waveguide mode in the graphene, and Gx is the reciprocal lattice vector of the grating (Gx = 2π/P). In the following discussion, the incident light angle of y-direction is zero (θ = 0°). The situation for other incident angles is discussed in Additional file m = 1 and the effective refractive index, ~ 33, is obtained by FDTD solutions, as shown in Fig. Ex distributions of F-P mode and graphene quasi-guided mode have been shown in Fig. where g, the near-field coupling and the quasi-guided mode become weaker, as the modulation of the reflectance dip becomes smaller. There are two possible ways caused the reflectance dip smaller what are the weaker coupling and the weaker quasi-guided mode excitation. Therefore, we used the coupled oscillator model to quantitatively understand the PIA system [In the coupling situation, the two eigenmodes will be strongly coupled when they get close to each other, and hence the reflection spectrum will be dramatically changed. A narrow, sub-linewidth dip of enhanced absorbance with an extinction ratio ~ 99.97% is observed on top of the broader reflectance dip, as shown in Fig. A system .2\documω1,2 and γ1,2 are the time-harmonic amplitudes, resonant frequencies, and damping constants of the bright mode and the dark mode, respectively. b is the coupling coefficient measuring how strong the bright mode couples with the incident electric field. φ is a phase shift, which is a key coefficient to determine the form of the interference between the two coherent pathways. When φ = 0 is a real parameter and the typical behavior of the PIT effect can be observed, and the interference between the two coherent pathways is destructive. For φ = π/2 is a pure imaginary parameter and the interference between the two coherent pathways is converted from destructive to constructive [Where tructive . The absentclass1pt{minimag, which have been shown in Fig. κ, φ, γ1, and γ2 have been shown in Fig. g yields a decrease in the coupling parameter κ, as shown in Fig. g), the phase φ is unchanged, and γ2 gradually decreases while γ1 changes slightly shown in Fig. κ exceed the damping constants of dark mode γ2 for the minimum gap distance, which confirms that the coupling from the bright mode to the dark mode is stronger than the dissipation processes in the graphene sheet.Then, we fit the numerical absorption spectra with the Eq.  for diffHz monitors have been placed 3 nm away from the center of graphene and 1000 nm away from the bottom of the silver groove, respectively. The oscillating phase difference between the two modes is 0.5π, as indicated in Fig. fq = 32.5 THz, where ωqt1 ~ 2.00 π and ωqt2 ~ 2.50π, as indicated in Fig. To visualize the constructive interference between the bright and dark modes, we investigated the structure’s magnetic field evolution with the time, and two P and depth of silver groove d to optimize our structural parameters. After calculating the reflection spectrum of different structure parameters P from 1900 to 2100 nm and d from 245 to 265 nm by FDTD, we earn a very high extinction ratio ~ 99.999% in P = 254 nm and d = 1980 nm. The reflectance spectrum of the PIA device under different refractive index environments is shown in Fig. In practical application, a narrow reflection band and high extinction ratio are highly desired. To achieve these two conditions, we can adjust period of unit f and I are the resonance frequency and the spectral intensity, respectively. When measuring the reflection intensity of a sensor, the sensitivity capability of the sensor can be quantified by the FOM* value. The higher the value means the higher the sensitivity of the sensor. From Fig. S = 11.2 THz/RIU and the associated FOM~94.1, with the full width at half maximum (FWHM) ~ 30 nm (0.12 THz). This FOM is larger than the value in metamaterial absorbers based on surface lattice resonance. Also, our PIA sensor can lead to an ultra-high FOM* value 3.5 × 106, as indicated in Fig. where In the PIA system, graphene plays another key role. The modulation of the resonant frequency can be achieved by tuning the gating voltage to adjust the Fermi level of graphene. The simulated spectra are shown in Additional files g is gradually increased, the resonance strength and linewidth will decrease. For the application, the FOM* in our system can achieve 106. Furthermore, the absorption window can be tuned by varying the geometrical parameter and the graphene Fermi level. These results could provide a new way toward the realization of nanoscale mid-infrared dynamical spectral control and ultrasensitive optical sensors.In summary, we have numerically demonstrated the perfect absorption induced by constructive interference between F-P resonance mode and graphene plasmonic quasi-guided mode. Through the introduction of graphene plasmonic quasi-guided mode, we obtain the spectral line with a narrower linewidth of the silver groove F-P resonance mode. When the distance Additional file 1:Table S1. The compare of the recent relevant works. (DOCX 19 kb)Additional file 2:Figure S1. The reflectance spectra of the graphene-silver groove structure at different incident angle θ. (PNG 236 kb)Additional file 3:Figure S2.The reflectance spectra of the structure of graphene-silver groove in normal incidence for different Fermi energy of the graphene sheet. (PNG 523 kb)


Additionally, the three rice microbial communities differed significantly in the metabolic utilization of carboxylic acids and miscellaneous (p < 0.05), and there were, however, no significant differences in the other four types of carbon sources. Furthermore, principal component analysis revealed that the microbial communities of stored rice had obviously different metabolic functions in different storage period. Therefore, the study indicated that the BIOLOG ECO microplate was applicable to evaluate the metabolic functions of rice microbial communities, and carboxylic acids and miscellaneous were two crucial parameters of carbon sources to identify the metabolic differences of microbial communities, a case in which it reflected the conditions of rice microbial contamination.Microbial contamination has been a pervasive issue during the rice storage and triggers extensive researches. The metabolism of microorganisms was proved as an indicator to mirror the degree of microbial contamination. It is necessary to develop a scientific method to analyze the metabolism of rice microbial communities, thereby monitoring the microbial contamination. In this study, the metabolism of rice microbial communities in different storing-year were investigated by BIOLOG ECO microplates. The three rice samples were respectively stored for 1–3 years. The related indicators of BIOLOG ECO microplates were determined, including average well-color development (AWCD) of carbon sources and three metabolic functional diversity indices. The results showed that there were significant differences in the AWCD of all carbon sources among the three rice microbial communities ( Rice is cultivated extensively in China, which is the staple food of Chinese. The storage period of rice is 3 years generally. During rice storage, however, microbial contamination is a common phenomenon, and it directly or indirectly affects the quality and safety of stored rice resulted from the production of mildew and rot as well as pigment secretion  was collected from the center and four corners of three layers of grain depot. Overall, three samples were analyzed. The three rice samples were respectively stored for 1–3 years and correspondingly marked as S1, S2, and S3. The rice was then stored in homogeneous bags at 4°C for the following experiment.-3 gradient, and each sample was processed in triplicate. Each diluent (150 μl) was added into wells of BIOLOG ECO microplates and the microplates were cultured at a constant temperature (25°C) for 10 days. During cultivation, the ECO microplates were read absorbance values at 590 nm wavelength each 24 h using a Multifunctional Enzyme Label Tester .The rice sample (25 g) was added into 225 ml of 0.85% stroke-physiological saline solution. The mixture was oscillated and shaken for a hour at 4°C, 200 rpm by Controlled Crystal Oscillator , and then stewed for 3 min. The three samples were separately diluted to a 10Metabolism of the substrate in particular well-results in Formosan production, producing chroma change in the tetrazolium dye . The capi is the absorbance value of each reaction well at 590 nm, R is the absorbance value of the control well, and n is the number of wells. (Ci–R) less than 0.06 of wells are calculated as zero (In formula (1), C as zero .(1) Shannon-Wiener diversity index (H′) i represents the ratio of the absorbance value in the ith (1 to 31) well to the total absorbance values of all wells.The P(2) Shannon evenness index (E) S represents the total number of utilized carbon sources (31 carbon sources), the number of wells that vary in color.(3) Simpson diversity index (D)The above indices reflected the metabolic functional diversity of microbial communities, which was similar to the measurements of diversity indices in general ecology.si value to perform principal component analysis (PCA). The calculation formula for the Rsi is:A BIOLOG ECO microplate contains 31 carbon sources, which are made up with six kinds of carbon sources, including carboxylic acids, carbohydrates, amino acids, polymers, miscellaneous, and amines/amides. In order to avoid the influence on statistical analysis results, we need to convert the absorbance data into Ri is the absorbance value of each reaction well at different incubation time at 590 nm excluding the control well, and R is the absorbance value of the control well.In formula (6), CIn this experiment, measuring the absorbance values of BIOLOG ECO microplates on day 8 were used for the PCA of microbial communities’ metabolic functional diversity.p-value was less than 0.05.All experiments were performed in triplicate, and the results were expressed as means ± standard deviations. Origin 8.5.1 was used to create pictures, and SIMCA 14.1 was used to conduct PCA. Results from one-way analysis of variance (ANOVA) were considered significant when the a  of the three rice microbial communities are shown in Figure 1. The results showed that the AWCDa of all stored rice samples exhibited an apparent lag phase in the first day. Then the average absorbance of all three samples in microplates increased significantly, demonstrating that the three microbial communities were capable of metabolizing organic substrates in BIOLOG ECO microplates. The period selected for metabolic activity analysis was from day 1 to day 8, and the slopes of AWCDa curves within this period represented average metabolic rates of the microbial communities , and the order was S3 > S2 > S1, which suggested that the longer storage length of time was, the higher metabolic capability of rice microbial community was. The similar research and method was reported by Among the three samples, the metabolic rate of S3 was faster than S1 and S2. The AWCDTable 2. The AWCD of different types of carbon sources were classified and analyzed in the experiment.According to the biochemical properties of carbon sources, the 31 substrates in the BIOLOG ECO microplates were assigned into six categories, including carboxylic acids, carbohydrates, amino acids, polymers, miscellaneous, and amines/amides , the detFigure 2 showed the AWCD of carboxylic acids, carbohydrates, amino acids, polymers, miscellaneous, and amines/amides. The results indicated that the utilization of six types of carbon sources by microbes presented an increasing trend with the prolongation of incubation time. For carbohydrates, amino acids, polymers and amines/amides, there were no significant difference in the utilization among the three microbial communities. The utilization of carboxylic acids and miscellaneous by the three rice microbial communities, however, differed significantly (p < 0.05), and the utilization capability order was S3 > S2 > S1, thereby clarifying that the microbial community of rice increased the utilization of carboxylic acids and miscellaneous with the extendence of storage period.The For different microbial communities, the capability utilizations of six-type carbon sources were different. The AWCD of carbohydrates was the highest, and the lowest was amines/amides, thereby illustrating that carbohydrates were the carbon sources with the highest degree of metabolic utilization, and the lowest degree of metabolic utilization was amines/amides. The results were similar to the findings reported by previous researches, the utilization of carbohydrates was relatively higher than other substrates among the six types of carbon sources, whereas the lowest utilization substrates differed from microbial communities .In general, through BIOLOG ECO microplates, the metabolic rates of carbon sources were determined by calculating a single value (AWCD) at a single time point, which demonstrated and made a clear comparison of stored rice microbial communities.Table 1. Shannon diversity index (H′) is greatly influenced by species richness of communities  of the rice microbial communities had significant difference (p < 0.05). The Shannon diversity and evenness index of S1 was highest, followed by sample S3 and S2, indicating that the species richness and evenness of microorganisms in stored rice were relatively high in the early stage of storage. It tended to decrease first and then increased during the storage. Table 1, there was no significant difference in Simpson index (D), which manifested that the most common species of the three rice microbial communities were similar, such as fungus of Aspergillums, Penicillium and Mucor, as observed in previous research (Duncan’s multiple range test was separately used in these indices. research . FurtherPrincipal component analysis was used to evaluate the carbon source metabolization of microbial communities in a certain environment . Some reFigure 3, the multivariate vectors were transformed into two uncorrelated principal component vectors. PC1 presented 43.1% of total variability, and PC2 described 20.42%. For the rice microbial communities, the point position of S1, S2, and S3 were certainly different. As As shown in Table 2 showed the loading scores of 31 carbon sources in the first two principal components. The higher loading scores were, the larger effects of carbon source on the principal components were. The sample distribution in the PC axis was relevant to carbon source substrates capability utilization of stored rice microbes (Table 2, it could be seen that there were 17 kinds of carbon sources had mainly impacts on the PC1. The 17 kinds of carbon sources included five kinds of carbohydrates, four kinds of amino acid, three kinds of carboxylic acid, two kinds of miscellaneous, polymers and amines/amides. These results indicated that the carbon sources with impacts on the PC1 were mainly carbohydrates, amino acid, and miscellaneous. Furthermore, there were mainly 14 types of carbon sources with effects on the PC2, including six kinds of carboxylic acids, three kinds of carbohydrates, two kinds of amino acids and polymers, a kind of miscellaneous. Thereby, the carboxylic acids made the major effects on the PC2. From the results of carbon sources analysis above, it was obvious that the carbon sources utilized by rice microbes were carbohydrates, amino acid, miscellaneous, and carboxylic acids. The similar analysis method was reported by microbes . From thIn present study, the metabolic functions of microbial communities of rice with different storage years were investigated and analyzed by BIOLOG ECO microplates. With the extension of storage period, the utilization of rice microbial communities for carboxylic acids and miscellaneous increased. Among the six types of carbon sources, the utilization of carbohydrates was relatively higher than other substrates. The metabolic functional diversity indices showed that there were differences among the three rice microbial communities, whereas the most common species of them were similar. Furthermore, carbon sources utilization of the three microbial communities were certainly different, and the carbon sources mainly utilized by stored rice microbes were carbohydrates, amino acid, miscellaneous, and carboxylic acids.Therefore, to draw a conclusion, this study illustrated that BIOLOG ECO microplates were capable of preceding a quick and accurate determination of metabolic functions of rice microbial communities, which provided a novel method for safe prevention and control of stored rice. However, BIOLOG ECO microplates are unable to evaluate the microbial communities’ structure of stored rice. The evaluation of dominant species as well as species distribution and difference of three stored rice microbial communities need to be studied in the next work.ZG: conceived and designed the experiments, performed the experiments, data analyses and wrote the manuscript, and contributed the experimental materials. ZG, HD, and YG: revised the manuscript, played an important role in interpreting the results, and helped perform the analysis with constructive discussions. WQ: approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Three different lateral sizes of FLG  were obtained from graphite  by a green, facile, low-cost, and scalable jet cavitation process. The corresponding characterizations, such as Raman spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), and transmission electron microscopy (TEM), verified the successful preparation of graphene flakes. Based on the results of four-point probe measurements, FLG20 demonstrated the lowest sheet resistance value of ~0.021 Ω/■. The optimized ECAs’ composition was a 60% solid content of FLG20 with the addition 2 wt.% of Al2O3. The sheet resistance value was as low as 51.8 Ω/■, which was a reduction of 73% compared to that of pristine FLG/polymer. These results indicate that this method not only paves the way for the cheaper and safer production of graphene, but also holds great potential for applications in energy-related technologies.In this study, we designed a three-dimensional structure of electrically conductive adhesives (ECAs) by adding three different kinds of nano filler, including BN, TiO Electronic products can be seen everywhere in daily life, since the new generation of electronic products emphasizes the needs of personalization and portability ,2. Amongt weight ,23,24. Tt weight ,26,27,28There are a lot of approaches for preparing graphene-based materials, such as chemical reduction , pyrolyt2 composite networks as flexible supercapacitor electrodes, lowering the electrical conductivity of the graphene/MnO2 composite due to the increase of MnO2 with its low electronic conductivity of 10−5~10−6 S/cm [−2 Ω-cm [−4 Ω cm [−7 to 1.02 × 10−3 S/m [2O3), titanium dioxide (TiO2), and hexagonal boron nitride (BN) in a graphene/polymer composite. Alumina is abundant in the world and it is low cost, exhibiting characteristics of anti-oxidation, corrosion resistance, and chemical and thermal stability. It not only prevents carbon from being oxidized in the air, but also has a high stability when combined with materials and polymers [The dispersion of FLGs in a polymer matrix is another important issue. In order to avoid the aggregation of FLGs in a polymer, nano-sized fillers are required to fill spaces between FLGs and the polymer matrix. He et al.  investig0−6 S/cm . Pu et a[−2 Ω-cm . Peng et[−4 Ω cm . Ghaleb 10−3 S/m . The abopolymers . Titaniupolymers . In termpolymers ,40.2O3, TiO2, and BN particles into the matrix resin to prepare ECAs in order to prevent FLG stacking. The corresponding characterizations, such as Raman spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), and transmission electron microscopy (TEM), as four-point probe measurements of FLG and FLG/polymer and FLG/fillers/polymer composites, were carried out in this research.In our study, we firstly synthesized three different sizes of FLGs by a low-temperature ultra-high pressure continuous homemade flow cell disrupter. Secondly, we incorporated Al®, d50 = 3 μm, namely G3), 8 μm graphite ), and KS-44 ), were dispersed in 500 mL of deionized (DI) water by sonication for 15 min (about solid content 5 wt.%). After dispersing them sufficiently, the solution was transferred into the tank of the low-temperature ultra-high pressure continuous homemade flow cell disrupter (LTHPD). As part of the work in designing the LTHPD, the suspension was poured into the device and the process was operated three times at each pressure . Therefore, the process was conducted nine times in total. Then, the graphite was separated by cavitation effects with different pressures. The course operated in a circulation cooling water bath, which kept the temperature at 14–16 °C The whole exfoliated experiment was carried out in room conditions. The suspension of graphene was vacuum-filtered to obtain graphene cake and was transferred to an oven at a temperature of 40 °C for 24 h. Finally, the cake of FLG was milled into powder with a grinder.First, three different lateral sizes of graphite, including KS-6 . First, A-polymer and B-polymer were mixed at the mass ratio of 1:1. Then, ethyl acetate was added to the resin drop by drop over 30 min, with stirring. The graphene and filler samples were added to the resin with various mass ratios, such as 95:5, 90:10, and 85:15, and the total mass fraction was 50%. After stirring for 24 h, the graphene/filler composite was dispersed into the resin. The slurry was coated on the PET  and cured at 150 °C for 2 h in the oven. Finally, the FLG/nano filler content was increased to 55% and 60% in order to improve the ECAs’ efficacy.The ECAs were mainly composed of a resin matrix, FLG, and nano fillers  by Hitachi S-4100  and high-resolution transmission electron microscopy (HRTEM) JEOL-JEM2000FXII. The height profile of the as-synthesized few-layer graphene was measured by using atomic force microscopy . Raman spectra were measured by a micro Raman spectroscopy system , with a laser frequency of 532 nm as the excitation source. The electrical conductivities of the graphene adhesives were measured using a resistivity meter  with a four-point probe.The morphologies of graphite and FLG were observed by SEM . Figure We used atomic force microscopy (AFM) to characterize the thickness and surface morphology of as-synthesized FLGs. 002 and the d101 reflection of the carbon atoms, respectively. Additionally, after exfoliating, the intensities of the diffraction peaks present a slight decrease in the few-layered graphene. The average crystallite size of G3, G8, and G20 was about 200,168, and 240 Å, respectively. However, the few-layered graphene obtained by LTHPD exhibited a slight decrease in crystallite sizes because the process wrecked the crystallinity. In −1, respectively, for visible excitation. The G peak is due to the doubly degenerate zone center E2g mode, while the D peak is a breathing mode of κ-point phonons of A1g symmetry [D/IG) of the D peak to G peak of the G3, G8, and G20 was about 0.226, 0.198, and 0.11, respectively. However, for FLG3, FLG8, and FLG20 which were obtained by LTHPD, the ID/IG was 0.231, 0.206, and 0.14, separately. The ID/IG ratio increased because the stripping process created defects. The results of graphite and FLG for testing the four-point probe are shown in symmetry . The intThe morphology of FLG20 were observed by TEM and HRTEM. 2, and Al2O3, respectively. The morphology of BN was two-dimensional and its lateral size was in the range of 30–50 nm, as shown in 2 and Al2O3 are granular materials, and had particle sizes of 20–30 and 10–15 nm, respectively  to prevent graphene stacking and generate a continuous conductive pathway. The results indicated that the solid content was 60% and the best condition was adding 2 wt.% Al2O3, for which the sheet resistance value was 51.8 Ω/■. The electrically conductive resin remained nearly the same after one thousand bending cycles. These results indicate that the formulated FLG/Al2O3/polymer composite adhesives have a great potential in conductive adhesives for flexible display applications.We succeeded in delaminating artificial graphite and natural graphite by jet cavitation to prepare few-layered graphene . The structure and morphology of few-layered graphene had a two-dimensional structure and few-layered graphene was composed of thinner sheets and smaller sizes than graphite. We used G20 and FLG20 as the foremost material to apply to ECAs with different nano fillers (BN, TiO


Comprehensive analysis of human TDP-43 knockout cells revealed that TDP-43 regulates hundreds of transcripts and is critical for maintaining the homeostasis of multiple subcellular organelles. knockout (KO) cells and subjected them to parallel cell biological and transcriptomic analyses. These efforts yielded three important discoveries. First, complete loss of TDP-43 resulted in widespread morphological defects related to multiple organelles, including Golgi, endosomes, lysosomes, mitochondria, and the nuclear envelope. Second, we identified a new role for TDP-43 in controlling mRNA splicing of Nup188 (nuclear pore protein). Third, analysis of multiple amyotrophic lateral sclerosis causing TDP-43 mutations revealed a broad ability to support splicing of TDP-43 target genes. However, as some TDP-43 disease-causing mutants failed to fully support the regulation of specific target transcripts, our results raise the possibility of mutation-specific loss-of-function contributions to disease pathology.TDP-43 is an RNA-binding protein that forms cytoplasmic aggregates in multiple neurodegenerative diseases. Although the loss of normal TDP-43 functions likely contributes to disease pathogenesis, the cell biological consequences of human TDP-43 depletion are not well understood. We, therefore, generated human TDP-43 Mutatiois (ALS)  that is is (ALS) . TDP-43 is (ALS) . Cytoplais (ALS) .The strong genetic and pathological links between TDP-43 and neurodegenerative disease have stimulated intense interest in elucidating the relationships between its normal and pathological functions . AlthougEfforts to define TDP-43 function in mice through knockout (KO) strategies revealed that TDP-43 is absolutely required for embryonic development and viability . Even TDAs a comprehensive understanding of TDP-43 functions is critical for understanding normal human cell biology as well as for deciphering disease mechanisms, we have developed the first human TDP-43 KO cells and used them to perform comprehensive cell biological and transcriptomic analyses of the consequences of TDP-43 depletion. The results of these experiments revealed that TDP-43 is required for the homeostasis of multiple subcellular organelles. Transcriptomic analysis of TDP-43 KO cells both confirmed the impact of TDP-43 on multiple known targets but also revealed new candidates. Given recent interest in the contributions of nuclear transport defects to neurodegenerative diseases associated with TDP-43 pathology , we highCollectively, this study takes advantage of a newly generated TDP-43 KO human cell line to shed new light on cellular functions of TDP-43 and establishes such cells as a platform for investigating the functionality of disease-causing TDP-43 mutations.Efforts to define TDP-43 function through genetic depletion approaches have previously been implemented in mice as well as in human cells in culture . AlthougTDP-43 sequence at the Cas9 cut site in all copies of the TARDBP gene and the presence of small insertions and deletions that result in frameshifts in our TDP-43 KO cell line , either alone or in combination, we analyzed polyclonal cell populations by immunoblotting and observed significant depletion of TDP-43 protein levels . Clonal ell line . In addiell line . To geneell line . It is sell line , the traPOLDIP3 mRNA splicing was further confirmed by RT-PCR analysis of RNA isolated from either WT, TDP-43 KO, or TDP-43–rescued KO cells  give rise to FTD with TDP-43 pathology  and the Lysosome impairment arising from loss of TDP-43 is of potential interest in the context of neurodegenerative diseases such as ALS-FTD, where human genetics and pathology have established roles for both TDP-43 and lysosomes as contributors to the disease process. This is particularly relevant in the case of GRN-linked FTD where GRN haploinsufficiency results in neurodegeneration that is accompanied by TDP-43 cytoplasmic aggregation . Our obsIn addition to its splicing functions within the nucleus, TDP-43 is also found in cytoplasmic stress granules and has been implicated in their formation . We obsePOLDIP3 splicing in such cells , whose levels fell by ∼6 fold in the absence of TDP-43 . This is, therefore, predicted to result in nonsense-mediated decay of this transcript and is consistent with the low levels of this Nup188 transcript in the TDP-43 KO cells.Amongst the list of genes whose expression changed dramatically in a TDP-43–dependent manner was nucleoporin 188  expression in TDP-43 KO cells (Table S1). CI-M6PR is a well-characterized sorting receptor for the trans-Golgi network to endosome trafficking of multiple lysosomal hydrolases , we notdrolases . Two facdrolases . In contdrolases  even thodrolases . This maf TDP-43 .POLDIP3, PFKP  splicing by TDP-43 , and NUot occur . For exa POLDIP3 . Consisty TDP-43 , we obse and were grown in DMEM, 10% FBS, and 1% penicillin/streptomycin supplement . 293FT cells (Thermo Fisher Scientific) were grown on dishes coated with 0.1 mg/ml poly-d-lysine (Sigma-Aldrich) in the media described above. For stress granule induction, the cells were incubated with 0.5 mM sodium arsenite  before fixation.Use of CRISPR/Cas9 genome editing to generate KO cell lines was previously described . The guiSummary of oligonucleotide primers used in this study.Table S3 TDP-43 cDNA sequence was PCR-amplified from a previously described TDP-43–tdTomato expression vector  were fixed with 4% paraformaldehyde (Electron Microscopy Sciences), washed with PBS, and permeabilized with either 0.1% TX100 or 0.1% saponin in a 3% BSA/PBS buffer. Subsequent primary and secondary antibody incubations were carried out in the permeabilization buffer. Coverslips were mounted in ProLong Gold reagent supplemented with DAPI (Invitrogen). Images were acquired with either a Zeiss LSM 800 laser scanning confocal microscope with a 63× Plan Apo (NA = 1.4) oil immersion objective and Zeiss Efficient Navigation software or an UltraVIEW VoX spinning disk confocal microscope (PerkinElmer) that consisted of a Nikon Ti-E Eclipse inverted microscope equipped with 60× CFI PlanApo VC, NA 1.4, oil immersion objective and a CSU-X1 (Yokogawa) scan head that was driven by Volocity (PerkinElmer) software. For live cell imaging analysis of microtubules and F-actin, the cells were labeled with SiR-tubulin and SiR-actin (Cytoskeleton Inc.) as per the manufacturer’s instructions. An automated analysis pipeline was developed with Cell Profiler  for the g. Immunoblotting was performed by standard methods using 4–15% Mini-PROTEAN TGX precast polyacrylamide gels and nitrocellulose membranes (Bio-Rad). Ponceau S staining of membranes was routinely used to assess equal sample loading and transfer efficiency. Blocking was performed in 5% milk in TBS with 0.1% Tween 20. Primary antibody incubations were performed in 5% BSA in TBS with 0.1% Tween 20. Signals were detected with HRP-conjugated secondary antibodies  and either Super Signal West Pico or Femto chemiluminescent detection reagents (Thermo Fisher Scientific) on a VersaDoc imaging system (Bio-Rad). ImageJ was used to measure band intensities. Antibodies used in this study are listed in Table S4.Cells were lysed in TBS supplemented with 1% Triton X-100 and protease/phosphatase inhibitor cocktails (Roche Diagnostics), the insoluble material was cleared by centrifugation for 10 min at 20,000Summary of antibodies used in this study.Table S4 Nup188 exon were as follows: 5′-GGAGCAGTAGAGAACTGTGG-3′ and 5′-GCTGATTCTTAAACCCAGTTC-3′. The primers to amplify POLDIP3 splice variant were: 5′-GCTTAATGCCAGACCGGGAGTTG-3′ and 5′-TCATCTTCATCCAGGTCATATAAATT-3′. The resulting PCR reactions were resolved on 2.5% agarose gels supplemented with SYBR green (Invitrogen) and visualized on a VersaDoc imaging system (Bio-Rad).Total RNA was extracted from WT, TDP-43 KO, and rescue cell lines using the RNeasy kit (QIAGEN). 400 ng of total RNA was reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad). Cryptic and alternatively spliced exons were PCR-amplified from the resulting cDNA with Q5 polymerase (NEB) and either Nup188 or POLDIP3 primers (IDT). The primers used to amplify the cryptic Six samples  were sequenced on Illumina’s HiSeq 2500 using 2 × 70-bp paired-end reads, generating 104.4–128.2 million reads (52.2–64.1 million pairs) per sample. The sequencing reads were first trimmed for quality, trimming to the last base with quality ≥20 and dropping any read pair where either of the trimmed reads was shorter than 45 bp. The trimmed reads were aligned to the hg19 reference genome using TopHat2 , an RNA-The Cufflinks and Cuffmerge software  was usedGSE136366.Transcript abundance calculation and differential gene expression analysis, using the assembled known plus novel transcripts, was performed using Cuffdiff . Heat ma