The preparation of mesoporous carbonaceous materials with particularly adjustable morphology is currently a hot area of research in mesoporous materials. Herein, a novel approach is reported for the construction of N-doped multicavity mesoporous carbon nanoparticles (NMMCNs) based on the “emulsion swelling–acid curing mechanism” using a nanoemulsion assembly method under a high-speed shearing force. Intriguingly, this approach adopted a novel acid (HCl) curing procedure. Impressively, the morphology evolution from an internal multicavity to a single cavity and then to a non-cavity interior structure could be accomplished by simply varying the synthesis parameters. Additionally, this synthesis approach ingeniously overcame the following problems: (i) technically, the employment of high temperatures and high pressures in traditional hydrothermal reaction curing environments is avoided; (ii) this approach removes the requirement for silicon coating, which provides a limited pyrolysis condition, to obtain a multi-chamber structure. Resveratrol (Res) is an insoluble natural medicine and was successfully loaded into NMMCNs, thereby the Res–NMMCNs delivery system was constructed. Importantly, the Res–NMMCNs delivery system could still retain the antitumor and antioxidant activity of Res in vitro. For many years, mesoporous carbon nanoparticles (MCNs) have played an irreplaceable role in catalysis, energy storage conversion, adsorption separation, drug storage and release and other fields due to their large specific surface area, large pore volume, unique construction and other fantabulous physicochemical properties ,5,6,7,8.Up to now, there have been various ways to synthesize mesoporous materials: the hard-templating method ,10, the Throughout the development trends of the synthesis of MCNs, the excellent characteristics of mesoporous carbon materials have evolved from an inherent single morphology and small mesoporous aperture to the synergistic effect of a unique morphology, small particle size, large pore size and rigid framework; thus, gradually realizing the improved application performance of MCNs as catalyst carriers, adsorption separators, drug carrier materials, etc. Even though extensive research has been conducted on the preparation of MCNs with multi-morphology and various structures ,22,23, cHerein, this research utilized a high-speed shearing force to create an emulsion that self-assembled with polybenzoxazine resin in an aqueous solution to prepare NMMCNs with cherimoya-shaped rough surfaces and changeable internal cavities under acid curing conditions. Moreover, the “emulsion swelling–acid curing mechanism” was also proposed to explain their growth mechanism. This formation approach not only avoided the high-temperature curing process and the employment of autoclave equipment, but also further supplemented the swelling mechanism of the emulsion strategy. Benzoxazines are an interesting class of monomers, which are produced by the Mannich condensation reaction of organic primary amine, phenol derivatives and formaldehyde ,25,26,27The F127 (PEO–PPO–PEO) was purchased from BASF. The diethyl phthalate , 3-aminophenol , formaldehyde , ethanol, ethylenediamine (EDA), hydrochloric acid  and Resveratrol  were purchased from the Guangzhou Chemical Company . The 2,2-Diphenyl-1-picrylhydrazyl (DPPH•), fluorescein isothiocyanate (FITC), dimethyl sulfoxide (DMSO) and 3--2, 5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Sigma . Deionized water was used for all experimental water. All chemicals were used in their original state without further purification.2 environment, the PBZ resin was carbonized at 350 °C for 3 h and then at 600 °C for another 3 h at a heating rate of 2 °C/min. The obtained black powder samples were labeled as NMMCNs-x, with x representing the sample number of the different synthesis parameters. The specific experimental parameters are presented in In the typical synthesis strategy, 0.4 g of F127 was dissolved in a mixture solution containing 5 mL of ethanol and 35 mL of deionized water under magnetic stirring at 40 °C. Subsequently, DEP was then dissolved in the above solution and agitated for 30 min at 600 rpm to create an oil-in-water primary emulsion. After that, the temperature was quickly lowered to 20 °C and a homogenizer was employed to prepare the secondary emulsion under a high-speed shearing force of 16,000 rpm. After 10 min, 0.25 mL of formaldehyde solution, 0.1 mL of EDA and 0.3 g of 3-AP were added and mixed for another 60 min. The reaction temperature was then elevated to 50 °C for 22 h and the pH value of the reaction solution was adjusted to around 5 with 2 M of HCl solution before continuing the reaction for another 10 h. Finally, the reaction solution was centrifuged and the resulting colloidal precipitate was washed three times with deionized water before being dried fully at 50 °C to yield polybenzoxazine (PBZ) resin. Under the protection of an N4 cells in each well and incubated in a constant-temperature carbon dioxide incubator at 37 °C and 5% CO2. When the cells grew to 80%, the materials that were dispersed in the complete medium were added according to the designed concentration gradient of 0, 6.25, 12.5, 25, 50, 100 or 200 μg/mL. The medium was withdrawn after 24 h of incubation and the cells were washed three times with fresh PBS. To generate formazan crystals, 20 μL of 5 mg/mL MTT solution was supplemented into each well and incubated with 3T3 cells at 37 °C for 4 h. The obtained formazan was then dissolved in 200 μL of DMSO and the cells were shaken slowly for 10 min. The microplate reader  was employed to measure the absorbance value at 490 nm [The cytotoxicity of NMMCNs-2 was evaluated by an MTT assay using 3T3 cells as cell line models. The cells were seeded in 96-well plates with a density of 1 × 10t 490 nm . The celIn order to test the blood biocompatibility of NMMCNS-2, blood samples were collected from the abdominal aorta of mice. Red blood cells (RBC) were collected by centrifugation at 4 °C and 3500 rpm for 10 min and were then washed with cold PBS three times until the supernatant was clarified. Then, the RBCs were prepared in a 2% red blood cell suspension. Next, 1 mL of PBS containing different NMMCNs-2 material concentrations  was combined with 1 mL of 2% red blood cell suspension and incubated at 37 °C for 3 h. Meanwhile, 1 mL of PBS was added into 1 mL of 2% red blood cell suspension as the negative control and 1 mL of deionized water was mixed with 1 mL of 2% red blood cell suspension as the positive control. After incubation at 37 °C for 3 h, the samples were centrifuged at 4 °C and 3500 rpm for 10 min. The absorbance value of the supernatant was determined at a wavelength of 541 nm ,29 by UVFITC-grafted NMMCNs-2 was obtained by grafting FITC onto the surface of NMMCNs-2 according to the literature method. Then, 3T3 cells in the logarithmic growth phase were harvested for the experiments. They were cultured in a complete medium after digestion with 0.25% trypsin (HyClone). After complete digestion, the isolated cell suspension was centrifuged and reconstituted with an equal fresh medium containing FITC-grafted NMMCNs-2 (100 μg/mL). After incubating for 1, 2, 4 and 12 h, the cells were fixed in 4% paraformaldehyde for 10 min and washed three times with a serum-free medium. Then, the cells were washed three times with a PBS buffer and further stained with 10 μg/mL of Hochest 33,258 for 10 min, followed by imaging with a confocal microscope .Res was chosen as the model drug and Res was loaded into NMMCNs-2 via the solvent evaporation method to construct the Res-loaded delivery system. Then, 40 mg of NMMCNs-2 was added to 10 mL of a Res–methanol mixed solution with a Res concentration of 4 mg/mL. To guarantee the best drug loading efficiency, the resultant mixture was swirled at an adequate speed for 30 h in a closed and dark environment at room temperature. Finally, the methanol solvent was allowed to evaporate for nearly 20 h at 50 °C, which fully obliterated the methanol solvent. The Res-loaded sample was denoted as Res–NMMCNs-2-(1:2). Notably, 1:2 represents the mass ratio of Res to NMMCNs-2. The Res-loaded delivery systems of Res–NMMCNs-2-(2:1), Res–NMMCNs-2-(1:1) and Res–NMMCNs-2-(2:3) were constructed by changing the dosage of NMMCNs-2. These Res-loaded samples were collectively termed Res–NMMCNs-2.0 refers to the mass of Res in Res–NMMCNs-2 and W refers to the total mass of Res–NMMCNs-2.The drug loading was determined by extracting 10 mg of Res–NMMCNs-2 with 25 mL of methanol under ultrasonic conditions. After ultrasonic treatment for 15 min and stirring for 18 h, the Res was completely released from the mesoporous channel of NMMCNs-2. Then, the mixture was filtered with a 0.22 μm filter to obtain a supernatant. Subsequently, the drug content was measured with UV-vis at a wavelength of 302 nm. The drug loading (DL) was then calculated according to the following formula: The release experiments of Res from the Res–NMMCNs-2 material were performed under four different conditions, which simulated gastric juice (HCl solution of pH 1.2), intestinal juice (PBS of pH 5.8), saliva (PBS of pH 6.8) and body fluid (PBS of pH 7.4). Firstly, Res–NMMCNs samples that were equivalent to 10 mg of Res were dispersed in buffers of different pH values and placed in dialysis bags (molecular weight cut-off of 3500). Then, the dialysis bags were immersed in 150 mL of different dissolved media and oscillated at 120 rpm. To simulate in vivo release characteristics, 5 mL of release medium was extracted at an appropriate sampling time, followed by the addition of the same amount of fresh medium. After filtering and appropriately diluting the extract, the absorbance was determined by UV-vis spectrophotometry at 302 nm to detect the concentration of Res in the released solution. For comparison, a control experiment was carried out with pure Res instead of Res–NMMCNs-2 under the same conditions. Furthermore, the release results were fitted to five mathematical kinetic models: zero order, first order, Higuchi model, Weibull model and Ritger–Peppas model.The antioxidant capacity of Res–NMMCNs-2 is based on its ability to scavenge DPPH•. So, 2 mL of different concentrations of Res–NMMCNs-2 (dissolved in ethanol) were added to 2 mL of DPPH• ethanol solution (0.1 mg/mL). Subsequently, the mixed solution was then thoroughly mixed and incubated at room temperature in the dark for 1 h and the absorbance at 517 nm  was recoAn ethanol solution of NMMCNs-2 with the same concentration was employed as the comparative sample to determine antioxidant capacity. The experimental procedure was the same as above.The in vitro antitumor activity of Res–NMMCNs-2 was evaluated using mouse cancer cells (Hepa1-6) as the tumor cell models. The same experimental procedure was followed as described in 2 sorption isotherms were performed on a Micromeritics Tristar 3020 (USA) at −196 °C. The sample powders were degassed at 200 °C in a vacuum for 6 h before measurement. The specific surface areas were calculated by employing the Brunauer–Emmett–Teller (BET) method. Correspondingly, the pore size distribution (PSD) and pore volume were calculated from the PSD curves in the adsorption branches of the isotherms using the Barrett–Joyner–Halenda (BJH) method. The surface compositions of the NMMCNs obtained with different amounts of EDA were measured by X-ray photoelectron spectroscopy (XPS) and recorded with an ESCALAB 250 X-ray photoelectron spectrometer (USA). Wide-angle X-ray diffraction (XRD) patterns were collected on a PAN Analytical Inc. X’Pert Pro with Cu–Kα radiation (50 kV and 300 mA). Raman spectra was recorded with a microscopic Raman spectrometer . Fourier-transform infrared (FTIR) spectra were recorded on an infrared spectrometer VERTEX 70 . The thermogravimetric (TG) characterization was performed on an STA449C thermogravimetric analyzer in the range of 25–800 °C under N2 protection at 20 °C min−1. The differential scanning calorimetry (DSC) was conducted on a DSC-4000  under the protection of N2 from room temperature to 500 °C.Transmission electron microscope (TEM) measurements were conducted on an FEI-Talos F200X , which was operated at 10 kV. Scanning electron microscopy (SEM) images were collected on a Zeiss Sigma 300  field emission electron microscope. Dynamic light scattering (DLS) was conducted at 25 °C on a ZS Nano S Instrument . The N2O secondary emulsion with a smaller and uniform particle size; (ii) the self-assembly of DEP–F127–PBZs SSUs; and (iii) the introduction of the acid curing procedure to enhance and strengthen the degree of cross-linking between the DEP–F127–PBZs SSUs.Innovatively, MCNs with distinct internal cavities were successfully produced by the self-assembly of benzoxazine resin with an emulsion prepared by a high-speed shearing force, which was based on the “emulsion swelling–acid curing mechanism”. In the synthetic system, a series of NMMCNs-x with cherimoya-shaped rough surfaces and adjustable internal cavities were obtained by employing the amphiphilic surfactant F127 as a template, 3-AP and formaldehyde as carbon sources and DEP as the swelling agent under EDA alkaline conditions. Notably, EDA was not only the primary amine compound involved in the construction of the benzoxazine resin, but it was also a basic catalyst and nitrogen source that could be used to realize the nitrogen doping of MCNs. The successful synthesis of NMMCNs-2 with adjustable internal cavities mainly underwent three essential processes: (i) the preparation of the DEP–F127–H1) that the obtained NMMCNs-1 exhibited a particle size of about 142 nm with a cherimoya-shaped rough surface. The partially magnified SEM image  provides a clearer view of the surface of the MCNs with many prominences resembling the surface protrusions of cherimoya fruit , which could have been derived from the accumulation of small F127–DEP–PBZs SSUs. As illustrated in 2, there was a cavity of about 35 nm inside the NMMCNs-1. With the amount of DEP continuously increasing, the obtained NMMCNs-2, NMMCNs-3 and NMMCNs-4 (1–d1) all exhibited the same surface structural characteristics  as NMMCNs-1. Surprisingly, the TEM image of NMMCNs-2 (2) depicts a multicavity structure with one to four chambers (about 60–72 nm) of different sizes in the case of 0.2 mL of DEP, which can also be distinctly observed in the enlarged TEM image . The mesopores were obviously visible on the surface of NMMCNs-2 with a particle size of 215 nm. As the quantity of the emulsion was brought to 0.3 and 0.4 mL, it can be observed in 2,d2 that the internal cavity enlarged to 78 nm and 87 nm, respectively, and that the particle size increased from 250 nm to 310 nm. Obviously, for the NMMCNs-1 to NMMCNs-4 samples, the cavity size and particle diameter gradually enlarged with the increase in the dosage of DEP and the internal structure changed from single cavity to multicavity, which also indicates that DEP was the “template of maternal emulsion droplet” that was formed by the cavity structure. The DLS characterization revealed that the average particle diameter of samples NMMCNs-1 to NMMCNs-4 were 192, 280, 292 and 331 nm, respectively. The results of the DLS data (3–d3) were slightly larger than the particle size of the SEM and TEM images, which could have been aroused by the hydration layer on the surface of the particle [In order to evaluate the effect of the oil phase (DEP) on the internal chamber structure of the NMMCNs, a series of NMMCNs were prepared by changing the amount of DEP. When initially trying to employ 0.1 mL of DEP as the nanoemulsion, it could be seen from the SEM image a1 that tNMMCNs-4 b1–d1 allNMMCNs-2 b2 depictEM image b2, insetDLS data a3–d3 werTo further demonstrate the distinction between the internal structures of the particles that was caused by the difference between the amounts of the DEP emulsion droplets, we measured the particle size of both the primary emulsion and the secondary emulsion (prepared by a high-speed shearing force) through a DLS experiment. As displayed in 2 adsorption–desorption experiments. The N2 sorption isotherms, shown in 0 < 0.8, which was consistent with the characteristics of mesoporous structures. Moreover, we could observe an H3 hysteresis loop at the relative pressure range of 0.85–1.0, confirming the existence of a hollow cavity inside the NMMCNs. Additionally, there were many types of mesopores , as revealed by the PSD curves (2 adsorption isotherm and pore size distribution (PSD) curve with DEP = 0  [− as an intermediary, the structure of the PBZs resin was further strengthened and stabilized by the interaction of the Coulomb force (I+X−S+ mechanism) [Taking PBZs-2 as an example, TG analysis was employed to explore the pyrolysis behavior of PBZs-2 with cherimoya-shaped rough surfaces. The TG analysis a curve oectively ,33. The ectively ,35. Addiibration ,35,36. Tn of N–H ,36. The n of N–H ,38. In tchanism) , the reachanism) .2–c2). This transformation can be explained by the fact that as the E/W ratio increased, the polarity of the solution decreased, which led to a higher solubility of DEP in water and offset the “emulsion swelling effect” that was produced by the DEP entering the PEO section of F127. Consequently, the contact area of the PBZs resin and DEP decreased, thereby making DEP unable to completely participate in the copolymerization of PBZs resin as an emulsion template. These results could be further verified by the N2 adsorption isotherm and the corresponding PSD curve (3–c3 and inset), in which the increase in the E/W ratio resulted in the decrease in the dual-mesopore size from 5.0 and 9.2 nm to 4.6 and 8.6 nm and also proved the reduction in the swelling effect of the emulsion. Hence, there were no chambers in the internal structure of the prepared NMMCNs-5 and NMMCNs-6. The morphology transformation could be attributed to the weakening of “the emulsion swelling effect”, which realized the internal mesoscopic structure transformation from multicavity to non-cavity mesoporous carbon nanospheres. Consequently, it can be concluded that an appropriate E/W ratio was an indispensable condition for the synthesis of particles with structural changes in the internal cavity.A set of NMMCNs samples was synthesized by varying the quantitative relationship of ethanol and water (E/W) within the reaction system to explore the influence of the polarity of the solution on the morphology of the synthesized samples. As described in 2= 0.99 was obtained. Therefore, the diameter of the NMMCNs could be predicted by the equation, making it possible to programmatically alter the particle diameter by adjusting the amount of EDA. DLS (3–c3) revealed the same results. More surprisingly, through careful observation, the SEM images (1–c1) display that adjusting the amount of EDA could not only control the particle size, but also exert a certain influence on the surface roughness of the particles. A high dose of EDA made the DEP–F127–PBZ SSUs assemble within a large range instead of accumulating together in the form of small particles; thus, this resulted in the transformation of the NMMCNs surfaces from rough to smooth. The TEM images (2–c2) illustrate that the amount of EDA also had a definite impact on the internal cavities of the NMMCNs and the thickness of the shells. As the amount of EDA reached 0.2 mL, the multicavity internal structures of the NMMCNs completely disappeared, the internal cavities changed from multicavity to a single cavity (110 nm) with the shell thickness of 175 nm. By increasing the EDA to 0.3 mL, NMMCNs-8 particles with a single cavity size of 195 nm and a shell thickness of 330 nm were obtained, which also proved the role of EDA as a basic catalyst. EDA was the key factor in realizing the adjustment of the morphology from a smaller multicavity to a larger hollow sphere with an increasing diameter. The nitrogen adsorption–desorption isotherms of all of the prepared NMMCNs-2, NMMCNs-7 and NMMCNs-8 (4–c4) exhibited a type-IV curve with a definite capillary condensation in the P/P0 range of 0.4–0.8. The PSD curves  that were calculated using the Barrett–Joyner–Halenda (BJH) method revealed two mesopore sizes: 5.0 and 9.2 nm. It is noteworthy that pores of 25 nm and 40 nm were absent from the PSD curves for NMMCNs-7 and NMMCNs-8, which also proves that the rough grooves on the particle surfaces were gradually disappearing. This was consistent with the results of the SEM and TEM observations. With the increase in the EDA amount, the surfaces of NMMCNs-7 and NMMCNs-8 became slightly smoother. The SBET values of NMMCNs-2, NMMCNs-7 and NMMCNs-8 were 374.0, 385.1 and 407.1 m2 g−1, respectively. The corresponding pore volumes were 0.42, 0.23 and 0.23 cm3 g−1, respectively.Serving as a catalyst to synthesize benzoxazine resin and a precursor for N-doping, EDA played an irreplaceable role in this synthetic system. In order to demonstrate the effect of EDA, NMMCNs-7 and NMMCNs-8 were synthesized by increasing the content of EDA. EDA. DLS a3–c3 revM images a1–c1 disM images a2–c2 illNMMCNs-8 a4–c4 exhTo further assess the N-doping degree of MCNs when using EDA as a nitrogen source, an element mapping analysis and XPS analysis were used to explore the elementary distribution of samples with different EDA contents. 2 bonds of the graphite crystallite [D/IG) provided qualitative information about the degree of graphitization in the carbonaceous materials. The larger the ID/IG ratio, the larger the structural defects in the carbonaceous material. As illustrated in D/IG values of NMMCNs-2, NMMCNs-7 and NMMCNs-8 were 0.87, 0.90 and 0.94, respectively. The increase in ID/IG ratio indicates higher amounts of N-doped content and the introduction of a higher disorder level. It is worth noting that the N-doped and oxygen-containing functional groups could improve the surface hydrophilicity and wettability of the material, thus promoting its adsorption performance. Furthermore, the wettability of the as-prepared samples  were evaluated by water contact angle measurements using JYSP-180 equipment. The results are displayed in Additionally, the N-doping of the sample also affected the degree of the graphitization of the material. As shown in stallite ,47. The 2O primary emulsion system under stirring. Significantly, the high-speed shearing force  of the homogenizer was employed to shear the primary emulsion into smaller emulsion droplets, thereby eventually constructing the secondary emulsion, in which the effect of Ostwald ripening on the merging of emulsion droplets was reduced by rapid temperature cooling [0S0 mechanism under the action of hydrogen bond, then induce the gathering and pre-polymerization of PBZs resin on the surface of nanoemulsion droplets, thus forming DEP–F127–PBZs SSUs. Subsequently, the prepared DEP–F127–PBZ SSUs aggregated into the precursors of multi-chamber structures with different sizes through the action of surface-free energy. It is worth noting that the emulsion played a significant role as the prerequisite for the formation of the internal cavities and that the DEP–F127–PBZ SSUs were the basis for the configuration of the multicavity structures. Then, the acid curing process was carried out by pH adjustment using HCl. The addition of HCl reduced the pH value of the system and increased the hydrophobicity of the PPO segments, resulting in the swelling of the nanoemulsion. Meanwhile, the PEO segments also played a stabilizing role in this process [+–Cl−–PBZ+. The participation of Cl− as an intermediary could stabilize the structure of the DEP–F127–PBZ SSUs through the interaction of the Coulombic force (I+X−S+ mechanism), meaning that the hydrogen bond forces (I0S0 mechanism) and Coulombic forces (I+X−S+ mechanism) could coexist in the system and continue to strengthen the degree of cross-linking between the polymers, thus making the polymer even more stable. The subsequent process of high-temperature carbonization could still maintain its original shape. By increasing the amount of DEP, the interior structures of NMMCNs were modified from a single cavity to multicavity structures and the diameter of the cavities were gradually enlarged, which was the result of the further swelling of the DEP oil phase. Increasing the E/W ratio weakened the swelling effect of the emulsion droplets that were acting as the cavity template, so it was easy to realize the transformation from a multicavity to a non-cavity structure by controlling the E/W ratio. Moreover, the dosage of EDA also played a vital role in determining the polymerization and fusion rate of the DEP–F127–PBZ SSUs, further altering the mesostructures and particle sizes of the prepared NMMCNs. A large EDA content promoted the polymerization rate of the DEP–F127–PBZ SSUs, which in turn affected its stacking mode. In this situation, the DEP–F127–PBZ SSUs were assembled in an extensive range instead of stacking together in the form of small particles, thus forming larger and smoother NMMCNs.Based on the above characterization results of NMMCNs with cherimoya-shaped rough surfaces and adjustable internal chambers, we propose an “emulsion swelling–acid curing mechanism” to explain its growth mechanism. As illustrated in  cooling . Crucial cooling . Consequ process . The PBZ−1), demonstrating the low cytotoxicity of NMMCNs-2. In addition, cell uptake experiments were performed on 3T3 cells and FITC was grafted onto NMMCNs-2 as a fluorescent agent. The results of the fluorescence spectroscopy  is a natural polyphenol compound that is known for its antioxidant, anti-inflammatory and anticancer effects . HoweverThe DPPH• scavenging capacity of the Res–NMMCNs-2 delivery system was measured c. The re2) and equations are presented in In order to investigate the dissolution and release of pure drugs through Res and the Res–NMMCNs-2 delivery system, in vitro drug release experiments were conducted in buffer solutions of pH 1.2, 5.8, 6.8 and 7.4 to simulate human gastric juice, tumor cell internal environments, intestinal juice and body fluids, respectively. The release profiles  manifestIn summary, we presented a novel synthesis strategy based on the “emulsion swelling–acid curing mechanism” to prepare NMMCNs. By using 3-AP, formaldehyde and EDA as the precursors of PBZs resin, a series of NMMCNs with cherimoya-shaped rough surfaces and adjustable cavities was synthesized by the polymerization of the PBZs resin with a secondary emulsion prepared under high-speed shearing force conditions. Impressively, the directional transformation of the internal structures and particle sizes of the NMMCNs could be accomplished by adjusting the synthesis parameters. For example, by adjusting the content of DEP, the multicavity size of NMMCNs could be tuned; varying the E/W ratio could accomplish the transformation of the internal multicavity structures to non-cavity; employing different doses of EDA achieved the conversion of multicavity to single cavity structures. Interestingly, the EDA dosage gradient experiment realized the prediction of the linear relationship between particle size and EDA amount, making it possible to programmatically alter the particle diameter. Furthermore, the traditional heat curing step was abandoned and a novel acid curing procedure was adopted to achieve the perfect morphology preservation of polymer colloidal particles during the subsequent high-temperature carbonization process. As a nano-delivery system, NMMCNs-2 exhibited low cytotoxicity, no hemolysis and could be completely taken up by 3T3 cells, indicating an outstanding biocompatibility. When loaded with the drug Res, the Res–NMMCNs-2 delivery system could exert the antitumor activity and antioxidant activity of pure Res. Moreover, the Res–NMMCNs-2 delivery system exhibited pH responsibility and could improve the release behavior of pure Res. Based on the calculations of five drug release models, the drug release rule of the NMMCNS-2 delivery system and pure Res under different pH conditions were most consistent with the Weibull model, including the release mechanism comprising a fast release stage and a slow release stage.


During the etching process, ions adhere to the graphene layer resulting in unintentional doping. We demonstrate that washing a CVD graphene layer in an aqueous ammonia flow bath after etching copper, removes the majority of the unintentional dopants. FETs fabricated from graphene after washing in DI-water display a large scattering in Dirac bias with lowered mobility. In contrast, FETs from graphene that is washed in ammonia furnish better performance with high geometrically normalized mobility exceeding 2.4 × 104 cm2 V−1 s−1, balanced transport and a Dirac voltage near zero. We attribute the improved FET behavior to effective removal of the ions with a typical density of 4 × 1012 cm−2 from the graphene layer.Doping-free transfer of graphene produced by catalytic chemical vapor deposition (CVD) on copper foil, is still a technical challenge since unintentional doping of the transferred graphene layer yields an uncontrolled shift of Dirac point in graphene-based field-effect transistors (FETs). Typically, CVD graphene is released from the growth template by etching of the template,  The paper addresses the technical challenge of producing doping-free transferred graphene layers produced by catalytic chemical vapor deposition (CVD), thereby preventing uncontrolled shift of the Dirac point in comprising field-effect transistors. To fabricate electronic devices, such as graphene field-effect transistors (FETs), the graphene/PMMA stack is transferred onto a secondary substrate. FETs are commonly realized by patterning the source and drain electrodes on top of the graphene layer, or by transferring, the graphene/PMMA layer onto a substrate with pre-patterned source and drain electrodes.Two-dimensional (2D) materials such as graphene have been extensively studied for various electronic and opto-electronic applications.8 Since FETs are sensitive interface devices, the presence of ions (or any ionic species) at the interface between the graphene layer and the gate dielectric leads to unintentional doping of the graphene layer, which drastically affects the electrical performance.9–11 As a result, the Dirac point shifts to voltages much larger than 0 V so that instead of an ideal ambipolar behavior, the graphene FET exhibits unbalanced electron/hole transport or even a seemingly unipolar behavior.Graphene transfer is one of the critical issues in the fabrication of graphene FETs and their upscaling.12,19 The FETs are fabricated using the conformal transfer technique.8 Ammonia effectively removes the majority of ions (nearly 4 × 1012 cm−2 dopants) that otherwise would unintentionally dope the graphene layer. The extra washing step also improves the mobility of the charge carriers and shifts the position of the Dirac point to biases close to zero volt. We attribute the improved FET behavior to effective removal of Cu2+ from the graphene layer, and quantify the density of ions.Herein, we demonstrate CVD-based graphene FETs with balanced ambipolar electron/hole transport. Towards that end, we have introduced an extra washing step of graphene in aqueous ammonia after etching away the copper.3 and the floating graphene/PMMA layer is thoroughly washed with DI water. The “ammonia” treatment is performed by placing the floated graphene/PMMA film in a continuous flow bath of NH3 : DI-water (1 molar) for 30 min followed by a drying step at 40 °C for 12 hours in a vacuum oven at 10−1 mbar. Hereafter, we use w-graphene and a-graphene to refer to graphene that is washed in DI-water or aqueous ammonia flow bath, respectively. Subsequently, the graphene/PMMA layers are transferred to a pre-patterned Si-wafer using the conformal transfer technique. After evaporation of residual water, the PMMA layer is removed in a fresh acetone bath. Subsequently, the exposed transferred graphene is washed in a deionized water bath. The final cleaning processes are repeated several times to minimize the PMMA debris. We note optimizing the transfer process toward full removal of the PMMA layer or minimizing structural defects were beyond the scope of this work.The process flow for graphene transistor fabrication is schematically shown in 2. The electrodes are fabricated by sputter deposition of Ti/Au (2 nm/150 nm), and patterned by conventional photolithography. The spacing between the source and drain electrode varies from 1 μm to 40 μm at a fixed channel width of 1000 μm, having a concentric geometry. A scanning electron microscopy (SEM) image of the transistor test structure is given in 2 surface is passivated with hexamethyldisilazane (HMDS) prior to conformal transfer of graphene/PMMA. The graphene transferred onto a pre-patterned substrate is inspected using SEM. Although the pre-patterned substrates have an uneven topography, the SEM image presented in 2 layer are identical and extrinsic defects such as cracks or film discontinuities are absent. Importantly, the same morphologies are observed for both w- and a-graphene. Both graphene layers showed some PMMA debris. A typical atomic force (AFM) topography image for the a-graphene layer is shown in The pre-patterned substrates are fabricated on 6-inch Si wafers with 250 nm of thermally grown SiO2 using a 0° take-off angle, defined as the angle between the surface normal and the axis of the analyzer lens. A charge neutralizer was used during spectra collection to compensate sample charging. Survey and detailed region XP spectra were collected by setting analyzer pass energy at 80 eV, C 1s and O 1s high-resolution spectra were collected with an analyzer pass energy of 20 eV. A neutralizer was always used during spectra collection. No X-ray damage was inferred to the sample during measurements. Atomic compositions were calculated by subtracting a linear background from the collected spectra with setting an analyzer pass energy at 80 eV. The peak areas were normalized by the manufacturer supplied sensitivity factors, and atomic compositions were calculated accordingly with CasaXPS software.Optical microscopy and atomic force microscopy (AFM) (Nanoscope Dimension 3100 Bruker) were used to analyze the surface morphology of the transferred graphene films before and after ammonia treatment. X-ray photoelectron spectroscopy (XPS) was conducted with a Kratos Axis Ultra DLD spectrometer  using Kα excitation of an Al source with a photon energy of 1487 eV. The data was acquired in the hybrid mode with a beam spot size of 300 × 700 μmThe sheet resistance of the graphene layer, before and after ammonia treatment is determined using a circular transfer line test method (CTLM), as detailed in 2. For each case, 25 MIS-diodes are fabricated and the results are averaged. All electrical measurements were performed in high vacuum (10−6 mbar) using a Keithley 4200 semiconductor characterization system and a Keysight 4980 precision LCR meter.Metal/insulator/graphene/metal (MIS-) diodes are fabricated by deposition of Cr/Au (1 nm/50 nm) on to thoroughly cleaned glass substrates. After deposition of the graphene/PMMA layer on the substrates and subsequent drying, 100 nm of a Au top contact are evaporated through a shadow mask. The PMMA layer thickness was 700 to 800 nm. The capacitors had a device area of 0.16 mmRsh, of the graphene layer after different washing processes are determined from the CTLM test structures.8 The Rsh for w-graphene amounts to 450 Ω □−1 and remains constant for different current levels whereas the a-graphene layer shows an increased Rsh of 500 Ω □−1. Observation of higher Rsh is due to the de-doping of the graphene layer.First, the sheet resistance, 13–16 This can be caused by copper, water or etchant (FeCl3 in our case) ions. A doping/de-doping mechanism will be discussed later.Transfer characteristics of the FET fabricated with w-graphene is presented in i.e. the Fermi level is near or at the Dirac point, which means that ions, or charged species are absent. It should be added both transistors presented in To eliminate the troublesome unintentional doping, and recover ambipolarity in the graphene FETs, we have introduced an extra washing step in ammonia flow bath, right before transfer of graphene/PMMA layer onto the FET substrate. The FET transfer characteristics of the resulting a-graphene FET is presented in μFE, of the graphene layer.17 Since μFE is a device parameter that is affected by the geometrical parameter W/L (channel width/length). For a fair comparison, here use a normalized mobility, μn, which is the μFE multiplied by the geometrical parameter W/L:where Cox is the capacitance of the gate dielectric. The geometrical normalization allows for a fair comparison between the mobilities obtained from the state-of-the-art paraffin-enabled graphene FETs and those presented in μn for the paraffin transferred graphene amounts to 6.5 × 103 whereas μn for a-graphene amounts to 2.6 × 104, which is by a factor of 4 larger.It has been recently demonstrated that using paraffin instead of PMMA for the graphene transfer yields four-fold increase in field-effect mobility, 3 is a well-known etchant for copper, and a strong Lewis acid. The chloride plays an essential role in dissolving copper since Cl− is a strong complexing agent for Cu2+ ions. The etching reaction is written in a simplified form as:The current in the a-graphene FET is lower than the one in the w-graphene FET. This indicates that the ammonia treatment de-dopes the graphene layer, and reduces the number of residual carrier in the graphene layer. We suggest a tentative de-doping mechanism as follows. Aqueous FeCl2 are soluble in water. As a result there can always be traces of Cl− or Cu2+ ions in w-graphene. The extra washing step of graphene in an ammonia flow bath is ideal to remove both ions. The NH3 molecules extract the residual Cl− ions from the graphene layer as ammonium chloride, which is soluble in water and is washed away. Furthermore, the Cu2+ ions form an ionic complex in water, namely hexaaquacopper(ii) ion [Cu(H2O)6]2+. The ammonia acts simultaneously as ligand for the [Cu(H2O)6]2+ ions to form solid precipitates of the neutral complex according to the following reaction:The Cu ions in the form of CuCl2+ ions that can potentially act as dopant.Therefore, the extra ammonia washing step would also remove Cu18 for C 1s, Cu 2p, Fe 2p and Cl 2p for both w- and a-graphene are presented in  PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C bonds. The Fe 2p spectra shows that washing the graphene layer with DI-water is sufficient to effectively remove Fe ions, as the XPS spectra does not show presence of Fe before and after ammonia flow bath treatment. The Cu 2p spectra clearly shows a weak signal near 930 eV, which could be assigned to Cu (2p3/2) electrons. After the ammonia washing step, the weak signal has disappeared in the background noise. Therefore, washing of the graphene in the ammonia flow bath has removed minute traces of Cu ions. The difference in XPS spectra is more pronounced for the Cl 2p survey of w- and a-graphene. While the a-graphene does not show presence of Cl, the w-graphene clearly shows a peak at ∼199–200 eV. The amount of Cl for the w-graphene and a-graphene is calculated, which amount to 0.27 ± 0.04% and 0.16 ± 0.04% respectively. Therefore using an ammonia flow bath treatment has effectively removed majority of the Cl ions.To unambiguously demonstrate the presence of ions and further estimation of their surface concentration we have performed XPS measurement for the graphene layer before and after ammonia transfer. A closer up X-ray photoelectron spectra12 cm−2 for w-graphene. The doping density is nearly the same as the intrinsic carrier density in graphene. As a result, a nearly two fold increase in the current of the transistors is expected when we assume that the mobility remains constant. Interestingly, the currents at Vg = −20 V for the transistors presented in To estimate the concentration of ions, we have fabricated MIS-diodes. A constant DC bias of +10 V superimposed with an AC signal of 0.1 V with variable frequency is applied to the Au electrode contacting PMMA. The capacitance–frequency responses of the MIS-diodes are given in In summary, washing of the graphene layer after etching copper in ammonia flow bath is a crucial step to achieve FETs with balanced transfer characteristics and with zero Dirac voltage. The ammonia can effectively remove both chlorine and copper ions. We note that the PMMA transfer technique is not ideal for the realization of high quality graphene FETs. Therefore, upon combination of ammonia flow bath with more advanced transfer techniques, high performance CVD-graphene based FET are within reach.There are no conflicts to declare.


The work deals with molecular dynamics (MD) simulationsof protonated,human telomeric i-motif deoxyribonucleic acid (DNA) with functionalizedgraphene. We studied three different graphene sheets: unmodified graphenewith hydrogen atoms attached to their edges and two functionalizedones. The functionalization of graphene edge consists in attachingpartially protonated or dissociated amine and carboxyl groups. Wefound that in all cases the protonated i-motif adsorbs strongly onthe graphene surface. The biased MD simulations showed that the worknecessary to drag the i-motif out from amine-doped graphene is abouttwice larger than that in other cases. In general, the system i-motif/amine-dopedgraphene stands out from the rest, e.g., in this case, the i-motifadsorbs its side with 3′ and 5′ ends oriented in theopposite to surface direction. In other cases, the DNA fragment isadsorbed to graphene by 3′ and 5′ ends. In all cases,the adsorption on graphene influences the i-motif internal structureby changing the distances between i-motif strands as well as stretchingor shortening the DNA chain, but only in the case of amine-doped graphenethe adsorption affects internal H-bonds formed between nucleotidesinside the i-motif structure. It contains information necessary for the reproduction, growth,and functioning of living organisms. In the sequence of nucleotides,DNA has genetic instructions with information necessary for the synthesisof all proteins. Moreover, each strand of DNA in the double helixcan serve as a pattern for duplicating itself. DNA molecules are verylong and are packed into more complex structures called chromosomes.An eukaryotic, i.e., human chromosome, is terminated by a specificsequence of bases called telomere which is normally folded at itsvery end into a loop. Inside chromosomes, DNA is coiled around histonesmainly in a double-helix form. Moreover, a wide spatial versatilityof DNA has been observed, like A-DNA, B-DNA, or Z-DNA in additionto the typical Watson–Crick duplex DNA. Also, a number of alternativestructures including four-stranded G-quadruplex and intercalated i-motifDNA have been found.n sequences,an individual DNA strand is ableto form a quadruplex structure. When the DNA region is cytosine-richit may fold into four-stranded, i-motif structure. It has been discoveredthat a telomeric i-motif can occur in vivo and plays an importantrole in the control of gene expression.5In the region of telomericDNA composed of (TTAGGG/CCCTAA)+, Na+, or K+ in solution.In addition, a few nanosized carbon materials can work as a stabilizerof the spatial i-motif structure.9 Due to its adsorption properties and sensitivityto pH, the i-motif can be considered as a bioconjugation factor8 and biosensor component.It is commonlyassumed that the i-motif structure is stabilizedat acidic pH. The stabilizing effect enhances the presence of monovalentions like Ag11 Almost unlimitedpossibilities of the practical use of DNA interactions with nanotubeshave been proposed.12The interactionof DNA structures with carbon nanomaterials hasgained increasing interest in the literature in the past few years.It concerns the interactions of carbon nanomaterials (CNMs) with differentstructures of DNA, e.g., single-stranded, duplex, triplex, and quadruplexones. As an example, it has been observed that single- and double-strandednucleic acids can adsorb onto carbon nanotubes. Single-stranded DNAcan helically wrap around a single-walled nanotube. In the case ofdouble-stranded DNA, its adsorption at the nanotube is weak. Nevertheless,it was reported that double-stranded DNA wrapped around nanotube isprotected from enzymatic cleavage.13 The binding affinity of nucleic acid to graphene oxideis dependent on the presence of a single-stranded region.14 The adsorption/desorption event of nucleic acidon nanomaterials is one of the key steps for effective DNA and RNAisolation.16In additionto carbon nanotubes, graphene is also the subject ofintense research. Most often it relates to graphene oxide which isconsidered as the component of biosensors, drugs, and gene nanocarriers.Similar to carbon nanotubes, single-stranded DNA shows stronger adsorptionaffinity to graphene. It has been found that cations, pH, and organicsolvent influence DNA binding to graphene oxide.17 Also, double-stranded DNA can adsorb at oxidized graphenebut in high ionic strength solutions. One of the most interestingapplications of graphene-DNA systems is molecular computing.18 Such biomolecular systems can play a role inlogic gates.19 Due to the outstanding physicalcharacteristics for DNA sequencing, graphene can also be consideredas a key component of modern virus tests.20Moreover, in the case of graphene oxide, it has been observedthatthe binding affinity depends on the DNA sequence.The aim of this study is a detailed analysis of the i-motifinteractionand adsorption on an edge functionalized graphene layer. We will investigatehow the functionalization style influences the i-motif adsorptionand how adsorption affects the i-motif spatial structure. In addition,we will carefully examine the physical properties of the investigatedsystems.221 and employing generalized amber force field(GAFF)22 for the functionalized graphenesheet and parm99 Amber force field with bsc1 modifications for thessDNA fragment.23 The structure of thei-motif considered in this study has been obtained by modifying thestructure published by Phan et al.25 (pdb ID 1EL2).The topology of the protonated i-motif has been built applying AmberTools20 package26 and ACPYPE script.27The studies werecarried out using Gromacs 2020 simulation suiteDuring simulations, we investigated thespatial configuration ofthe i-motif by monitoring the distances between selected phosphorusatoms from the backbone located at the characteristic points of thestructure, as shown in 28 Thus, we studied the structurein which half of the cytosines are protonated. The sequence of nucleobasesin the DNA chain is then (CCCTAA)2C+C+C+TAAC+C+C+T  charge model and the Gaussian09 quantum mechanics program. Next, the information obtained fromthe R.E.D. Server has been applied to the whole graphene sheets bymanual edition of MOL2 files. Finally, using ACPYPE, we have builtthe force field topology according to the GAFF parameterization. Graphenesheets are almost rectangular and only two opposite edges were functionalized(2), its total chargeis +9, whereas for the carboxyl-doped one (GR-COOH), the total chargeof the sheet is −9. Carbon atoms at the graphene edge are saturatedwith hydrogen atoms.The functionalization of graphene sheets assumedattaching amineand carboxyl groups to their edges. Half of them are in charged form:amine groups are protonated and carboxyl groups are dissociated. Thesefunctional groups are present in a lot of biological systems. Becauseof the large number of atoms in graphene sheets applied in the presentstudies , only theircharacteristic fragments were used to build topologies. These characteristicfragments included about 50 atoms and were about 9 nm × 9 nmin sizes. This procedure is due to the fact that R.E.D. Serveronalized3. In theNPT ensemblein the cubic box with sizes approximately 11 nm × 11 nm ×11 nm. Boxes were composed of one i-motif and one graphene sheet,about 40,000 tip3p water molecules and a dozen of Na+ andCl– ions to imitate 0.145 mol/dm3 ionicstrength.Simulations were carried out in the 31 or even 1000ns32 but we think that in our case it ispossible to simulate a little shorter. This is confirmed by the valuesof parameters monitored during the simulation, e.g., the root of meansquared displacement (RMSD) of the i-motif from its initial state.As we will show in the work later, after a maximum of several nanoseconds,the values of different parameters monitored during simulations shortlyreach an approximately constant value.The temperature of simulation systems was maintainedat 310 K usinga Nose–Hoover thermostat. Periodic boundary conditions wereapplied. For long-range electrostatic interactions, the smooth particle-meshEwald algorithm was used. Cutoff distances for Lennard–Jonesand Columbic interactions were set to 1.2 nm. All particles were placedrandomly in the simulation box. Equations of motion were integratedusing the Leap-Frog algorithm with the time step of 2 fs. Each simulationwas equilibrated for 10 ps. Based on our previous experience in studyingthe interaction of DNA with carbon nanotubes, e.g., in ref , we deci32 and GR-COOH, which are positively andnegatively charged, respectively, the charge of graphene sheet wherefunctionalization has not been applied is neutral. It could be assumedthat the electric charge of graphene is one of the most importantfactors which influences the interactions with the protonated, i-motifDNA fragment. This thesis confirms the minimum distance between thecenter of mass (COM) of the i-motif structure and the closest atomfrom the graphene surface, calculated for investigated systems. 2 and COM of the i-motif is reached at about 1 ns. In the caseof the system with GR-COOH, this time is 7 ns. For the simulationsystem which includes graphene without functionalization, GR-H, theprocess in which the i-motif approaches the graphene surface is themost time consuming, as it takes approximately 15 ns. Also, the minimumdistance between these two structures depends on the type of graphenefunctionalization. In the case of GR-H and GR-COOH, when the i-motifreaches the graphene surface, the minimum distances between thesetwo structures are initially a bit different. The distance of theCOM of the i-motif and GR-H is ca. 1.8 nm, whereas the distance fromthe GR-COOH surface is about 1.6 nm. After 35.5 ns of simulations,the i-motif molecules get closer to GR-H and reach almost the sameposition as in the i-motif/GR-COOH. It is worth noting that the fluctuationsof distance for both pairs are similar but for the system with GR-NH2 they are a little bigger. After reaching the equilibriumconfiguration, the minimum distances of i-motif/GR-H and i-motif/GR-COOHvary in the range of ca. 0.25 nm, whereas the minimum distance betweenthe DNA fragment and GR-NH2 varies in the range of ca.0.3 nm.The studieswere carried out for three systems that differed inthe method of graphene functionalization. Instead of amine- and carboxyl-dopedgraphene, GR-NHBecause the starting configuration of simulatedsystems was generatedin a random way, the time consumed for the DNA fragment to approachthe graphene plane says little about the physical affinity betweenthese two structures. Nevertheless, it is clearly seen that, in comparisonto other systems, the amine-doped graphene interacts with the i-motifin a different way. Detailed examination of the positions of P1–P5points shown in 2 system, as showed in the part (b) of 2 surface is approximately 0.6 nm, whereasthe analogous distances from GR-H and GR-COOH are approximately 3and 2.7 nm. Further comparison of the distances presented in 2 is oriented “upside down,”i.e., the DNA fragment is adsorbed to graphene with the side containingP2 and P4 atoms. Moreover, the fluctuations of minimum distances shownin 2 dimer is the most stable among the consideredsystems. For the record, P1, P3, and P5 phosphorus atomsare located onthe i-motif’s side with 3′ and 5′ ends, whereasP2 and P4 are on its opposite side. 30 When studying i-motif interactions with single-walledcarbon nanotubes, we also observed its different orientation withrespect to the nanotube surface. While using the same terminology,the arrangement of the i-motif in the case of adsorption on GR-NH2 can be called as “top” orientation, whereasin other cases we have the “bottom” orientation.As seen in One of the most interesting effects is the influence of grapheneplane on the i-motif structure. The quantitative analysis of suchan effect can be done through the investigation of mutual distancesbetween P1, P2, P3, P4, and P5 phosphorus atoms. 2 and decreases when it is adsorbed at GR-COOH . It is dueto the fact that a part of carboxyl groups is in anionic form. Finally,the i-motif structure adsorbed at nonfunctionalized graphene GR-Hin some period of simulation trajectory also reaches the grapheneedge. This is due to charge distribution between carbon atoms in grapheneand hydrogen atoms at its edge. While carbon atoms in the center ofthe sheet have neutral charge, the carbon atoms near the grapheneedges are increasingly negative charged. The total charge of the graphenesheet is balanced by positively charged hydrogen atoms. Thus, whenthe i-motif comes close enough to the edge of the GR-H sheet, it fallsinto a potential well where it is attracted by positively chargedhydrogen atoms that are on that edge.It can be concluded that in the case of GR-NHr and α. One of them is the distance r between two strongly electronegative atoms D and A from hydrogenbond donor D–H and hydrogen bond acceptor A. The second oneis the angle α between three atoms D–H···A.The bond becomes stronger when defined in such a way that distance r and angle α become smaller. Here, we applied themost commonly used values of H-bond parameters: r ≤ 0.35 nm and α ≤ 30°. Thus, when bothcriteria are fulfilled, the H-bond bridge is formed. Figures 13The next feature investigatedis hydrogen bonds between nucleotidesinside the i-motif molecule. It is worth recalling that, in the MDsimulations, hydrogen bonds are defined by two geometrical parameters 2).It is worth noting that only in the case of GR-H,we have a relativelylong simulation trajectory for the nonadsorbed i-motif at disposalsee 4. Inoth2. While H-bond lengthsbetween i-motif nucleobases remain unchanged, their mutual orientationchanges slightly. The most probable H-bond angle changes from about8° to 11°. Although it is not a big change, the shapes ofthe angle distribution curves shown in 33 Nevertheless,angles change in a relatively narrow range from 0° to 20°.A much more narrow range of changes occurs in the case of H-bond distancedistributions. It is always a very narrow peak with the maximum atabout 0.29 nm. These values indicate that hydrogen bonds between nucleotidesinside the i-motif structure have, in most cases, a strong characterwith dominant strongly covalent interactions.What we can seefrom 2, the i-motif adsorbs “upside down,” i.e., withits 3′ and 5′ ends on the opposite side to the graphenesurface . It seems that this effect may mainly be responsiblefor the different properties of the i-motif/GR-NH2 system.In comparisonto other investigated systems, we observed that whenthe i-motif adsorbs at the amine-doped graphene, the fluctuationsof distances between its selected phosphorous atoms are the smallestand, simultaneously, the directionality of i-motif internal H-bondsin this system decreases. Thus, it seems that the influence of amine-dopedgraphene on the stability of the DNA fragment is much more complex.It is worth noting that among the investigated systems, only at GR-NHThus, we can state that the DNA fragment is more strongly adsorbedby amine-doped graphene see 4. Theref34 In such a case, it is necessary to average a lot of trajectoriesdetermined from various initial configurations. Unfortunately, sucha procedure is numerically complicated even for simple systems. Forthis reason, the work profiles shown in 34 ensures that exponential averaging of many work versusdistance/time profiles can lead to the exact free energy profile inthe limit of infinite number of such trajectories determined. Becausewe used only a single trajectory, the obtained profiles are probablyfar from a true free energy of binding. Nevertheless, applicationof the same settings during the enforced detachments allows us tocompare properties of these three systems and draw reliable conclusionsabout their stability.It is worth mentioning that the constant velocitydragging canformally be used to determine the free energy.Nevertheless, the analysis of 2, the required work is approx. twicelarger than in the case of GR-H and GR-COOH. It is about 427 kJ/mol,whereas the work necessary to drag i-motif out from GR-H and GR-COOHis 265 and 252 kJ/mol, respectively. Such results are consistent withour previous observations and confirm the significant role of electrostaticsinteractions in the i-motif/GR-NH2 dimer. Another factorinfluencing the value of this work can be the reverse i-motif orientationon the GR-NH2 surface.First of all, we cansee a huge difference between the work necessaryto remove i-motif from various functionalized graphene surfaces. Inthe case of GR-NHsc of the i-motif draggedout from graphene surfaces.35Regardless of the type offunctionalized graphene, the time necessaryto drag the DNA fragment out is about 3 ns. In the case of i-motif/GR-Hand i-motif/GR-COOH, the extreme  parts of workprofiles are similar. Visible difference can be seen in the rangeof 1.0 to 3.5 ns. It suggests that the spring attached to the i-motifCOM during biased simulations causes not only its desorption, butalso influences the i-motif spatial structure. This is one of thereasons why calculated work is not the same as the free energy ofinteraction. To check it more carefully, in 2. In this case, up to 1 ns of simulation, the similarity parameterρsc varies in the range of 0 to 0.7. Following Maiorovand Crippen,35 when ρsc > 0.5, it can be interpreted as antisimilarity from the initialstructure. Next, when the DNA fragment is dragged out from GR-NH2 surface (times in the range of 1 to 4 ns), the similarityparameter remains constant. Finally, when the i-motif is desorbed,its structure randomly changes during some relaxations connected withshape optimization. In other cases, the similarity parameters ρsc of the i-motif adsorbed on GR-H and GR-COOH are much lower.Their values are in the range of 0.3 to 0.5 and can be interpretedas visually recognizable similarity of the i-motif structure to thereference state.35As can be expected, the spatial structure of thei-motif is modifiedto the greatest extent in the case of dragging it from GR-NHAlthough the mainaim of the paper is the investigation of thechanges in the spatial i-motif structure caused by adsorption, itis worth seeing the effect of DNA adsorption on the graphene sheet. It can be stated that the changes of RMSD are smalland basicallydo not exceed 0.2 nm. This indicates a high degree of similarity ofgraphene sheets to their initial states when DNA was far from thesurface. The changes in RMSD are caused by thermal fluctuations. Itis worth noticing that RMSD plots shown in 42 system is much more different from the rest. In the caseof i-motif adsorption on GR-H and GR-COOH, the DNA fragment orientedits 3′ and 5′ ends toward graphene, whereas on the GR-NH2 surface it is inversely oriented.The presented studies enableto recognize new physical insightsinto the interaction of the i-motif with functionalized graphene.First of all, the i-motif is very stably bonded to the graphene surface.The minimum distance between these two structures depends on the typeof graphene functionalization, but the shortest one is in the caseof amine-doped graphene. Further analysis showed that the i-motif/GR-NH2 causes the stabilization of the upstream part ofthe DNA strand, and the mobility of thymine and adenines is very low.In the case of GR-H and GR-COOH, the opposite effect is observed.While consideringthe influence of graphene plane on the i-motifstructure, our investigations suggest the changes of its internalstructure due to adsorption interactions. Such changes consist ofthe stretching or shortening of the DNA chain as well as changingthe distances between i-motif strands. These observations were confirmedby i-motif’s RMSD curves. The contact between the DNA fragmentand graphene sheet always results in a change of the i-motif spatialstructure. The analysis of fluctuations of i-motif atomic positionsshows that the adsorption on graphene does not affect strongly bondedsemiprotonated cytosine pairs. The fluctuations of 3′ thymineare large when it is adsorbed on GR-H and almost the same in othercases. When the DNA fragment is adsorbed on GR-COOH, a notable flexibilityof thymine and adenines appears. The adsorption of the i-motif onGR-NH2 is also distinguished fromthe others by the location of the adsorbed DNA fragment on the graphenesurface. For almost all simulation trajectories, the i-motif is adsorbedvery close to the amine-doped graphene edge. Moreover, only in thecase of the i-motif/GR-NH2 dimer, the adsorption influencesthe internal H-bonds formed between nucleobases. These hydrogen bondsbetween nucleotides inside the i-motif structure have a strong characterwith the dominant of strongly covalent interactions.The system i-motif/GR-NH2 dimer is the most stable among the considered systems.The biasedMD simulations confirmed the strongest i-motif adsorptionon the amine-doped graphene. The work necessary to drag it out fromthe surface is almost twice larger than in other cases. Dragging DNAfrom the graphene surface causes significant changes in its shape.Thus, one of the most important conclusions is that the i-motif/GR-NHThe observed features of investigated systems are very promisingand encourage further experimental and theoretical research.


Glioma is a malignant tumor that often occurs in the adult central nervous system. Metadherin/astrocyte-elevated gene-1 (MTDH) is involved in the development of cancer, but its relationship with glioma remains unclear. This study is aimed at clarifying the role of MTDH in glioma. GEPIA was employed to find the difference of the expression level of MTDH and MYB protooncogene-like 2 (MYBL2) in glioma tissues and normal tissues, and real-time quantitative reverse transcription PCR (qRT-PCR) and western blot (WB) were applied to verify the differential gene expression of MTDH and MYBL2 cells. After knocking down of MTDH, the expressions of forkhead box M1 (FoxM1), MTDH, and MYBL2 were detected by WB cells. Cell counting kit 8 (CCK-8) was used to detect cell proliferation, and flow cytometry was applied to measure cell apoptosis. The transwell assay was utilized to investigate the ability of cell migration and invasion. The results showed that MTDH and MYBL2 were overexpressed in glioma cells compared with normal cells. The knockdown of MTDH would inhibit the expression of MYBL2 through decreasing the expression of FoxM1 and further reduce glioma cell proliferation and cell migration and invasion. The present study showed that knockdown of MTDH inhibits glioma proliferation and migration and promotes apoptosis by downregulating MYBL2, which suggests that MTDH is a potential gene in clinical treatment of glioma. Glioma is a common malignant primary brain tumors in the adult central nervous system . Gliomasin vivo and in vitro experimental studies, overexpression or knockdown of MTDH can affect a series of cell activities such as gene expression and cell proliferation. Moreover, the invasive and metastatic capabilities of tumor are also altered by MTDH [Metadherin/astrocyte-elevated gene-1 (MTDH)  is an abnormally activated oncogene found in a variety of cancers. It is a multifunctional protein, and the gene encoding MTDH is located on chromosome 8q22. Its main function is to promote tumor growth and migration, and it is also related to drug resistance. In addition, MTDH can also regulate oxidative stress, immune aging, and other functions . MTDH is by MTDH . However by MTDH –12.The MYB protooncogene-like 2 (MYBL2) gene is a member of the myeloblastosis transcription factor family . MYBL2 p2.Human brain astroglial normal cell HEB and glioma cell lines U251, U87, T98G, and LN-229 were obtained from Zhejiang University . Cells were cultured in DMEM  supplemented with 10% FBS  and 1% of penicillin-streptomycin . Cells were cultured at 37°C in a cell incubator containing 5% COhttp://gepia.cancer-pku.cn/index.html) is an online database. It was applied to compare and study the expression level of MTDH and MYBL2 in glioma and normal tissues. Boxplot with disease status  was used as a variable to calculate the differential expression of MTDH and MYBL2.GEPIA (μg) and the transfer plasmid (15 μg), the GV115 plasmid (20 μg) was cotransfected into 293T cells using Lipofectamine® 2000 . Following transfection with the lentiviruses for 48 h (MOI = 10), the transduced cells were selected with 5 μg/ml puromycin . The grouping scheme was as follows: (1) the HEB group, (2) the U251 group, (3) the U87 group, (4) the T98G group, and (5) the LN-229 group. After knockdown of gene, the grouping scheme was as follows: (1) the siNC group, (2) the siMTDH group, (3) the siMTDH+NC group, and (4) the siMTDH+MYBL2 group.MTDH lentiviral-based gene silencing particle was purchased from Beyotime  and was cloned into GV115 plasmids. The sequences of MTDH silencing RNAs were as follows: siMTDH-1, 5′AGGAATAAAGGATTCTGAT3′; siMTDH-2, 5′-AAGTCAAATACCAAGCAAA-3′; and siMTDH-3, 5′-AACTTACAACCGCATCATT-3′, as well as a negative control siRNA (siNC), 5′-TTCTCCGAACGTGTCACGT-3′ . The groβ-actin was used as an internal control. The grouping scheme was as follows: (1) siNC+pc-control, (2) siMTDH+pc-control, and (3) siMTDH+pc-FoxM1. All primers used are listed in A SteadyPure Plant RNA Extraction Kit  was used to lysate cells to extract total RNA. Then, cDNA was synthesized by a universal RT-PCR kit . SYBR Green Master Mix  was employed for the qRT-PCR, and g and 4°C for 15 min. To quantify the protein concentration, a bicinchoninic acid (BCA) protein assay  was performed. Denaturation of total protein was processed in a metal bath at 95°C for 5 min, and then, 30 μg of total proteins was separated by SDS-PAGE (7.5%). After that, proteins were transferred to PVDF membranes. 5% skimmed milk was dissolved in TBST to block the PVDF membrane for 2 h at room temperature. Then, the membranes were washed and incubated with the primary antibodies overnight.Protein was lysed from cells by using RIPA buffer  followed by centrifugation at 16,000 × β-actin (1 : 2000)  antibodies. Then, the membranes were incubated with the horseradish peroxidase- (HRP-) conjugated secondary antibody (1 : 1000)  for 2 h at 37°C. To measure the chemiluminescence signals, the EasySee Western Blot Kit  was utilized. ImageJ version 1.53 software was used to quantify the proteins.The primary antibodies included anti-MTDH (1 : 1500) , MYBL2 (1 : 1500) , FoxM1 (1 : 1500) , and CCK-8 assay  was maneuvered to measure cell viability of cells with indicated transfections. Cell proliferation rates were measured after 24 hours. The absorbance value could be immediately read at 450 nm by a microplate reader . At each concentration, each group was repeated for three times, followed by data analysis.To evaluate the apoptosis rates of cells, a BD Accuri™ flow cytometer  was employed to flow cytometry. An Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit  was applied for detecting the proportions of cell cycle and cell apoptosis, and then, results were analyzed according to manufacturer's protocols. The ratio of cell apoptosis was computed and calculated using FACS scan software .When cells grown to logarithmic growth period, cells were subcultured and evenly planted in a plate and cultured in an incubator at 37°C. Colonies were formed and observed, and the culture was terminated and the supernatant was discarded, followed by immersing twice with PBS. 4% paraformaldehyde was added to fix cells for 15 minutes. Then, fixing solution was removed, and the cells were stained with proper amount of Giemsa solution for 10-30 minutes. Then, cells were slowly washed off in dyeing solution with running water and air dried. The number of cells was calculated under microscopy.μl solution. The transwell had two chambers including the upper and the lower. The upper was placed at 37°C for 4-5 hours; this would induce Matrigel to form gel. 2 × 105 U251 and U87 cells in 200 μl serum-free medium were plated into the 6.5 mm transwell chamber, and the complete medium was added into the lower chamber. Cells were then incubated at 37°C for 24 hours. Methanol was applied to fix cells for 15 min, and then, 0.1% crystal violet was employed to stain cells for 40 min. The cell migration from the upper chamber to the lower chamber is induced due to the permeability of the membrane and the composition of the medium in the upper and lower chambers. The superfluous, i.e., nonmigrated, cells in the upper layer are erased, while the cells in the lower layer, i.e., cells that have migrated, are stained with crystal violet. The migration rate was calculated. Invading cells will invade the extracellular matrix, which in turn determines cell invasion. Cell numbers were counted under a light microscope.Matrigel  was diluted and prepared in 100 t-test or one-way analyses of variance (ANOVA) were utilized to analyze results followed by Tukey's test to compare each group. Data were analyzed, and graphs were plotted by using GraphPad Prism version 6.0 software . p < 0.05 indicated a significant difference.All data were presented as the mean ± standard deviation (SD) for experiments performed in triplicate. Unpaired or paired Student's The predicted results are shown in To investigate the role of MTDH in glioma cells, the expression of MTDH in U251 and U87 cells was knocked down. WB results showed that , in bothThe effect of knockdown of MTDH on the proliferation of glioma cells was then examined. As shown in The cell migration and invasion of glioma cells U251 and U87 were detected after knockdown of MTDH. The results showed that the knockdown of MTDH decreased the cell migration compared to the siNC group, but in the siMTDH+MYBL2 group, the addition of MYBL2 enhanced cell migration compared to the siMTDH+NC group . The celGlioma is a common primary tumor in the brain. Although many cancer therapies have been developed in the past few decades, the unique structure of the central nervous system in the brain has brought challenges to clinical treatment. Targeted therapy presents certain opportunities for cancer treatment . In thisMYBL2 is a member of the myeloblastosis transcription factor family that is activated in tumorigenesis and progression as Rachel's systemic review summarised . In breaThere are limitations to this study. MTDH has been shown to be aberrantly expressed in glioma and has been shown to regulate EMT during cancer invasion. The mechanism involved in this process may be through regulation of certain miRNAs and activation of Akt or coactivation of key signaling pathways with target genes. MYBL2 has also been proved to be associated with poor prognosis of cancer by pan-cancer analysis, and some literatures have shown that MYBL2 can be upregulated by activation of Akt/FoxM1 to promote the development of glioma. However, it is still unclear through which biological microevents/processes MTDH affects MYBL2, thus affecting the behavior of cells. These will be explored in the future studies. It is still not clear whether MTDH acts directly on MYBL2 or through other proteins that mediate it. But it is worth affirming that the decreased expression of MTDH will mediate the downregulation of MYBL2. Moreover, this study has not established an animal glioma model, and a good animal model will provide some evidence for targeting MTDH in the treatment of glioma, which will be conducted in the following studies.This study showed that MTDH and MYBL2 were overexpressed in gliomas, and knockdown of MTDH inhibited the expression of MYBL2. Further experiments showed that knockdown of MTDH inhibited the proliferation, migration, and invasion of glioma cells and promoted cell apoptosis. Overexpression of MYBL2 could reverse the knockdown effects of MTDH in glioma cells. These results suggested that downregulation of MTDH inhibited the progression of glioma cancer.


Terahertz spectroscopy is a perfect tool to investigatethe electronicintraband conductivity of graphene, but a phenomenological model (Drude-Smith)is often needed to describe disorder. By studying the THz responseof isotropically strained polycrystalline graphene and using a fullyatomistic computational approach to fit the results, we demonstratehere the connection between the Drude-Smith parameters and the microscopicbehavior. Importantly, we clearly show that the strain-induced changesin the conductivity originate mainly from the increased separationbetween the single-crystal grains, leading to enchanced localizationof the plasmon excitations. Only at the lowest strain values explored,a behavior consistent with the deformation of the individual grainscan instead be observed. It is especially in the terahertz(THz) frequency range that graphene plasmonics and magnetoplasmonicsdeserve special attention, thanks to the small plasmon confinementlength and high Faraday rotation power, enabling applications suchas modulators, nonreciprocal elements, coherent absorbers, polaritoniccomponents, detectors, and metasurfaces.15 Considering strained graphene, THz can be regarded both as an applicationdomain and as a diagnostic tool. From the first perspective, THz manipulationcan benefit from appropriate graphene strain engineering;16 in addition, pseudo-LL could be employed forultrastrong coupling experiments, without the need for high magneticfield environments.20 From the second, one may leverage on the various THz spectroscopictechniques that proved powerful in understanding the micro- and mesoscopicstructure of materials.22 Indeed, technologiesto fabricate graphene samples of sufficient quality and size withan engineered strain are currently still challenging. On the otherhand, the use of THz as a diagnostic tool has proven often extremelypowerful. In the present article we investigate the THz conductivityof a variably strained polycrystalline graphene sample, unveilingthe origin of the observed Drude-Smith behavior.25 We analyzedour experimental data by means of an atomistic technique, ωFQ,28 highlighting the presence of strain-induced carrier reflection atthe grain boundaries and strain-induced mutual separation betweengrains. The ensuing localization of the plasmon excitations confirmsthat the conductivity trend of the macroscopic Drude-Smith model findsits roots in the creation of actual atom-size gaps between graphenegrains of realistic size. Our study extends prior knowledge on theTHz conductance of strained graphene where uniaxial strain was considered,30 but, most importantly, reports for the first time the applicationof a fully atomistic approach to quantify the deviation from the Drudemodel and, at the same time, provides a novel microscopic interpretationof the Drude-Smith behavior, which is a widespread tool for the analysisof THz conductance in nanostructured disordered materials.Extremely simple in its crystal structure,graphene has revealedan outstandingly complex and rich spectrum of fundamental physicalphenomenologies, with unique perspectives toward technological applications.31 was used to deposit the graphene film on a thin (≈8 μmthick) circular polyvinyl chloride (PVC)-based membrane of about 20mm diameter. The membrane was cut from a foil by means of a hollowpunch in order to provide the desired geometry. The graphene samplewas spin-coated with a protective layer of poly(methyl methacrylate)(PMMA), and the copper was etched away using iron chloride (FeCl3). The remaining PMMA/graphene film was rinsed in deionizedwater several times and was picked up from water using the PVC membrane.The sample was dried under ambient conditions. A reference sampleconsisting of the sole PVC membrane was also fabricated. The sampleholder is a manual chuck with four jaws, on which the membrane samplecan be accommodated and fixed by means of glue. By actuating the chuck,the four jaws move apart from one another, thus applying a biaxialdeformation field to the sample, as illustrated in Figure S1 in the Supporting Information (SI). The usage ofa four-jaw chuck allows to deliver a mostly homogeneous and isotropicstrain distribution, avoiding position-dependent strain profiles thatcould give rise to specific PMF effects. In any case, it should behighlighted that the polycrystallinity of our sample prevents thecreation of directionality-dependent effects, as the unstrained sampleis isotropic. The sample is then inserted in the main optical pathof two kinds of spectrometers: (i) a THz-TDS instrument, operatingin the 0.3–2 THz spectral range, (ii) a FTIR instrument, operatingeither in the FIR (2–10 THz) or in the MIR (10–100 THz)spectral ranges depending on the employed beam splitter and detector.In 1 As a first attempt, we employed a pure Drude form for thegraphene conductivity; with this approach, however, it was not evenpossible to fit the zero-strain spectrum with reasonable parameters.The simple Drude form would be the (asymptotic) form expected forthe conductivity of graphene in the frequency range below the onsetof interband transitions, i.e., in the case of a sample grown withour technique, for frequencies smaller than around 150 THz.33 A deviation from the Drude behavior is howeverexpected in polycrystalline samples; indeed, the Drude model is unableto capture the backscattering of carriers at the grain boundaries,as it only describes a time-domain Poissonian process of inelasticscattering events that is suitable for describing bulk scatteringonly. We thus moved to the Drude-Smith model, that, for graphene,is given by the expression0 = 2e2/h ≈ 7.75 × 10–5 Ω–1 is the conductance quantum, EF is the Fermi energy, τDS isthe Drude-Smith scattering time , and c ∈  is a parameterthat quantifies the deviation from the pure Drude model.23 The choice c = 0 recovers theDrude model; the effect of c > 0 is to suppresstheDC conductivity and to move the maximum of Re(σ) from ω= 0 (as in the Drude model) to a finite value ω > 0. Sucheffectsare interpreted, in the original view of the model given by Smith,as a “memory effect” experienced by the carriers, whosevelocity distribution is not fully randomized after the first collision,and rather retains a net backward component.23 Otherwise, it can be interpreted as the consequence of a finitereflectivity felt by the carriers while moving across the sample,modeled as a disordered assembly of grains with partially impenetrableboundaries.24To perform the aforementionedstudy, we have designed a specialsample and sample holder. Sample fabrication began with chemical vapordeposition (CVD) synthesis of a polycrystalline monolayer graphenefilm on copper foil  using an AixtronBM Pro cold-wall CVD reactor. Wet transferSI, section 2. The slightincrease with frequency in the 0.5–2 THz range (of the orderof just a few percent) in the experimental transmission for the unstrainedspectrum is likely due to deviations from flatness of the membranein the absence of any applied tension  or to residuals of the polymer absorption.Consistently with the general behavior of the Drude-Smith model withnonzero c, the real part of the conductance has amaximum at finite values of the frequency , the initial increasing τDS trend is not unambiguous and the data could also be consistentwith a global decrease, albeit with different slopes for strains belowor above ≈3.5%. A microscopic interpretation of this effectis given later. The data of 30 or uniaxial strain was employed.29The transmittance spectra following from the fitto experimentaldata using our multilayer model and  strains 1d. A pecarameter 1e. Here,24 where the carriers motion in a multidomain sample is studiedby a Montecarlo approach and by considerations on the diffusion current.In this model, new parameters are involved: the diffusion time ineach domain (t0), the domain wall reflectivity(R), and the bulk scattering time (τ). However,the two models are connected, since relatively simple algebraic relationsbetween the above-mentioned parameters and the “original”Drude-Smith parameters can be derived. Exploiting such relations, we report in the SI (section 3.6) details on the analysis of ourdata in view of the model by Cocker et al. In summary, the domaindiffusion time decreases with increasing strain, the reflectivityincreases with increasing strain, and the bulk scattering time remainsalmost constant. These observations support the vision that the strainapplied to the polycrystalline sample leads to a progressive separationbetween the monocrystals (connected to reflectivity increase) andto the creations of cracks within each crystal . Effects connected to the stretch ofcarbon–carbon bonds seem instead negligible34 (see section 3.8 in the SI),and the consequent opening of a bandgap, yet expected at even largerstrains, would rather affect mostly the optical spectral region.35 It should be recalled, however, that the modelby Cocker et al. was derived for massive charges, which is not thecase of graphene.To move toward a microscopic interpretationof the trends observedin 36 which is able to correctly describe how the grain boundaries morphologyaffects the electron conduction across the sample  nature. In fact, the charge’simaginary part enters the definition of the dipole moment of the wholesystem and, as a consequence, the calculation of the complex polarizability(α(ω)), that can be easily related to the conductivity(see section 3.1 in the SI). Also, thecharge exchange between nearest neighbor atoms is described in termsof the Drude model of conductance, which is further modulated by meansof a Fermi-like damping function, which introduces an exponentialdecay, typical of quantum tunneling  by modulatingboth the intrinsic dimensions of the considered substrate and theFermi energy by the same numerical factor.38 In this scenario, a graphene-based nanostructure showing a PRF inthe THz regime, which is unfeasible from a computational point ofview due to the prohibitive number of atoms to be described, can bemodeled as a smaller system by decreasing the Fermi level to makethe electron density equal to that of the real-size structure. Remarkably,ωFQ retains this important physical feature;27 therefore, in the present work, we adopt a special parametrization,which is able to correctly describe the initial experimental systemat rest (see section 3.2 in the SI). Inthis way, graphene samples characterized by a PRF in the THz regimecan be described by actually computing much smaller structures  and the results of calculations can be directly comparedwith experimental data. We model the experimental unstressed polycrystallinegraphene as a multidomain graphene sheet constituted of 6 grains ofdifferent size as the starting geometry at rest (see section S3.7 in the SI. Such data showthat similar trends are obtained independently of the number of grains.Drude-Smith parameters are extrapolated from conduction trends witha fitting procedure (see section 3.3 in the SI). Also, in order to allow for a direct comparison between our modelingand the experimental data, we impose the Drude-smith parameter c = 0.53 at 0% strain (see sections 3.2 and 3.4 in the SI). ωFQ isthen tasked to explain at the microscopic level experimental macroscopictrends as a function of the applied strain (see d2) for eachstrained geometry  are reported in To provide a more solid analysis of DiracFermions dynamics ina multidomain graphene sample, we then exploit a fully atomistic,yet classical, model, called ωFQ,see also .37 In ωFling see  section.rest see 2 a. Obvirain see 1 d, by train see 1. We firrain see 2a,b. Comd2) maximumfor graphene at rest falls at about 0.7 THz, which differs from theexperimental result. However, such a shift does not affect the overalldescription of the behavior of the conduction peak as a function ofthe applied strain. In fact, as reported in The ωFQ Re. At the grain boundaries amorphous C phasescould indeed be present, and they might prevent a clear separationof the islands until the strain is large enough, leading to a complexinterplay of mechanisms. If grains deform without moving away fromeach other, an overall increase of the system size, and, as a consequence,of the scattering time, is expected, without major changes in theplasmon confinement. In fact, the computed ωFQ trend of τDS as a function of the applied strain shows in this case asmall linear increase (see DS are relatively unchanged (within the errors).To support this vision, we consider the alternativesituation producedby the strain: the multidomain unstressed graphene is elongated, stretchingthe chemical bonds without detaching the grains  are determined through the following equation:27qi(ω) is a complex-valued charge placed at atom i, oscillating at frequency ω. vF is the Fermi velocity, n2D isthe 2D-density of graphene, and τ the scattering time. lij is the distance betweenatoms i and j, whereas Aij is the effective area connecting i and j atoms. The electrochemical potentialacting on each atomic site is labeled as ϕel. Finally, f(lij) is aFermi-like damping function mimicking quantum tunneling effects, whichreads as the following:lij0 is the equilibriumdistance between two adjacent carbon atoms at rest . The position of the inflection point and the steepness of thestep function are determined by the parameters d and s, respectively.In this work, we describe the optical propertiesof multidomaingraphene nanostructures, with an emphasis on structural defects createdby a biaxial strain applied to a sample initially at rest. We modelthe optical response of such structures by using a classical, fullyatomistic approach, called ωFQ.


There is growing interest in retained introns in a variety of disease contexts including cancer and aging. Many software tools have been developed to detect retained introns from short RNA-seq reads, but reliable detection is complicated by overlapping genes and transcripts as well as the presence of unprocessed or partially processed RNAs.We compared introns detected by 8 tools using short RNA-seq reads with introns observed in long RNA-seq reads from the same biological specimens. We found significant disagreement among tools (Fleiss’ Short-read tools detect intron retention with poor recall and precision, calling into question the completeness and validity of a large percentage of putatively retained introns called by commonly used methods.The online version contains supplementary material available at 10.1186/s13059-022-02789-6. During RNA transcription, multiple spliceosomes may act on the same transcript in parallel to remove segments of sequence called introns and splice together flanking exons . Most spnylation , 6  is a form of alternative splicing where an anticipated intron remains after transcript processing is complete. IR occurs in up to n humans  and may n humans –20 as wen humans –23. Trann humans . In cancn humans –27 can gn humans . It is un humans , 30, butn humans –35.Despite its biological relevance, detection of IR from bulk RNA sequencing (RNA-seq) data remains challenging for two principal reasons: (1) a short RNA-seq read  is almost never long enough to resolve a full intron or its context in a transcript, particularly in genome regions with multiple overlapping transcripts, and (2) RNA-seq data may contain intronic sequence from unprocessed or partially processed transcripts, DNA contamination, and non-messenger RNA such as circular RNAs (cRNAs) , 36, potExisting tools designed specifically for RI detection make simplifying assumptions to address the above issues. These tools include keep me around (KMA) , IntEREsTo generate a dataset to test RI detection, we identified two human biological specimens on the Sequence Read Archive (SRA) with RNA-seq data from both Illumina short-read and PacBio Iso-Seq RS II long-read platforms Fig. . These wmentclass2pt{minimi and transcript t, we defined persistence r is a read among the set of all reads t, information density i, the binary variable r provides evidence for the retention of i, and the spliced fraction i’s persistence i:We sought to quantify IR in each biological specimen using long-read data. To account for random splicing and sample contamination that may lead to noisy splicing patterns, we developed a novel variant of intron percent spliced in (PSI) . For a gmentclass2pt{minimAcross the union of transcripts from both samples, a substantial majority  and their relationships with the set of RIs called by each short-read tool and their relative expression levels Fig. : Fig. S7entclass1pt{minimaentclass1pt{minimaWe tested performance  of RI detection by eight short-read tools, comparing sets of called RIs against persistent introns identified from long-read data  vs. the corresponding subset of introns called as RIs by that tool. Rather than improving overall performance by retaining persistent RIs and removing false positive ones, stringency filters improved precision at the expense of recall, with typically slight corresponding improvements in F1-score across tools Fig. .Fig. 4a We next compared the distributions of seven intronic properties  between the sets of true positive (TP), false positive (FP) and false negative (FN) RIs for each tool (defined as described in es) Fig. a, b. FPsWe also probed how much distributional uniformity of mapped read coverage across an intron  andSRSF7 gene, which has previously been shown to exhibit post-transcriptional splicing  ). Calculation of LWMs is shown for an example intron in Additional file For each short-read tool, we used the tool-specific called RIs and the LR persistent introns, as described above, to categorize introns as either true positive , true negative , false positive , or false negative .  We then identified subsets of TP introns identified among at least N tools , most recently updated in 2017. We extracted all cRNAs labeled with the “intronic” flag in the annotation column and performed a liftover of genomic coordinates for these cRNAs from hg19 to GRCh38 using the UCSC Genome Browser liftover tool (https://genome.ucsc.edu/cgi-bin/hgLiftOver). For each sample, we determined the percent of introns overlapping at least one cRNA for the We downloaded a database of human circular RNAs from circbase  (http://LBR and AP1G2, but only one sample each for remaining genes.In order to test introns in this study against experimentally validated RIs, we identified wet-lab studies in the literature that had first predicted and then validated intron retention. We identified 4 such studies , 52–54 tAdditional file 1. This file includes all supplementary figures and tables referenced in the article.Additional file 2. This file includes performance benchmarks applying tool-specific filtering criteria.Additional file 3. Review history.