The yield of bilayer and few-layer graphene obtained was quantified by Raman spectroscopic analysis, and the adsorption of the pyrene derivative on the graphene surface was studied by thermogravimetric analysis and X-ray diffraction. The whole procedure was rationalized with the help of molecular modeling.The search for graphene or few-layer graphene production methods that are simple, allow mass production, and yield good quality material continues to provoke intense investigation. The present work contributes to this investigation through the study of the aqueous exfoliation of four types of graphene sources, which are namely graphite and graphite nanoflakes with different morphologies and geographical origins. The exfoliation was achieved in an aqueous solution of a soluble pyrene derivative that was synthesized to achieve maximum interaction with the graphene surface at low concentration (5 × 10 The hydrophobic nature of graphene leads to its re-stacking and agglomeration in aqueous dispersion. In fact, solvents with surface tensions of near 40 mJ m−2 [Graphene has emerged as an exciting material that has been intensively studied throughout the last decade, revealing potential applications in various fields ,2,3. How0 mJ m−2 .−) and pyrene sulfonic acid salt, as well as a pyrene-terminated poly (PDMAEA) and poly(acrylic acid) (PAA) have been investigated for use in solar cells, as well as electrochemical and composites applications [The performance of water for the LPE of graphite can be greatly improved with the aid of amphiphilic molecules that strongly interact with water and with graphene simultaneously. These molecules help the dispersion of graphite flakes and graphene, preventing their agglomeration. Polycyclic aromatic hydrocarbons such as pyrene derivatives have been studied for the exfoliation and stabilization of graphene and FLG in water ,7. The aications ,9,10. Inb-poly((polyethylene glycol) acrylate) (polyPA-b-polyPEG-A) and a pyrene-terminated polyethylene glycol (pyrene-PEG) were used by Liu et al. [Other approaches, based on the direct exfoliation of graphite in water, have been reported. A pyrene-functionalized amphiphilic block copolymer, poly(pyrenemethyl acrylate)-u et al.  and Zhenu et al. , respectBiocompatible polymers have been functionalized with pyrene moieties to prepare aqueous graphene suspensions for biomedical applications ,14,15. PIn this paper, the synthesis of a pyrene derivative through a simple and low-cost functionalization methodology is presented. This synthesis presents a high yield of the modified pyrene that may be scaled up for large-scale production. The synthetized pyrene derivative is soluble in water, and it was tested for the exfoliation and stabilization of three different types of graphite nanoplatelets (GnP) , which w2/g, while GnPH5 has a nominal equivalent diameter of 5 µm, thickness of 15 nm, and an average surface area of 60–80 m2/g. GnP Micrograf, provided by Nacional de Grafite , is a micronized graphite with an equivalent diameter of about 10 µm, and Graphexel is a natural crystalline graphite with a large equivalent diameter of approximately 180 µm that was provided by Graphexel Ltd. . Copper (II) nitrate trihydrate (Cu(NO3)2·3H2O), anhydrous copper (II) sulphate (CuSO4), and potassium hydroxide pellets (KOH) were purchased from Sigma Aldrich . Maleic anhydride 99% pellets and pyrene 98% were purchased from Acros Organics . 4-nitrobenzaldehyde was obtained from Merck . Acetic anhydride (Ac2O) and sodium borohydride (NaBH4) were purchased from VWR chemicals . Ethyl acetate (EtOAc), petroleum ether 40–60 °C, ethanol absolute (EtOH) and acetonitrile (ACN) were purchased from Fisher Scientific . Anhydrous magnesium sulphate (MgSO4), dicholoromethane (CH2Cl2), and diethyl ether were obtained from Panreac . Deuterated dimethyl sulfoxide  was purchased from Euriso-top .Two grades of GnP were purchased from XG Sciences , grade C (GnPC), and grade H5 (GnPH5). GnPC has, according to the manufacturer, a size distribution ranging from very small (100 nm) to relatively large flakes (1–2 µm), an average thickness of approximately 2 nm, and a typical average surface area of 750 m1H and 13C nuclear magnetic resonance (NMR) ). The detailed information about the synthesis of the pyrene derivatives and its spectral characterization . Graphite suspensions were prepared by mixing approximately 5.0 mg of graphite in 10 mL of the pyrene derivative solution using an Ultrasonic processor UP100H from Hielscher  equipped with a sonotrode MS7D. Ultrasound energy was applied to the suspensions for 1 h at maximum power. The suspensions were centrifuged  to remove larger aggregates, and the supernatant was collected. These stable suspensions were analyzed by UV-visible spectroscopy on a Shimadzu UV-2401PC, using quartz cells with a 10-mm pathlength. The concentration was determined using the Beer–Lambert Law, and the extinction coefficient for these materials was calculated using a procedure reported elsewhere [The suspensions were sprayed on a glass slide using a XL2000 Airbrush  with a 0.6-mm nozzle and analyzed by Raman spectroscopy. Raman spectra were obtained on a Horiba LabRAM HR Evolution confocal microscope  using a laser excitation of 532 nm (2.33 eV). A 100× objective lens was used to focus the laser onto the sample. For each sample, 60 spectra were collected randomly over a sample area of 50 × 20 mm, and analyzed using the LabSpec 6 software . Lorentzian functions were used to fit the characteristic peaks of the spectra.2.Scanning transmission electron microscopy (STEM) samples were prepared by the deposition of liquid suspensions of exfoliated graphite samples on 400 mesh carbon coated copper grids  and dried using a hot plate. The samples were analyzed using a NanoSEM FEI Nova 200 microscope . The dried samples, which were obtained by the solvent evaporation of the liquid suspensions of exfoliated graphite, as well as the graphite starting material, were analyzed by scanning electron microscopy (SEM) using the same equipment. The thermogravimetric analysis (TGA) of these powder samples was performed on a Modulated TGA Q500 from TA Instruments , heating the samples at 10 °C/min under a constant flow of NX-rays diffraction (XRD) measurements were performed on a PANalytical X´Pert PRO XRD System  using Cu–Kα radiation with the X-ray tube operated at 45 kV and 40 mA. The XRD spectra were obtained by continuously scanning 2theta/omega axes in steps of 0.0098 2theta°, using a scanning line detector with Soller slits and a Ni filter. The X-ray beam was incident over the sample in line focus type using a 1° divergence slit and also Soller slits. The XRD samples were obtained from solvent drying from the bulk solution, powder samples, and by spraying the solution over a heated silica wafer.In the present work, an amphiphilic pyrene derivative was synthesized for the purpose of graphite exfoliation, combining the pyrene moiety for an effective π-π interaction with the graphene layers and a hydrophilic carboxylic group for affinity with water , which is typical for this isomer [13C NMR spectrum at δ 164.46 ppm (for the amide) and δ 166.84 ppm (for the carboxylic acid). The FT-IR spectra are also presented as −1 and 1331 cm−1, corresponding to the –NO2 asymmetric and symmetric stretching vibrations. After reduction to 2, the characteristic symmetric and asymmetric stretching vibrations of the primary amino group can be seen at 3445 cm−1 and 3379 cm−1. The reaction of product 2 with maleic anhydride leads to the final product 3 showing the characteristic intense bands for the stretching vibration of the carbonyl groups at 1711 cm−1.The structure of compound 3 was confirmed by the presence of the amide and the carboxylic acid protons at δ10.84 and 13.26 ppm, respectively, in the s isomer . The two2O). The pyrene derivative 3 (PY) was used to exfoliate the graphite nanoplatelets GnPH5, GnPC, Micrograf and the natural graphite Graphexel in water, and stabilize the FLG formed. The same exfoliation/centrifugation procedure was applied to the different graphite grades using distilled water without PY , and the concentration of FLG was determined by UV-visible spectroscopy. The results are presented in The TGA curves of the graphite starting materials Graphexel, Micrograf, GnPH5, and GnPC show thermally stable materials with a weight loss at 600 °C of 0.6 wt%, 0.1 wt%, 2.4 wt% and 5.4 wt%, respectively. GnPC presents a larger weight loss at 600 °C, which may be due to its smaller flake size and decomposition of the oxygen-containing groups. The weight loss of the corresponding FLGs, also at 600 °C, are 39 ± 2 wt%, 35 ± 2 wt%, 34 ± 1 wt%, and 12 ± 2 wt% respectively, and these large values are due to the decomposition of the PY molecules adsorbed on the FLG surface.−1, is present in all of the materials with conjugated C–C double bonds and reflects the in-plane bond stretching motion of pairs of C sp2 atoms [laser), but is sensitive to temperature, strain, and doping of the graphene sample. The D mode, which is observed near 1350 cm−1, is a breathing mode of A1g symmetry that is forbidden in perfect graphene, becoming active in the presence of disorder [2 carbon network disturbed by chemical bonding such as that observed adjacent to a graphene edge or a defect. The D band is highly dispersive as a function of the Elaser. The two-dimensional (2D) band is a second-order mode that is sometimes referred as an overtone of the D band, although it is not related to graphene defects, and is always strong in graphene, even when the D band is absent. The shape and position of the 2D band varies with the number of graphene layers [−1, while bilayer graphene requires four characteristic Lorentzian functions, each with a FWHM of ~24 cm−1. As the number of layers increases, the 2D band shifts to a higher wave number and becomes broader and more asymmetrical in shape. Its deconvolution becomes more complex, and for few-layer graphene (less than 10 layers), it may be fitted with three Lorentzian functions with a FWHM higher than 24 cm−1 each. For more than 10 layers, the 2D band becomes similar to that of graphite, and may be fitted with two Lorentzian functions [Raman spectroscopy is an important tool for the characterization of graphene and carbon-based materials. The Raman spectrum of single-layer graphene presents three main characteristic bands, associated with the G, D, and 2D (or G′) modes. The G mode, which is observed near 1580 cmp2 atoms . Due to disorder . Its pree layers . For sinunctions ,38. Figu−1 is almost absent compared to the G band (at 1581 cm−1), which indicates the good structural quality of the graphite. The 2D band position of Graphexel and GnPH5 occurs at approximately 2722 cm−1, and its shape is asymmetric, which is typical of the graphite 2D band [−1), which is typical of a more exfoliated material. The 2D band of all of these starting materials can be fitted with two Lorentzian functions, as shown in −1, which was deconvoluted with four Lorentzian functions , which may be related to the smaller flake size. Thus, it also has a higher edge-to-basal plane ratio, as well as a higher oxidation level, which is in agreement with the producer specifications and the TGA analysis. The 2D band position occurs near 2692 cm−1, showing high symmetry, although with lower intensity compared to the G band. The 2D band of the GnPC starting material can be fitted with two Lorentzian functions, as shown in The Raman spectra of the GnPC starting material that was exfoliated with PY are presented in A statistical analysis of the flakes formed in suspension by exfoliation of graphite in water using PY was performed by Raman spectroscopy. The analysis was based on the Raman spectra collected for 60 different flakes of each exfoliated material , and the spectra of bilayer and FLG were identified. The FLG and bilayer graphene spectra were identified based on the position of the 2D band and its deconvolution, and are illustrated in It should be pointed out that the GnPC starting material is itself a highly exfoliated material, as its 2D spectrum is quite different from that of graphite d. Jang eThe morphology of the graphite materials and corresponding PY exfoliated materials is illustrated in The Raman spectroscopy studies reported above demonstrated that the application of energy to graphite in a PY aqueous solution produces a GnP stable suspension with more that 70% of the suspended GnP flakes exfoliated into FLG. The question arises now concerning the nature of the interactions between PY and the FLG surface. In particular, how PY is adsorbed at the FLG surface, how many PY molecules can be accommodated on a given area, and if they form a monolayer or stacks of several adlayers? Also, does PY tend to stack and form organized structures itself? In order to answer these questions, we used X-ray diffraction (XRD) and molecular modeling studies. X-ray diffraction studies were performed on a set of materials produced from FLG/PY suspensions by water evaporation; molecular modeling studies were performed that helped understanding the nature of the graphene/PY interactions.A GnPH5 PY suspension was prepared as described in the experimental section, and the GnPH5 PY powders were obtained by water evaporation from the bulk suspension in an oven at 90 °C. A PY solution with the same concentration used for exfoliation was prepared, and the PY powder was collected by solvent evaporation. The XRD profiles presented in The PY in powder form, which was obtained after dissolution in water and drying, does not display any peaks in the XRD difractogram, showing that this pyrene derivative does not form organized structures upon solvent evaporation. Thus, the results obtained indicate that when FLG and PY molecules are present in solution, an ordered structure is formed additionally to that observed for the original GnPH5. If these structures originate on PY organization/stacking, then they must be formed from several PY adlayers in order to produce a 3D crystal, and grow from the FLG surface.The same GnPH5 PY suspension was used to prepare a sample by spray drying that was also analyzed by XRD. The deposition/solvent drying method may strongly influence the molecular organization in the powder formed. Here, the additional XRD peaks observed on GnPH5 PY from slow drying, which are absent on the GnPH5 PY obtained by fast drying, indicate that PY stacking occurred from solution only under slow-drying conditions.A graphene hexagonal flake possessing a closed-shell electronic ground state, 546 carbon atoms, and 5-nm radii was chosen as the substrate. The use of flakes, as opposed to periodic systems, allows the explicit study of edge effects, the possibility of side switching, and the possibility of solvated systems. The adsorption of a variable number of the functionalized pyrene molecules, namely 1, 2, 4, 6, 7, 8, 9, 10, 11, and 12, was computed. For this, we performed molecular dynamics (MD) simulations using the molecular mechanics MMFF94 force field as implemented in the software Tinker. For simplicity and computational efficiency, the graphene substrate was held fixed during the classical simulations. A simulated annealing technique with initial and final temperatures of 500 K and 300 K respectively was chosen in order to improve the sampling of the potential energy surface during the simulations. Simulations were run for 20 ns with a RESPA integrator that allowed the use of a time step of 2 fs. Snapshots of the trajectory were selected at 2 ps intervals, yielding 10,000 different adsorption geometries that were optimized with the MMFF94 force field. Two representative geometries for each system were selected; geometries with the lowest total energy and geometries with the lowest intermolecular energy and optimized at the PM6-D3H4 level with the program MOPAC16 in vacuum, giving comparable results . The relative position of the adsorbate with respect to the graphene flake was monitored through the MD simulations by following the Cartesian coordinates of the nitrogen atom of PY, as nitrogen atoms with positions up to 5 Å from the flake plane were found to correspond to PY molecules directly adsorbed onto the surface. The typical number of adsorbed neutral pyrene molecules during the MD simulations was found to be eight. The maximum number of molecules that this flake can accommodate in one adlayer was found to be 10, but this was seldom found during the MD simulations. The geometries for the lowest energy minima found at the MMFF94 level were optimized at the PM6-D3H4 level (not shown) and at the GFN-xTB level methods for PY in its neutral (carboxylic) and anionic deprotonated (carboxylate) states; these are presented in For neutral PY in vacuum, the binding energy changes considerably, from −38 kcal/mol for 1 adsorbed PY to ~50 kcal/mol for 4 PY and 8 PY adsorbed molecules, respectively. This is due to the attractive interaction between PY molecules, which peaks for 4–8 PY molecules. When the carboxylate form of PY is considered, the binding energy for one molecule, −39 kcal/mol, is similar to the binding energy for neutral PY. However, in this case, it does not change with the number of adsorbed molecules, as in negatively charged PY adsorbates and the electrostatic repulsion counterbalances the hydrogen bonding and dispersion attractions. The differences between the neutral and the anionic form of PY are clear when the graphene flake is saturated with 10 PY molecules. In the neutral form, the lowest energy structure accommodates 10 PY molecules in more than one adlayer, while in the anionic form, the opposite is observed, i.e., one adlayer is more stable, as electrostatic repulsion inhibits the deposition of more PY adlayers.The optimized geometries at the GFN-xTB level for four, eight, and 10 deprotonated PY molecules in water are presented in The experimental TGA weight loss (WL) results have been compared with the estimate calculated considering the mass of PY in solution and the exfoliation yield of carbon in suspension (from UV-visible spectroscopy); these are presented in The calculations showed that the experimental WL is close to the estimated WL for all of the materials, considering that all of the pyrene derivative remains in solution. During solvent evaporation, the PY molecules may deposit randomly or in a regular manner, with the latter leading to the formation of stacked PY adlayers at the surface of the exfoliated material ,25. ThesA pyrene derivative was synthetized through a simple and low-cost functionalization methodology that leads to a high yield of the final compound, and can potentially be scaled up for large-scale production. The solubility of this pyrene derivative in water allowed its use for the exfoliation and stabilization of different types of graphite in aqueous media. The statistical Raman spectroscopic analysis showed the formation of bilayer and few-layer graphene after the exfoliation process of all of the types of graphite, and the STEM images showed the formation of thin flakes, in agreement with Raman spectroscopy. The XRD results showed the d-spacing of the larger FLG materials, similar to the graphite, and the molecular modeling demonstrated the maximum number of PY molecules that can be adsorbed to the surface of graphene. The estimated weight loss in the TGA analysis is similar to the experimental values, suggesting an excess of PY molecules adsorbed on the exfoliated material in more than one adlayer. The very low concentration of pyrene derivatives used in this work  to exfoliate graphite in water opens up the possibility of an efficient and cost-effective process to produce FLG. The application in biomedical areas requires further studies on the toxicity and biocompatibility of this new pyrene derivative. The FLG may be deposited directly from the aqueous suspension using techniques such as spray or spin-coating, or in the form of composites with water-soluble polymers or stable aqueous suspensions of polymers. The FLG may be collected from the aqueous suspensions by solvent evaporation and used in powder form in the production of polymer composites by melt-mixing methods.


Herein, hybrid k-carrageenan bio-nanocomposite films were fabricated by using two types of nanofillers, organically modified montmorillonite (OMMT), and cellulose nanocrystals (CNCs). Hybrid bio-nanocomposite films were made by casting techniques employing 4 wt% of CNCs, OMMT, and hybridized CNCs/OMMT in a 1:1 ratio. The rheological and morphological properties and thermal stability of all composites were investigated using rotational rheometry, thermogravimetry analysis, differential scanning calorimetry, field emission scanning electron microscopy, and transmission electron microscopy (TEM). The results showed that the hybrid CNC/OMMT bio-nanocomposite exhibited significantly improved properties as compared to those for the bio-nanocomposites with single fillers due to the nanosize and homogenous nanofiller dispersion in the matrix. Rheological analysis of the hybrid bio-nanocomposite showed higher dynamic shear storage modulus and complex viscosity values when compared to those for the bio-nanocomposite with individual fillers. The TEM analysis of the hybridized CNC/OMMT bio-nanocomposite revealed that more particles were packed together in the CNC network, which restricted the matrix mobility. The heat resistance and thermal stability bio-nanocomposite k-carrageenan film enhanced rapidly with the addition of hybridized CNCs/OMMT to 275 °C. The hybridized CNCs/OMMT exhibited synergistic effects due to the good affinity through interfacial interactions, resulting in the improvement of the material properties. Eucheuma cottonii) and consisting of repeating 4-linked 3,6-anhydro-D galactose and 4-linked β-D-galactose-4-sulphate units with anionic sulfate groups (OSO3−) disaccharide units [Biopolymers formulated with reinforcement materials such as natural fibers, nanoparticles, and synthetic fibers are currently being developed because of their unique properties to reduce the usage of synthetic plastics, particularly for short-term packaging and disposable materials ,2. K-carde units ,6. The ide units ,8,9. BioNanocellulose that can be extracted from abundant natural fiber exhibits properties such as relatively large surface areas, low density, high specific stiffness, high strength, and easy processability ,11,12. CNanocomposites based on polysaccharide and nanoclay reinforcements are widely used across various research fields as clay minerals are cheaper, and biodegradable nano additives used to enhance the properties of polymers materials are known as polymer–clay nanocomposites ,17,18,19In prior studies, k-carrageenan bio-nanocomposite materials were studied with a focus on the effects of the CNC and OMMT on the mechanical properties to determine the optimum nanoparticle loadings. Morphological studies show the well distribution of CNC and OMMT nanoparticles in the matrix. Hybridized CNC/OMMT nanofillers exhibit synergistic effects resulting in excellent mechanical properties, good water resistance, with decreasing absorption of water of the bio-nanocomposite in contrast to the bio-nanocomposites with single fillers . PreviouThe ultimate aims of this current work are to show how the hybridization of different organic and inorganic nanofillers affect the rheological behavior and thermal stability properties, and to extensively investigate the entanglement of the nanoparticles inside the matrix for applications depending on the location distribution of the nanofillers. Rheological properties of the materials are important during polymerization as well as downstream processing of the product . The measurement of the viscosity of the bio-nanocomposite can provide direct evidence for changes in the product quality due to the destabilization in a polymer .®; Hoffman Estates, IL, USA). Sulfuric acid (98%), Glycerol (99.5%), glacial acetic acid (99.5%), sodium chlorite (80%), and sodium hydroxide (99%), were obtained from SYSTERM-chemAR  and Sigma-Aldrich . Membrane tubes  were used for dialysis. Kenaf fiber was purchased from Kenaf Fiber Malaysia Sdn. Bhd.  and k-carrageenan was provided by TACARA Sdn. Bhd. . Methyl dihydroxyethyl hydrogenated tallow ammonium (OMMT 25–30 wt%) was purchased from AMCOL International Corp. , k-carrageenan–organically modified montmorillonite (k-carr–OMMT), and hybrid k-carrageenan–cellulose nanocrystal/organically modified montmorillonite (k-carr–CNCs/OMMT) bio-composite films was accomplished by the solution casting technique. K-carrageenan was first mixed with the 4% (w/w) nanofillers in distilled water. The suspension was heated in the range of 70–90 °C. The suspension was continuously stirred for another 30 min after the heating was removed. The thickness of the bio-nanocomposite film was controlled using a casting knife before being dried at room temperature to obtain the final film thickness in the range of 30–50 µm.The morphological observation of the fibers after each treatment was examined via field-emission scanning electron microscopy (FESEM) at a magnification of 1.00 K× and 15.00 K× . The morphology of the CNCs particles and bio-nanocomposite film in the water medium was analyzed by using transmission electron microscope , as described by Zakuwan and Ahmad .The rheological behavior of the polymers was carried out using a dynamic rotational rheometer  as described by Noranizan et al. . The sto−1. The thermal stability and degradation studies of the bio-nanocomposite films were performed using a thermogravimetric analyzer  under a nitrogen atmosphere. Each sample was heated from 25 °C to 600 °C at 10 °C·min−1. Thermal stability characteristics of the k-carrageenan bio-nanocomposite were determined using a differential scanning calorimeter . The heating processed up to 200 °C at a rate of 10 °C·minThe scanning electron microscopy (SEM) micrograph of the kenaf raw fiber after different chemical treatments are shown in The morphologies and sizes of the obtained CNCs were determined using TEM. TEM images of the CNCs produced after the acid hydrolysis in water are shown in The morphology of the hybrid CNCs/OMMT bio-nanocomposite films were analyzed using TEM. 3−) on k-carrageenan enable its interaction with the inorganic cations, with polymer chain intercalation [A rheological study of the bio-nanocomposites showed high complex viscosities and dynamic shear storage moduli for the CNCs, OMMT, and hybridized CNCs/OMMT. This study is useful for understanding the relationship between the nanofillers. calation . A decrecalation . Figure The variation in the complex viscosity values for the neat matrix and bio-nanocomposite films is shown in The thermal stability of the k-carrageenan matrix and bio-nanocomposite with the addition of CNCs, OMMT, and hybridization was investigated by using TGA. 42−) [Further degradation of the k-carrageenan matrix film occurred at approximately 228–236 °C. The addition of CNCs to the k-carrageenan matrix increased the decomposition temperature to approximately 264–278 °C at 4-wt% filler loading. The maximum degradation occurred at 270 °C. Furthermore, the addition of CNCs to k-carrageenan significantly increased the mass loss because the CNCs were less thermally stable compared to OMMT. Higher thermal decomposition with the incorporation of CNC due to high crystallinity, and had a higher aspect ratio of the CNC that was well dispersed within the matrix. This result shows that CNCs interacted with the matrix k-carrageenan and enhanced the thermal degradation of the corresponding bio-nanocomposites to a greater extent. Meanwhile, a drastically degradation rate due to the presence of negative sulfate groups (SO42−) . The add42−)  showed so, and the peak temperature, Tp, is important to determine whether a material is close enough to lose its structure during the heating process [p of 94 °C. A factor that influences the sealing strength is the hydroxyl groups in the plasticizers. Endothermic peaks for k-carr/CNCs were observed at 54–120 °C with a Tp of 94 °C. This proves that k-carr/CNCs show a higher transition temperature when compared to the neat matrix, with the lowest onset temperature and afforded better seal strength. The K-carr/OMMT bio-nanocomposite exhibited a transition temperature in the range of 54–115 °C with a Tp of 93 °C, which was lower than that of the neat matrix. Based on the TGA results, k-carr/OMMT only affords an increase in thermal stability  data  for the  process . All filtability . The DSCCellulose from kenaf and CNCs with average diameters and lengths of 12–15 nm and 101–260 nm, respectively, were obtained using alkaline treatment, bleaching, and acid hydrolysis. K-carrageenan bio-nanocomposite films reinforced with 4-wt% CNCs and OMMT were prepared via a casting technique. The hybrid bio-nanocomposite was prepared with the addition of 4-wt% CNCs/OMMT (1:1). Different types of nanofillers enhanced the thermal stabilities and rheological properties of the bio-nanocomposites. Hybrid bio-nanocomposite films showed a significant enhancement of various properties when compared with those for the k-carrageenan with CNC and k-carrageenan with OMMT bio-nanocomposite. Bio-nanocomposite films with CNCs/OMMT showed better rheological characteristics among the three formulations due to the entanglement of CNCs/OMMT inside the matrix phases, and the development of a network filler structure leading to the enhanced filler–matrix interactions. TEM data revealed that the OMMT particles were packed together in the CNC network, which restricted the matrix mobility. The hybrid bio-nanocomposite films exhibited synergistic effects with higher thermal degradation temperature when compared to the bio-nanocomposite films with single fillers. This improvement was associated with the homogeneous and well dispersed CNCs and OMMT, high aspect ratio for different dimensions, and the interfacial and ionic interactions between CNCs and k-carrageenan functional groups. It can be concluded from this study that a uniform mixing of the hybridization of CNC/OMMT with different polarities like CNCs and OMMT can increase the rheological behavior and that the thermal stability properties have the ability to make a fully biodegradable nanocomposite, and hence present a wide range of potential in packaging and coating applications.


Background: Applications of two-dimensional (2D) materials in electronic devices require the development of appropriate measuring methods for determining their typical semiconductor parameters, i.e., mobility and carrier lifetime. Among these methods, contactless techniques and mobility extraction methods based on field-effect measurements are of great importance.Results: Here we show a contactless method for determining these parameters in 2D semiconductors that is based on the photomagnetoelectric (PME) effect . We present calculated dependences of the PME magnetic moment, evoked in 2D Corbino configuration, on the magnetic field as well as on the intensity and spatial distribution of illumination. The theoretical predictions agree with the results of the contactless investigations performed on non-suspended single-layer graphene. We use the contactless PME method for determining the dependence of carrier mobility on the concentration of electrons and holes induced by a back-gate voltage.Conclusion: The presented contactless PME method, used in Corbino geometry, is complementary to the mobility extraction methods based on field-effect measurements. It can be used for determining the mobility and diffusion length of carriers in different 2D materials. The application of two-dimensional (2D) materials in electronic devices –6 requirB, perpendicular to the photogenerated carrier concentration gradient, the diffusing electrons and holes are deflected in opposite directions. Their current flows in a third, mutually perpendicular direction. The PME response decreases with the increase of recombination rate. Of course, the PME signal is stronger for higher μ. Therefore, investigations of the PME effect are used to determine carrier recombination and transport [There are a few phenomena which are or may be called PME effects. For example, in semiconductors the simultaneous action of light and magnetic field evokes specific electromotive forces . In the ransport –14 as weransport –17. The ransport –20 whichMette  proposeds) of the illuminated (front) and unilluminated (back) surfaces were previously developed in [s [s was possible only after calibrating the apparatus with standard samples of known semiconductor parameter values [The formula describing the PME magnetic moment, evoked in a bulk semiconductor wafer illuminated by weakly absorbed radiation as a function of τ, sample thickness, and surface recombination velocity  is taken into consideration. To test the theoretical predictions, we apply the induction technique in PME investigations of single-layer graphene on polyethylene terephthalate (PET) foil. The main aim of our work is to use the PME method for determining the dependence of μ on the concentration of electrons and holes induced by a back-gate voltage in graphene.00 light with photon energy greater than its optical energy gap. The transport of electrons and holes through 2D samples in the presence of a steady magnetic field under photogeneration and recombination is described by the solution of Maxwell's equations, the Poisson equation, the continuity equations and the transport equations  is the sum of azimuthal components of electron and hole current densities,  is the position vector, and r is the distance from the center of the light spot.where  as a function of parameters describing the experiment: magnetic field (B), beam radius (Rb) and maximum intensity (IV0) of the radiation incident upon a sample. These calculations were done for different values of τ and μ.Assuming an absorption coefficient of light appropriate for graphene (α = 1/137 ), the thMb = ) on the illumination intensity for different values of the beam radius. The Mb is a nonlinear function of Rb  and for μ smaller than about 1 m2V−1s−1, the  is proportional to the square of the carrier mobility  cm2V−1s−1 and sheet carrier concentration ne = 4.65(6)·1016 m−2 in the graphene were determined using the Van der Pauw method. For these measurements of resistivity and the Hall coefficient, the samples were equipped with 150 nm thick Au electrodes deposited using a Q150R ES rotary-pumped sputter coater with a film thickness monitor. For the PME-Corbino investigations of graphene with electrostatically tunable carrier density, a 150 nm thick Au film was also deposited on the back surface of the PET foil.The investigated graphene samples were supplied by Graphene Supermarket. The single-layer graphene films were grown by CVD processing on a cooper foil and transferred onto a 170 μm thick polyethylene terephthalate (PET) foil. The polycrystalline graphene films covered about 90% of the foil, with occasional holes and cracks. The presence of single-layer graphene was confirmed by Raman spectroscopy using an NTEGRA Spectra (NT-NDT) device with a wavelength of 532 nm. The carrier mobility μP = 100 mW). The laser diodes were biased with an ITC510 (Thorlabs) laser diode controller. In our experiments, the laser diode bias was sinusoidally modulated around a DC bias chosen to ensure that the waveform of the light output signal is sinusoidal with negligible distortion. The radiation intensity was changed using neutral density filters . The intensity was monitored using a Hamamatsu S3399 photodiode whose short-circuit current was measured using an EG&G 5110 lock-in amplifier. The time dependence of the illumination was determined using a Rigol Technologies DS1202CA oscilloscope. The spatial distribution of illumination  in a weak magnetic field [e = 1256(25) cm2V−1s−1 determined in the Van der Pauw measurements.nduction  and to tnduction . Such deic field . These ric field –28 and wVPME on the frequency (f) of sinusoidal modulation of the illumination presented in 2f2τ2 << 1 (see Equation S19 in The linear dependence of VPME voltage (induced in a measuring coil) and the investigated PME magnetic moment. However, the relative changes of a material parameter due to an external factor can be determined. Therefore, in this work, the PME-Corbino investigations of the dependence of carrier mobility on concentration of electrostatically tunable carrier density (N) were performed on a back-gated sample of graphene (e = 1256(25) cm2V−1s−1 and ne = 4.65(6)·1016 m−2 determined using Van der Pauw method).Unfortunately, the observed linear dependences of the PME response on experimental parameters –d have ngraphene  with knoVg, induces a sheet carrier concentration approximated by [0ε/we)Vg, where ε0 and ε are the permittivity of free space and the used dielectric, respectively; e is the electron charge; and w is the thickness of the dielectric. The authors of [It should be underlined that one of the most important properties of graphene –35 and omated by  (ε0ε/we). Therefore, the left axis in In our case, the energy position of the Fermi level of graphene was modulated by the field effect through the 170 μm thick PET foil. The relative permittivity of PET is ε = 3.5 at 71 kHz . Figure It was experimentally shown that the carrier lifetime only decreases slightly with carrier density in graphene . Taking The electron–hole asymmetry is concluded from the observed PME response in non-suspended graphene. It agrees with theoretical predictions  based onThe presented PME method is restricted to photogenerated carriers and does not account for several groups of carriers with distinct mobilities, and in particular, hot electrons. A brief review  presenteThe investigation of complex phenomena in 2D materials requires the combination of multiple experimental techniques. The contactless PME method, used in Corbino geometry, is complementary to the mobility extraction methods based on field-effect measurements. In the case of small carrier mobility and weak magnetic field, the PME magnetic flux is proportional to the fourth power of the diffusion length of carriers. The contactless PME method can be used to obtain scan maps of local values of 2D semiconductor parameters, i.e., diffusion length, mobility or lifetime of carriers, in future investigations.Possible difficulties in applying the proposed method may result from disturbances through the Corbino–Nernst effect from radial temperature gradients that are caused by heating of the center spot in the sample with the incident light beam. However, the distorting photovoltage and thermovoltage evoked due to illumination of the near-contact regions are eliminated in the proposed PME method. The existence of photogenerated hot carriers which can interact with the equilibrium free carriers must be taken into serious considerations in future experiments.File 1Theoretical description of the photomagnetoelectric effect in 2D materials in Corbino configuration.


Mutations in minor spliceosome components such as U12 snRNA (cerebellar ataxia) and U4atac snRNA ) result in tissue-specific symptoms. Given that the minor spliceosome is ubiquitously expressed, we hypothesized that these restricted phenotypes might be caused by the tissue-specific regulation of the minor spliceosome targets, i.e. minor intron-containing genes (MIGs). The current model of inefficient splicing is thought to apply to the regulation of the ~ 500 MIGs identified in the U12DB. However this database was created more than 10 years ago. Therefore, we first wanted to revisit the classification of minor introns in light of the most recent reference genome. We then sought to address specificity of MIG expression, minor intron retention, and alternative splicing (AS) across mouse and human tissues.We employed position-weight matrices to obtain a comprehensive updated list of minor introns, consisting of 722 mouse and 770 human minor introns. These can be found in the Minor Intron DataBase (MIDB). Besides identification of 99% of the minor introns found in the U12DB, we also discovered ~ 150 new MIGs. We then analyzed the RNAseq data from eleven different mouse tissues, which revealed tissue-specific MIG expression and minor intron retention. Additionally, many minor introns were efficiently spliced compared to their flanking major introns. Finally, we identified several novel AS events across minor introns in both mouse and human, which were also tissue-dependent. Bioinformatics analysis revealed that several of the AS events could result in the production of novel tissue-specific proteins. Moreover, like the major introns, we found that these AS events were more prevalent in long minor introns, while retention was favoured in shorter introns.https://midb.pnb.uconn.edu.Here we show that minor intron splicing and AS across minor introns is a highly organised process that might be regulated in coordination with the major spliceosome in a tissue-specific manner. We have provided a framework to further study the impact of the minor spliceosome and the regulation of MIG expression. These findings may shed light on the mechanism underlying tissue-specific phenotypes in diseases associated with minor spliceosome inactivation. MIDB can be accessed at The online version of this article (10.1186/s12864-019-6046-x) contains supplementary material, which is available to authorized users. RNU4ATAC, which encodes for U4atac snRNA, has been linked to microcephalic osteodysplastic primordial dwarfism type I (MOPD1), Roifman syndrome and Lowry-Wood syndrome [Splicing of introns is required for the expression of most eukaryotic genes. A subset of mammalian genes contains both major and minor introns, therefore requiring both the major  and the minor  spliceosome, respectively [syndrome . One wayE2f4, which is part of the E2F family that already had E2f1, E2f2, E2f3, E2f5 and E2f6 listed as MIGs in the U12DB  . HoweverNext, we investigated the terminal dinucleotides of all the identified minor introns, and found that ~ 72% of the minor introns were of the GT-AG type, whereas only ~ 24% and ~ 22% was of the AT-AC type in mouse and human, respectively Fig. d-e. The Finally, we wanted to compare the identity of MIGs between mouse and human. To this end, we curated the orthologs listed in the Ensembl and NCBI database, and found that 586 genes contained a minor intron in both mouse and human genome  in one tissue compared to all other samples to generate the UpSignature  that often serves a physiological function . HoweverCoa3. This suggests that splicing and indirectly expression of these MIGs solely relies on the minor spliceosome, yet retention of this single minor intron was tissue-specific  in the cortex and the kidney  in bone marrow, kidney and thymus by RNAseq  was often coincident with a cryptic 3′SS CAT7)  was ofteTo test whether these isoforms could also result in the production of tissue-specific proteins, we performed bioinformatics analysis to predict the fate of alternatively spliced transcripts. RNA transcripts are under tight quality control, such that aberrant splicing can result in activation of several degradation pathways. For instance, intron retention can result in nuclear retention of the transcript and subsequent degradation by the exosome . AlternaFinally, we also wanted to determine the prevalence of AS around minor introns in humans. Again, we found that cryptic splicing of minor introns (CAT6 and CAT7) was used at the highest levels in all tissues  . The inhrow Fig. d. In allSNRPE, E2F2, INSIG1), minor introns were retained at a higher level than the flanking major introns [We favor such a model, which would mean that splicing and/or retention of minor introns can behave like the splicing of major introns. This model is in contrast to the previous model of stochastic or inefficient minor intron splicing resulting in prevalent retention of minor introns. The latter ideas were based on comparison of minor intron retention levels to that of the flanking major introns. Briefly, Patel et al., employed this comparison to test the hypothesis that minor introns are excised more slowly than the upstream and/or the downstream major introns . They sh introns . Afterwa introns . However introns . While wCBP20 became progressively more efficient by increasing the length of the intron [P120 has served as a golden standard for studying the efficiency of minor intron splicing. Thus, the confluence of a naturally occurring preference for retention of shorter minor introns and the choice of minor introns that were studied for technical reasons might have led to the minor intron retention as a model for regulating all MIGs.The apparent differences in our findings and the subsequent models could be explained by the choice of MIGs that were interrogated. We found that retention of shorter minor introns was detected at a higher level than the longer introns Fig. a. This fe intron . Togethee intron . The elee intron . Since tRnpc3 resulted in leukopenia and a severely reduced thymus lacking medullary or cortical regions [Given our observation that splicing of minor introns is highly regulated, it stands to reason that it is also tissue-specific. Indeed, we found dynamic levels of minor intron retention across different mouse tissues  for the 5′SS and 3′SS of minor introns (AT-AC and GT-AG type) and major introns (GT-AG and GC-AG type) were downloaded from SpliceRack . MoreoveThe curated splice sites of all introns were then scored against the PWMs for minor introns and major introns by adding the scores of each individual element  would be considered the canonical translation. If these were not present, then any transcript with the longest CDS would be considered the canonical isoform. If there was no CDS, then the longest non-protein coding transcript was assigned as the canonical transcript.Data analyzed in this study was downloaded from the DDBJ and EMBL database. They can be accessed using the following accession numbers: Mouse WT tissues  ; Human tReads from human tissue samples were aligned to the hg38 genome (UCSC genome browser) using Hisat2 as described previously , 46. ReaP < 0.01. Using this method, 55 pairwise comparisons were performed between all mouse tissues. Afterwards, the intersect was taken of all the upregulated genes. Genes that were binned as upregulated in all pairwise comparisons, were classified as being part of that tissue’s UpSignature. Conversely, genes that were binned as downregulated in one tissue compared to all other tissues were classified as being part of that tissue’s DownSignature  and utilized for the splicing efficiency analysis in Fig. To detect AS across minor introns, we extracted all uniquely mapped spliced reads spanning the minor intron. Reads mapping with only one nucleotide on either side of the junction were excluded. The remaining spliced reads were then binned into one of 9 splicing categories Fig. a using BP-value< 0.05 for DAVID; FDR-corrected P-value< 0.05 for ToppGene; g:SCS threshold< 0.05 for g:Profiler) were isolated. Only GO-Terms that were significant according to all three functional annotation tools are reported in Fig. UpSignature lists for each tissue were submitted for functional enrichment analysis to DAVID, ToppGene and g:Profiler –16. SignFor all alternatively spliced MIGs, we determined the new exons of the canonical transcript depending on the specific AS event that was observed. If multiple AS events were observed around a minor intron, these were treated as separate events. Moreover, if the read filter was passed for an AS event, as result of multiple coordinates being employed, but no single coordinate passed the read filter separately, the event was excluded from this analysis. An example would be the use of a cryptic 5′SS in a minor intron (CAT6) which was supported by 20 reads, however this was the sum of reads all supporting different coordinates being employed as a cryptic 5′ SS. The transcripts of the remaining protein-coding genes were then translated in silico and the open reading frame was predicted. The location of the new stop codon was compared with the annotated stop codon to determine whether the AS event had resulted in a truncation or extension of the ORF. If the new stop codon was found > 50 nt upstream of an exon-exon junction, the alternatively spliced isoform was predicted to be degraded by NMD. If no stop codon was identified, then the transcript was predicted to be degraded by NSD. In all other cases, the transcript was predicted to give rise to a protein.To determine whether AS events across minor introns had been annotated, we downloaded the exon coordinates of all MIG transcripts from Ensembl (v.95). Exon coordinates of each transcript were then compared to the minor intron coordinates and transcripts were binned into one of four categories: 1) transcripts that supported constitutive splicing of the minor intron, 2) transcripts that were truncated before the minor intron coordinates, 3) transcripts with their first/last exon within the minor intron coordinates, and 4) transcripts with AS across the minor intron.To determine whether the median length of retained vs. spliced minor introns were statistically different, we performed a Kruskal-Wallis rank sum test, followed by post-hoc Dunn test. A one-way ANOVA, followed by post-hoc Tukey test was employed for determining dynamic levels of retention for individual minor introns across tissues. To determine whether minor introns were retained at significantly higher levels than their flanking major introns, we also performed one-way ANOVA, followed by post-hoc Tukey test. Finally, to determine whether there was a significant difference in the median length of alternatively spliced vs. canonically spliced minor introns, we performed a Mann Whitney U-test.N = 3) were obtained from The Jackson Laboratory and anesthetized using isoflurane, and perfused using 0.125 M NaCl as described previously [Eight week old C57/Bl6J male mice (eviously . All mouN = 3). Cortex, heart, kidney, liver, lung, and testis were homogenized in 1 mL TRIzol (#15596018); skin, spleen, thymus and tibialis anterior  were homogenized in 500 µL TRIzol. RNA was extracted using the DirectZOL RNA Miniprep kit , per the manufacturer’s instructions. 1μg of total RNA was then used for cDNA synthesis, as described previously [Ten tissues were dissected from 8-week-old C57/Bl6J perfused male mice  analyses. For AS analysis in pan tissue, cDNA from three replicates was pooled. MSI values were determined by quantification of band intensity by ImageJ.Additional file 1:Figure S1.SignatureCalc pipeline. Schematic describing the pipeline to determine the UpSignature and DownSignature for bone. This pipeline was then run to determine these signatures for the other ten mouse tissues. NonDE = non-differentially expressed. (TIF 599 kb)Additional file 2:Figure S2. Functional enrichment of DownSignatures. (A) Dendogram showing the hierarchical clustering of mouse tissues based on overall gene expression. (B) Venn diagram showing the number of uniquely downregulated  MIGs in each tissue. (TIF 3875 kb)Additional file 3:Figure S3. Bioinformatics strategy for detection of retention and AS of minor introns. (A) Schematic describing the pipeline to detect minor intron retention, including filtering criteria. (B) Schematic describing the pipeline to detect novel AS events across minor introns, including filtering criteria. Adopted from Madan et al., 2015 [l., 2015 . See alsAdditional file 4:Figure S4. Minor intron retention is not dependent on consensus sequence. Frequency plots of the annotated 5′SS, BPS and 3′SS of minor introns that are retained in 3 (top), 1 or 2 (middle), or 0 (bottom) replicates of at least one tissue. (TIF 5672 kb)Additional file 5:Figure S5. Minor intron retention is tissue-specific in human tissues. (A) Piechart with number of minor introns that show retention in number of replicates of at least one tissue. Boxplots reflect the 5th–95th percentile of minor intron length in each of the three categories. Significance was determined by Kruskal-Wallis rank sum test, followed by post-hoc multiple comparison using Dunn method. ** = P < 0.001; n.s. = not significant. (B) Histogram of the number of tissues in which minor introns were retained. (C) Venn diagram showing the overlap of retained minor introns across eight human tissues. Only minor introns that passed filtering criteria in at least three replicates of a tissue were included. (D) Stacked bargraph showing the number of retained minor introns in each tissue in mouse (blue), human (red), or both (grey). (TIF 10985 kb)Additional file 6:Figure S6. Location of minor introns within canonical transcript. Piechart showing the percentage of minor introns that have flanking major introns. (TIF 2247 kb)Additional file 7:Figure S7. Alternative splicing across minor introns is annotated in the Ensembl database. (A) Bar graph showing the number of annotated isoforms in the Ensembl database which resulted from AS across the minor intron. (B) Venn diagram showing the combined usage of AS events in annotated isoforms that are alternatively spliced across the minor introns. (C) Boxplots reflect the 5th–95th percentile of minor intron length in minor introns that are and are not alternatively spliced. Significance was determined by Mann Whitney U test. ** = P < 0.01. Transcr. = transcription. (TIF 11148 kb)Additional file 8:Figure S8. Alternative splicing across minor introns in human tissues is dynamic. (A) Bubbleplot reflecting AS usage across minor introns in 8 human tissues. Size of the circle represents the number of introns that passed the filtering criteria, the colour represents the type of AS. (B) Venn diagram revealing the overlap of MIGs that are alternatively spliced in at least one tissue between mouse and human. MSI = mis-splicing index. (TIF 6629 kb)Additional file 9:Table S1. Minor intron coordinates. Contains the coordinates of the identified minor introns in mouse and human, as reported on MIDB. (XLSX 85 kb)Additional file 10:Table S2. MIG orthologs. Contains information on MIG orthologs between mouse and human. (XLSX 81 kb)Additional file 11:Table S3. MIG expression. Contains the expression of MIGs across mouse and human tissues in TPM. (XLSX 208 kb)Additional file 12:Table S4. Functional annotation. Contains the functional annotations enriched for by the UpSignature and DownSignature of mouse tissues. (XLSX 15 kb)Additional file 13:Table S5. Minor intron retention. Contains the MSI values of retained minor introns across tissues. (XLSX 23 kb)Additional file 14:Table S6. Flanking major intron retention. Contains the MSI values of retained upstream and downstream major introns flanking the minor intron. (XLSX 53 kb)Additional file 15:Table S7. Alternative splicing around minor introns. Contains the MSI values of alternatively spliced minor introns in mouse and human. (XLSX 58 kb)Additional file 16:Table S8. RNAseq sample information. Contains information on sequencing depth and mapping percentage of all RNAseq samples used. (XLSX 13 kb)


PD-L1 or PD-L2, driving elevated expression in the corresponding gene. In addition to the previously described upregulation of the PD-1 immunosuppressive pathway in Epstein-Barr virus (EBV)-positive stomach tumors, we also observed upregulation of the PD-1 pathway in cytomegalovirus (CMV)-positive tumors. Furthermore, we found signatures of T-cell and B-cell response in HPV-positive HNSC and EBV-positive stomach tumors and HPV-positive HNSC patients were associated with better survival when T-cell signals were detected. Our work reveals that viral infection may recruit immune effector cells, and upregulate PD-1 and CTLA-4 immunosuppressive pathways.Viruses drive carcinogenesis in human cancers through diverse mechanisms that have not been fully elucidated but include promoting immune escape. Here we investigated associations between virus-positivity and immune pathway alteration for 2009 tumors across six virus-related cancer types. Analysis revealed that for 3 of 72 human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSC) the HPV genome integrated in immune checkpoint genes  Cao et al. show that human papillomavirus-positive, head and neck squamous cell carcinoma patients are associated with better survival when T-cells are activated. This study suggests that viral infection may recruit immune effector cells and that it may activate PD-1 and CTLA-4 immunosuppressive pathways. These genomic biomarkers may aid in therapy selection6. Although large-scale sequencing projects such as The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) continue to catalog variants across cancer types, only a minority of patients harbor tumors with genomic aberrations associated with sensitivity to targeted therapy.Since the sequencing of the first genomic DNA from a leukemia patient8. PD-L1 and PD-L2 on tumor cells or antigen-presenting cells suppress T-cell immune response by binding to PD-1 on T-cells10. To escape attack by immune cells, tumor cells overexpress PD-L1 by gene amplification, utilization of an ectopic promoter, and disruption of 3′ untranslated regions (3′ UTRs)11, in addition to PTEN loss-of function12 and EGFR mutations13. Other studies indicate that EGFR mutations are not associated with an increased PD-L1 expression and a better clinical response of PD-L1 immune checkpoint inhibitors15. Elevated PD-L1 expression creates an immunosuppressive microenvironment that facilitates tumor progression16. Anti-PD-1 and anti-PD-L1 immune checkpoint blockades show favorable clinical outcome for treating patients with high PD-1 and PD-L1 expression20. Another important immune checkpoint pathway involves CTLA-4 and its ligands CD80 and CD86. CTLA-4 serves as a negative regulator of T-Cell activity. The anti-CTLA-4 blockade is also an effective therapeutic strategy to kill tumor cells22.Complementary to targeted therapy, cancer immunotherapy utilizes the host immune response to kill tumor cells24. Despite the importance of immune infiltrates and their theoretical associations with viral-positivity25, there is no systematic study of associations between virus-positive samples and the immune response except for some limited studies on human papillomavirus (HPV) in head and neck squamous cell carcinoma (HNSC)29. Our previous large-scale study demonstrated viral positivity in multiple cancer types30. Here, using TCGA RNA-Seq data for six virus-associated tumors, we systematically study the associations between virus-positivity and the tumor microenvironment, as measured by expression of PD-L1, PD-L2, PD-1, CD80, CD86, CTLA-4, Tim-3, LAG3, and 4-1BB and the prevalence of infiltrating immune cells across multiple types of human cancers. Specifically, we found the enrichment of a T-cell immune signature in HPV-positive and EBV-positive tumors compared to non-viral tumors. The increase of T-cell immune response is associated with a better prognosis in HPV-positive patients. In addition, we found HPV integrations at PD-L1 and PD-L2 are associated with high expression of these genes. Higher levels of PD-L1, PD-L2, PD-1, CD80, CD86, CTLA-4, Tim-3, LAG3, and 4-1BB expression were found in Epstein-Barr virus (EBV)-positive stomach adenocarcinoma (STAD) and cytomegalovirus (CMV)-positive colon and rectum adenocarcinoma (COADREAD) tumors compared to virus-negative tumors, providing the rationale for treating virus-positive tumors by anti-PD-1, and anti-CTLA-4 immune therapy. Besides PD-L1, PD-L2, PD-1, CD80, CD86, CTLA-4, Tim-3, LAG3, and 4-1BB, we found the increase of an inducible co-stimulator (ICOS) expression in both HPV-positive head and neck squamous cell carcinoma and EBV-positive stomach adenocarcinoma tumors. ICOS is the immune checkpoint protein, functionally and structurally related to CD2831. A positive ICOS signature may indicate a better clinical outcome of anti-CTLA-4 immune therapy in HPV-positive head and neck squamous cell carcinoma and EBV-positive stomach adenocarcinoma patients32.Immune infiltration of the tumor microenvironment correlates with improved survival in cancer patients30. The 413 HNSC tumors with clear HPV status were most common in two ethnicity groups: 364 Caucasians and 30 African Americans. There was no significant difference in HPV status between the two ethnicity groups. We additionally found that HPV-positive HNSC tumors were mostly from males (92%). See Supplementary Table PD-L1 or PD-L2 by using discordant read pair analysis . Although different integration patterns and anatomic sites were observed in the three tumors, HPV integrations at PD-L1 or PD-L2 were all accompanied by higher expression levels of these genes compared with those in virus-negative tumors .We analyzed 498 TCGA HNSC tumors using VirusScan and identified 72 HPV-positive tumors (numbers of virus-supporting reads per hundred million reads mapped (RPKM) > 100) and 341 virus-negative tumors (RPKM < 5)ors Fig. , and PD-PD-L1 or PD-L2 in other tumor types, we analyzed 229 TCGA HPV-positive cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) tumors and found no putative HPV integrations at either gene. We also checked for integrations of viruses, including HPV, hepatitis B virus (HBV), EBV, and cytomegalovirus, in other cancer types and found no integration events at PD-L1 or PD-L2. This result indicates that high PD-L1 or PD-L2 expression induced by HPV integration is a phenomenon that is selectively associated with HPV in HNSC tumors, with PD-L1 or PD-L2 integrations occurring in 4.2% of HNSC HPV-positive tumors.To examine the prevalence of HPV integrations at NR4A2, TBC1D1, BTNL9, DTX1, FOXP1, INPP4B, PDE4D, and STAT4 with HPV integration events are expression outliers (see Methods) and all these events are associated with the increase of expression.We further looked the relationship between HPV integration and expression in T-cell and B-cell genes. Supplementary Fig. PD-L1 and PD-L2 expression in HPV-positive HNSC, we next correlated any viral infection with PD-L1, PD-L2, PD-1, CD80, CD86, CTLA-4, Tim-3, LAG3, and 4-1BB expressions. In Fig. PD-L1 or PD-L2, i.e., TCGA-CV-5443 and TCGA-T2-A6X0 with 11.8 and 9.2 for PD-L1 and TCGA-HL-7533 with 10 for PD-L2 . RSEM stands for RNA-Seq reads by expectation maximization, which is widely used for quantifying gene expression34. Overall, no significant difference in PD-L1, PD-L2, CD80, or CD86 expression between HPV-positive and virus-negative HNSC tumors was found. A similar observation was also made for HBV , CD80 (p = 0.01), and CD86 (0.04) associated with EBV or cytomegalovirus positivity  between HPV-positive and virus-negative HNSC tumors  genes such as HLA-DQB1, HLA-DQA1, HLA-DMA, HLA-DMB, HLA-DRB5, and HLA-DOA. These genes play an important role for the formation of major histocompatibility complex (MHC) class II/peptide complex, which recognizes microbial antigens and cancer neoantigens45.Next, we identified 78 T-cell genes with significant differential expression  between EBV-positive and virus-negative STAD tumors Fig. . In the tus Fig. . B-cell tus Fig. , 4d. ThiICOS increases about two-fold in EBV-positive STAD and HPV-positive HNSC compared to virus-negative samples. ICOS is an immune checkpoint gene in CD28 and CTLA-4 family, which plays important role in regulating the immune response and enhances the antitumor immune response in anti-CTLA-4 blockade32.From our analyses, 78% of T-cell signature genes in EBV-positive tumors overlapped with those from HPV-positive HNSC, indicating that similar T-cell immune responses are associated with EBV and HPV. For instance, an ICOS signature was identified in EBV-positive STAD  passed the cut-off. We note that for PLAC8, a gene that is associated with pancreatic cancer progression46, was highly elevated in HPV-positive samples with about 16-fold increase compared to the virus-negative HNSC samples. However, in EBV-positive STAD sample, the expression of PLAC8 was downregulated by about four-fold compared to virus-negative STAD sample  and CD45 compared with virus-negative HNSC and colon and rectum adenocarcinoma tumors. Also, HPV-positive HNSC tumors and EBV-positive tumors had a higher expression of CD103, a marker for resident T-cells, compared to virus-negative samples, but not in cytomegalovirus-positive colon and rectum adenocarcinoma and HBV-positive liver hepatocellular carcinoma tumors.We further compared the T-cell differentiation phenotype in virus-positive and negative tumors for different viruses by using markers such as CD28, CD27, CD45, CD103, perforin, GMP-17, and granzymeAp < 0.01, Fisher’s exact test, two-tailed)  with frequency greater than 10% in HNSC tumors with elevated immune response. The HPV-positive cohort contained no variants in either TP53 or CDKN2A and only one in FAT1; in the HPV-negative cohort, these genes were frequently mutated. HNSC tumors with wild-type TP53 are more sensitive to radiation therapy than tumors with TP53 mutations51. In addition, we examined differentially expressed genes in the p53 signaling pathway between the two cohorts and found 4 of 16 p53 pathway genes showing substantial expression alteration  compared with virus-negative tumors  (ed) Fig. . We thened) Fig. ; in totaed) Fig. . Notablysee Fig. . Levels p = 0.01, Fisher’s exact test) between HPV integration status at PD-L1 or PD-L2 in HPV-positive HNSC (N = 72) and CESC (N = 229) tumors. Specifically, we found three integrations among the HNSC samples with high expression of PD-L1 or PD-L2 and no integrations among the CESC samples. It is likely that HPV has co-evolved to target PD-L1 or PD-L2 to create an immunosuppressive tumor microenvironment in some head and neck cancers. Further investigation with a large sample set is important for leveraging the observed relationship between virus integration at PD-L1 or PD-L2 in HNSC and the increased expression. We also found samples with HPV integrations in other immune-related genes  have an increased expression of these genes. Previous studies show that high expression of NR4A2, BTNL9, FOXP1, or PDE4D can antagonize immune response or is associated with tumor progression57. In EBV-positive STAD, cytomegalovirus-positive colon and rectum adenocarcinoma and EBV or cytomegalovirus positive stomach and esophageal carcinoma, we found that viral infection associates with high expression of PD-L1 or PD-L2, CD80, CD86, PD-1, CTLA-4, Tim-3, LAG3, and 4-1BB without integrating into the human genome. Moreover, our study indicates that EBV and cytomegalovirus elevate PD-L1 or PD-L2, CD80, CD86, PD-1, CTLA-4, Tim-3, LAG3, and 4-1BB expression in multiple cancers along the gastrointestinal tract including stomach and esophageal carcinoma and colon and rectum adenocarcinoma.In this study, we systematically investigated associations between virus infection or integration and alteration of the tumor microenvironment. We found a significant difference (58 demonstrated elevated PD-L1 expression in both tumor and immune cells across a large number of tumor samples by using immunohistochemistry (IHC) assays. For example, in 101 head-neck tumors, they found 28 and 19% of immune cell and tumor cell, respectively, were positive for PD-L1 in their samples. The anti-PD-L1 antibody works well on PD-L1 positive tumors to neutralize PD-L1 and make the tumor susceptible to attack by the immune system58. Our studies show an elevated PD-L1 expression in EBV and cytomegalovirus-positive samples, suggesting clinical trials of PD-L1 immunotherapy in these patients may be beneficial. Also, in a subset of HPV-positive HNSC, PD-L1 is highly expressed when HPV integrates into the PD-L1, suggesting these patients may have responded to anti-PD-L1 therapies. The previous clinical trial on HNSC has shown longer overall survival in both HPV-positive and PD-L1-positive tumors when treating with ant-PD-1 monoclonal antibody than the standard single-agent therapy59. Furthermore, we found that the expression of PD-L2, CD80, CD86, and CTLA-4 are also elevated in cytomegalovirus and EBV-positive tumor patients, suggesting anti-PD-L2 and anti-CTLA4 immunotherapy may be effective in patients with these types of tumors.A previous study62. In our previous large-scale study, we found a higher abundance of cytomegalovirus in tumors than in adjacent normal samples30. In the current study, we discovered a high level of PD-L1/PD-L2 in cytomegalovirus-positive tumors across the gastrointestinal tract suggesting that cytomegalovirus mediates the tumor microenvironment, which helps tumor cells to avoid the attack of immune cells.In contrast to the clear association between EBV and stomach adenocarcinoma, some controversy exists about the association between cytomegalovirus and colorectal cancerICOS and CTLA-4 in both HPV-positive HNSC and EBV-positive STAD, suggesting these tumors may have had an effective clinical response to anti-CTLA-4 immune therapy. Survival analysis shows high immune response is associated with favorable survival in HPV-positive but not HPV-negative HNSC samples. The different mutational status and expression patterns may lead to different clinical outcomes of the immune response. For instance, we found different expression alteration in key genes involved p53 signaling pathway in two cohorts. The complete retention of wild-type TP53 in HPV-positive HNSC tumors is another key factor driving the difference, as previous studies show better radiotherapy sensitivity in HNSC patients with wild-type TP5351. For cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), we separated tumors into low and elevated immune infiltration cohorts according to HPV-positive T-cell signatures  will enable us to investigate the virus-mediated tumor microenvironment at the protein level30, which is available from Github64. For the identification of virus integration sites in human genome, we first used BWA65 to align RNA-Seq data to the human plus viral reference. From the re-aligned bam file, we extracted the discordant read pairs, where one read of a read pair maps to human, the other to virus. Pindel66 was used to identify exact breakpoints for all samples with ten or more human-virus discordant reads. The breakpoints in Fig. 67.We discovered viruses in the tumor samples by using the VirusScan pipelinet-test to extract differentially expressed genes in virus-positive and negative samples, using FDR = 0.05 as the cut-off. The FDR value was obtained by p.adjust with Benjamini and Hochberg correction from R package. The heatmap figure was generated by using Heatmap.3R package with default parameters.Survival analysis was implemented by using R package survival. We used the Student’s To investigate if genes with virus integrations are expression outliers, we used the Tukey’s standard formula to quantify the outlier score:x − Q3)/IQR for upper tail and (x − Q1)/IQR for low tail,Outlier score =  are constructed from HPV16 protein sequences. We use NetMHC3panFurther information on experimental design is available in the Supplementary InformationReporting SummarySupplementary Data 1Description of Additional Supplementary Files


Highly sensitive electrostatic doping of ReS2 enables fabrication of gate-tunable NAND logic gates, which cannot be achieved in bulk semiconductor materials because of the absence of gate tunability. The vdW heterostructure NAND gate comprising a single transistor paves a novel way to realize “all-2D” circuitry for flexible and transparent electronic applications.Two-dimensional (2D) semiconductors, such as transition metal dichalcogenides (TMDs) and black phosphorus, are the most promising channel materials for future electronics because of their unique electrical properties. Even though a number of 2D-materials-based logic devices have been demonstrated to date, most of them are a combination of more than two unit devices. If logic devices can be realized in a single channel, it would be advantageous for higher integration and functionality. In this study we report high-performance van der Waals heterostructure (vdW) ReS Among them, transition metal dichalcogenides (TMDs) have been actively studied as channel materials because of absence of short-channel effect7. It has been anticipated that a scale-down limit can be overcome by 2D semiconductors for higher integration of electronic devices. In this regard, “all-2D” devices comprising only 2D materials, i.e., van der Waals heterostructure (vdW) devices, have been demonstrated11. As emerging 2D materials, rhenium-based TMDs, such as ReS2 or ReSe2, have exhibited promising properties. Typically, few-layer TMDs outperform monolayer in terms of carrier mobility, while the band structure of TMDs transforms from direct to indirect band structure as the thickness changes from monolayer to few-layers, which limits their use for optoelectronic applications12. However, ReS2 and ReSe2 exhibit direct band structure at all thickness because of the distorted 1 T’ structure and weak interlayer coupling13. Despite the unique properties of ReS2 and ReSe2, there have been only limited studies on them, which reported preliminary results of transistors and photodetectors based on ReS2 and ReSe2 with conventional device geometry19.Ultrathin two-dimensional (2D) semiconducting materials are useful for a number of electronic applications because of their unique properties originating from their atomically thin nature20, which makes it difficult to utilize the 2D semiconductors for practical applications required by the industry. In addition, because the band gap of TMDs increases with decreasing thickness, the Schottky barrier height for monolayer TMDs is significantly higher than that for few-layer TMDs, leading to a lower field-effect mobility22. The use of graphene electrodes has been considered as a solution because of the de-pinning of the graphene Fermi level and absence of chemical bonds at a stacked heterointerface of 2D layers23. In addition, it has been shown that hexagonal boron nitride (hBN), used as an ultraflat dielectric, suppresses charged impurity scattering, resulting in enhanced carrier mobility of hBN-encapsulated 2D materials24.To improve the device performance of 2D semiconductors, contact resistance and carrier scattering should be reduced. Significant contact resistance between deposited metal and 2D semiconducting channels severely deteriorates device performance due to the Fermi level pinning at the interface28. Even though the NMOS inverters using ReS2 have been demonstrated27, all of the logic devices reported so far are made of double devices (two connected devices). Therefore, for higher integration and functionality, a novel device geometry is required for logic devices. Because atomically thin 2D materials are highly sensitive to electric fields with no screening effect, it is possible to locally tune the densities and types of carriers, leading to electrically controlled p-n junction devices comprising graphene or WSe233. Therefore, by locally tuning the Fermi level of a 2D channel, a multi-functional logic device can be realized in one 2D channel.A number of logic devices based on 2D materials have been demonstrated by combinations of two different 2D transistors: n-type and p-type2 channel with a graphene split gate. We first demonstrate ReS2 field-effect transistors (FETs) on an hBN substrate with graphene electrodes. The field-effect mobility (μFE) of an n-type ReS2 FET is 35 cm2/V·s and the on/off ratio is as high as 106 at room temperature. When the graphene split gate is used to locally modulate the channel, two regions, separately tuned by the split gates, act as independent transistors, which can be used as a gate-tunable NAND logic gate.Here we report a NAND gate based on a single ReS2, graphene, and hBN was fabricated by stacking the exfoliated nanosheets, as shown in Fig. 2 substrate22. The ReS2 channel was placed on the hBN to reduce scattering from charged impurities of the substrate and interfacial impurities. As ReS2 has a smaller effective mass along the a-axis, it has anisotropy in conductance, leading to higher carrier mobility along the a-axis27. Therefore, the ReS2 flake, which has an elongated shape along the a-axis, was chosen as a channel. Graphene was used as an electrode to lower the Schottky barrier. Two pieces of graphene were placed on the ends of the ReS2 channel to form source and drain electrodes. After stacking, e-beam lithography and metal deposition (1 nm Cr/50 nm Au) were used to define the metal electrodes. Figure For “all-2D” devices, a vdW heterostructure of ReS2 FET on the hBN substrate with graphene electrodes is shown in Fig. IDS − VDS) at different back gate voltages (VBG) clearly exhibit a gate-dependent linearity, as shown in Fig. 2-graphene interface forms Ohmic contact even at low carrier concentrations. As shown in the transfer curves (IDS − VBG) in Fig. 2 exhibits n-type transport as typically observed in MoS2 or WS234. Because of the low contact resistance and reduced carrier scattering in the vdW heterostructure device, higher mobility (μFE = 35 cm2/V·s) and a high on-off ratio (~106) were extracted, compared with the same ReS2 FET with metal electrodes on a SiO2/Si substrate  of ReS2 devices with metal or graphene electrodes and SiO2 dielectric19. We also fabricated ReSe2 devices using the same procedure and device structure. As ReSe2 exhibits a smaller mobility of 6.6 cm2/V·s than ReS2, ReS2 was used for the logic device.  etching for cutting. The patterned graphene split gates are annealed at 350 °C for 4 hrs in forming gas (5% H2 in Ar) to remove residue. After cleaning, other 2D flakes of hBN dielectric, ReS2 channel, and graphene electrodes were sequentially transferred by the PDMS stamping technique as described in Fig. 2 device with graphene split gates.We modified the previous device structure to demonstrate the logic devices comprising a single 2D channel. To locally tune the ReSVGG1 and VGG2), the corresponding band alignments are shown in Fig. VGG1 and VGG2 are higher than the threshold voltage (Vth), the whole channel region is conductive and the Schottky barrier is lowered because of the simultaneous up-shift of the graphene Fermi level, which results in n-type conductance of the device. When the VGG1 is higher than the threshold voltage and VGG2 is smaller, carrier transport through the ReS2 channel is blocked because of the depletion of the negatively gated ReS2 region, while the Schottky barrier is still small, resulting in carrier injection of the positively gated channel  of the ReS2 device with graphene split gates when VGG1 is varying while VGG2 is fixed. The transfer curves are plotted on a semi-log scale in Fig. VBG < 3 V) and low subthreshold swing (SS) of <300 mV/dec were obtained, which were an order of magnitude smaller in the ReS2 device on the 300 nm-thick SiO2 substrate which is calculated from the device described in Figs VGG1 > −1.5 V), conductance of the whole channel increases with increasing VGG1. However, in the off-state (VGG1 < −1.5 V), the off-current remains at a low level of 10−12 A at different VGG1 because the region where VGG1 is applied is completely off. Therefore, the on-current can be strongly modulated by VGG1 and VGG2. The on-off ratio modulation ranges from 102 to 106, which is beneficial for multifunctional applications  used in this study is 1 V and the dielectric constant (εr = 3–4) of hBN is relatively small compared to previous studies38. Therefore, the gain can be further enhanced by increasing VDD as shown in the inset in Fig. VDD was set as 1 V because the VTC exhibits a mirror reflection and a gain larger than 1 (unity gain), as shown in Fig. VDD.The logic device characteristics of the ReS2 devices as NAND logic devices, output voltages were measured under different combinations of input states, as shown in Fig. VDD, a logic state of 1 can be realized when either Vin1 or Vin2 is −3 V (defined as an input logic state of 0). For Vout of state 0, positive input voltage of 3 V was applied to both graphene split gates of GG1 and GG2. If a gate voltage smaller than −1.5 V is applied to one of the two gates, the whole device is non-conducting. Therefore, ReS2 devices with graphene split gates can be used as a NAND gate, one of the universal logic gates. It should be noted that a NAND gate was fabricated on a single 2D channel material, which is distinct from other reports using two transistors regardless of the carrier type. Output voltages measured at different combinations of the two input signals are shown in Fig. 2 device with graphene split gates operates well as a NAND gate. Unlike bulk materials, gate tunability of 2D semiconducting materials enables independent control of the separated channel regions by split gates. Therefore, NAND logic device can be fabricated within single 2D channel, which is clearly different from conventional logic gates that require more than two separate transistors.To utilize ReS2 transistors by using graphene and hBN as electrode and dielectric, respectively. The high field effect mobility of 35 cm2/V·s and high on-off ratio of 106 were obtained by suppression of Fermi level pinning at the graphene-ReS2 junction and reduction of charged impurity scattering. For the “all-2D” integration, we also fabricated ReS2 devices with graphene split gates for logic devices. The graphene split gates and bottom hBN enables us to locally and effectively modulate channel regions. By independently changing gate voltages from the two split gates, the device conductance can be controlled with a higher degree of freedom: the whole device is turned off by applying a negative voltage to only one gate, while a positive gate voltage is applied to both split gates to turn-on the device. By utilizing local tunability of the channel by split gates, it was shown that the ReS2 device with graphene split gates can be used as an efficient NAND gate, which is one of the most essential elements in integrated circuits. The vdW heterostructure NAND gate comprising a single transistor fabricated in this work provides a further step toward realization of “all-2D” circuitry for flexible and transparent electronic applications.In conclusion, we fabricated high performance ReS2/Si chips, and exfoliated other 2D materials, such as graphene and ReS2, separately onto each cured PDMS flake on glass slides. After careful alignment with a micromanipulator in the transfer stage, the PDMS with 2D materials were attached and detached, as carefully and slowly as possible, at room temperature.PDMS transfer First, the PDMS base (Sylgard 184) was cured with 10 wt% of curing agent at 120 °C for 2 h. We then mechanically exfoliated hBN, which is the bottom of the heterostructure on SiOTo pattern metal electrodes on the as-stacked heterostructure or gap on the graphene for the split gate, we performed e-beam lithography with a Raith Pioneer 2 nanolithography system.−8 Torr, to create the interconnection to the pads for the electrical measurement from the graphene electrodes, where 1 nm of Cr is an adhesive layer. For the lift-off process, the samples were soaked in acetone overnight.We deposited metal electrodes (Cr 1 nm/Au 50 nm or Cr 1 nm/ Pd 20 nm/Au 30 nm) with an e-beam evaporator (Korea Vacuum Tech.) in a high vacuum, of approximately 102 RIE (Femto Science) for only 5 s to prevent etching of e-beam resist.  We then put the samples in acetone to remove the e-beam resist.We etched the developed region after the e-beam lithography using OAll electrical measurements were carried out by semiconductor parameter analyzers at room temperature under ambient condition. (Keithely 4200, Keithely for FET characterization in Figs Supplementary information for “All-2D ReS<Subscript>2</Subscript> transistors with split gates for logic circuitry”


Graphene nanobubbles (GNBs) have attracted much attention due to the ability to generate large pseudo-magnetic fields unattainable by ordinary laboratory magnets. However, GNBs are always randomly produced by the reported protocols, therefore, their size and location are difficult to manipulate, which restricts their potential applications. Here, using the functional atomic force microscopy (AFM), we demonstrate the ability to form programmable GNBs. The precision of AFM facilitates the location definition of GNBs, and their size and shape are tuned by the stimulus bias of AFM tip. With tuning the tip voltage, the bubble contour can gradually transit from parabolic to Gaussian profile. Moreover, the unique three-fold symmetric pseudo-magnetic field pattern with monotonous regularity, which is only theoretically predicted previously, is directly observed in the GNB with an approximately parabolic profile. Our study may provide an opportunity to study high magnetic field regimes with the designed periodicity in two dimensional materials. Presence of nano-bubbles within an atomically thin material generates potentially large pseudo-magnetic fields. Here, the authors report an innovative technique to induce nano-bubbles in graphene with desirable features and high precision through energized AFM tips, and experimentally measure three-fold symmetric pseudo-magnetic fields up to 120 T. The introduced strain can induce local changes of Dirac point position at low-energy, and always together with lattice distortions that will change the electron hopping in graphene. That phenomenon can be equivalence of gauge field imitating the electron behavior in real magnetic field, unveiling the signatures of Landau quantization3. The strain-induced PMFs conserve the time-reversal symmetry unlike real magnetic field and have opposite signs in the K and K’ valleys of graphene forming the low-energy electronic band structure4. In consequence, the gauge field can be used as a building block for valleytronic device5 or the realization of the Aharonov–Bohm effect by building STM interferometer in strained graphene6. The creation of graphene nanobubbles (GNBs) has been proven as an efficient strategy for strain engineering of graphene, thus generating the enormous PMFs8. For instance, the unconsciously introduced GNBs with the triangular shape on a Pt(111) surface exhibit large uniform PMFs up to 300 T at room temperature7, which was not ever obtained previously. Diverse methods had been proposed to create graphene nanobubbles including gas ion irradiation11, electric field stimulus14, water splitting15, or intense laser irradiation16. However, all proposed methods have the limited capability in the manipulation of GNBs including position definition, size control, and shape design, thus greatly impeding the exploration of the relevant physics phenomenon and the practical application of GNBs, though the enormous PMFs exist.Strain engineering in graphene has been proven as an effective method for modifying its electronic structure due to the remarkable feature of Dirac fermions, such as bandgap opening and pseudo-magnetic fields (PMFs) generationHere, rendered by the accuracy of AFM, the programmable GNBs with the tunable size and shape have been successfully obtained on germanium substrate at the pre-defined locations. With controlling the voltage applied on AFM tip, the contour of bubbles can gradually transform from parabolic profile to Gaussian profile, and the diameter of bubbles can be tuned from tens to hundreds of nanometers. Moreover, higher-resolution scanning tunneling microscopy/spectroscopy (STM/S) reveal that the strain-induced three-fold symmetric pseudo-magnetic field with monotonous regularity exists in GNB with approximately parabolic shape, which is only theoretically predicted previously.17, as characterized by phase-sensitive sum-frequency vibrational spectroscopy  surface (see Methods), as shown in Fig. 10 surfacell Fig. . Besides28. Figure R < 50 nm) can be well fitted by a parabolic curve written ashmax is the maximum height of bubble and R is the radius of bubble base. Our fitting results agree well with the membrane model proposed by Yue et al.29 and the unified power form proposed by Lu et al.31. However, the large GNBs (R > 50 nm) exhibit a more complex profile: the bottom part of the bubble can be still fitted by a parabolic curve, but the top part of the bubble changes into a Gaussian contour. The Gaussian contour part of GNBs can be depicted as29. The bending stiffness can be described by the bending rigidity, κ, and the in-plane stiffness is always described by Young’s modulus Y. Beyond the characteristic parameter 10, the stiffness is dominated by in-plane stiffness. For the GNBs in our study, the dimension is much larger than 4 Å, so the in-plane stresses contribute the majority of the elastic energies, while the contribution of the out-of-plane bending stiffness can be neglected.In addition to the tuning of height and radius of GNBs, their contour can also be customized by the applied voltage, thus further regulating the strain distribution of GNBs10 concludes a universal law of hmax/R considering the stress γ is the adhesion energies between graphene and the germanium, the coefficient c ≈ 0.7. Figure hmax/R behaves approximately as a constant, 0.08, on a wide range of radius or volume. This value is comparable to the aspect ratio hmax/R ≈ 0.11 for circular GNBs (R > 50 nm), hmax/L ≈ 0.07 for triangular GNBs (1000 nm > L > 500 nm) obtained on hBN substrate experimentally10, or 0.18 for small GNBs filled with helium gas in the graphene interlayer11. The similar aspect ratios hmax/R suggest the adhesion energy between graphene and hydrogen terminated Ge surface is quite small17. Besides, the transition of GNB from the parabolic profile to the combination of parabolic and Gaussian profile may attribute to the change of vdW interactions, which strongly depend on the distance, h, between the graphene and substrate, and could be trivial in experimental scales for large GNB. For small GNB with the height hmax < 5 nm, the vdW force on the center deflection of GNB may exist though is not comparable to that at the edge of round base of GNB31. Due to the clamping effect at the bubble edge and the internal hydrogen gas pressure, the GNB with parabolic profile is formed. However, when the height of GNB hmax > 5 nm, the vdW force on the center deflection of GNB is negligible33 and the corresponding constriction effect disappears. However, the clamping effect at the bubble edge still exists. Therefore, the profile of central part of large bubble transits from the parabolic profile to Gaussian profile, but the parabolic profile near the edge of round base of GNB preserves, as observed in Fig. In addition, the shape of bubbles is determined by the adhesion energies, which is related to the vdW force between graphene and the substrate. ref. 1. Figure I/dV spectra collected at different rotational angles  caused by strain-induced pseudo-magnetic fields14. In strained graphene, lattice deformation can create PMFs, which affect the behavior of massless Dirac fermions, thus yielding zero-field pLLs quantization. Similar to the influence of a real magnetic field, the PMF gives rise to a sequence of quantized pLLs:The lattice distortion in GNB creates non-uniform strain distribution, which will modify the low-energy electronic band structure of graphene and change the electron hopping amplitude between carbon atoms, in an equivalent way to the effect of real magnetic fields applied perpendicular to the graphene plane, as known as PMFsEDirac is the energy of Dirac point, e is the electron charge, ℏ is the reduced Planck’s constant, n is the level index, 40. The positions of the pLLs peaks indexed as n = 0, 1, 2, 3, and 4 are marked on Fig. Here, BS| data corresponding to each tunneling spectrum can be extracted by linear fitting of the PMFs using Equation  surface7. Moreover, intriguingly, the maxima of the field occur near the angles of which the interval is nearly 60° at both the edge and the center of GNB, such as 30°, 90°, 150°, 210°, and 270°, and the values diminish and even vanish to zero at the angles between two adjacent ones, such as 60° and 180°. Such the evolution has a six-fold (every 60° in circle lines) symmetry, which can be described by the function |BS| = |121sin(3θ)|. Similar |Bs| distribution have also been observed in different GNBs with distinct heights and radii  with alternating signs42. Here it should be noted that, the distribution of PMF in our experiment only change as the rotational angle changes, but keep constant for the certain rotational angle even when the distance between the measured position and the bubble edge varies, which is different from the predicted three-fold symmetric PMF occurring only at the bubble edge27. The discrepancy may be due to the fact that, unlike the theory prediction, the GNBs with the strictly parabolic shapes could not been obtained in the real experiment, and the configuration complexity of the approximately parabolic GNB including the loss of axisymmetry and the anisotropy of strain may play a crucial role in the formation and modulation of the local PMF distribution. Moreover, GNBs can also be created by AFM tip on other substrates pre-hydrogenated, e.g., SiO2 or Si substrate . During the graphene bubbles fabrication, Pt/Ir coated silicon AFM tips with radius of curvature ~30 nm  were chosen.Intrinsic Ge(110) wafers  were used in the experiments. The graphene film was synthesized on Ge(110) substrate by chemical vapour deposition in a horizontal tube furnace with H−11 Torr) with constant-current scanning mode at liquid-nitrogen temperature of ∼77 K. The STM tips were obtained by chemical etching from a wire of Pt0.80Ir0.20 alloys. Lateral dimensions of the STM images were calibrated using a standard graphene lattice as well as a Si(111)-(7 × 7) lattice, and, the STS spectra were calibrated using Ag(111) surface. The STS spectra, i.e., the dI/dV curves, were collected with a standard lock-in technique by turning off the feedback circuit and using a 793-Hz 5 mV a.c. modulation of the sample voltage.An ultrahigh vacuum scanning probe microscope (USM-1400S) from UNISOKU was utilized for STM and STS measurements. Both STM and STS measurements were performed in the ultrahigh vacuum chamber (∼10Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Source Data