Since the production of ferromagnetic graphene as an extremely important matter in spintronics has made a revolution in future technology, a great deal of efforts has recently been done to reach a simple and cost-effective method. Up to now, controlling the magnetic properties at extremely low temperature have been investigated only by adding and removing atoms in graphene lattice. In this regard, the effect of strain on the magnetic and electronic properties of graphene has been probed. Here, the ferromagnetic properties are what have been created by strain, magnetic field, and temperature along with observation of the parallel magnetic domains in ferromagnetic graphene for the first time as a great achievement. In this way, we have represented the following: First, introducing three novel methods based on temperature, magnetic field, and strain for producing ferromagnetic graphene; Second, obtaining ferromagnetic graphene at room temperature by significant magnetization saturation in mass-scale; Third, probing the electronic systems and vibrational modes by Raman and IR spectroscopy; Fourth, introducing stacking and aggregation as two types of gathering process for graphene sheets; Fifth, comparing the results with leidenfrost effect-based method which the temperature, magnetic fields, and strain are simultaneously applied to graphene flakes (our previous work). In the honeycomb structure of graphene, the presence of one free electron per atom is responsible for high electron mobility7and the carriers transport is described by Dirac equation2. Moreover, other electrical properties such as room-temperature quantum Hall effects8, unique band structure, and ambipolar electric field effects2, covers the electronic application.Graphene, as a 2D material9. Indeed, graphene is suitable for spin logic devices due to its properties such as room-temperature spin transport with long spin-diffusion lengths10. Although the magnetic properties do not naturally exist in graphene, it is valuable in spintronics. Recently, the magnetic property is created in graphene by manipulating electronic systems. Interestingly, magnetic domains structure directly effects on magnetic properties. Generally, ferromagnetic materials are composed of one magnetic domain, with all dipoles aligned in the same direction which is a consequence of a strong driving force for parallel alignment caused by exchange energy11 . This energy is a quantum mechanical effect which tends to align electron spins, and in consequence their magnetic dipole moments, simultaneously. Noticeably, magnetic domains formation would certainly minimize the exchange energy; hence, it reduces total magnetic energy of ferromagnetic materials formation and raises their stability. By the same token, production of these domains in graphene during magnetization can thus make a revolution in future technology. Essentially, magnetism in graphene can be created by many methods such as functionalization, doping, and adding atoms. In fact, the symmetry of the electronic structure is locally broken around the new bonds and the magnetic moments are created in this area30. Additionally, vacancy and edge defects, Introduced as another method in magnetization. By removing one carbon atom and rearrangement of others in vacancy defect, the magnetic moment is created due to remaining a dangling bond which is theoretically estimated 1 μB. Experimentally, vacancy defects are created by ionic bombarded and reduction of graphene oxide41. Among these methods, the highest magnetization is occurred in the doping methods by sulfur27 and nitrogen24 but at extremely low temperatures.Apart from electronics, graphene is also attractive material in spintronics, where in addition to charge, the spin of electrons is considered44. Indeed, the creation of the pseudo-magnetic field in highly strained nanobubbles of graphene is one of the extreme approaches in graphene magnetism42. In our previous study, Leidenfrost effect-based (LFE) method for preparation ferromagnetic graphene are introduced45. Evaporating graphene droplets under high temperature and external magnetic field leads to magnetism in graphene. In this method, temperature, magnetic field, and strain is simultaneously applied to graphene flakes and magnetization occurred. Here, all these parameters are considered separately and determined their contributions.Observation of the pseudo-magnetic field in strained graphene without damaging the lattice structure is the huge gap in the magnetization process47. After first isolation of graphene by Scotch tape1, many methods are proposed to produce graphene, from exfoliation50 of graphite to growing graphene on the substrate by nucleation of carbon atoms51. On the other hand, most mechanical exfoliation methods49 such as the Liquid-phase exfoliation53 produce high-quality graphene flakes in mass-scale. Therefore, in this study, the graphene flakes are obtained by the Liquid-phase exfoliation method by using of ethanol solution54.Graphene properties are extremely influenced by the preparation methods54. To follow the instruction, pristine graphite was added to the ethanol solution in which the graphite concentration was 5 mg/ml and the ethanol/water ratio was 20:80. Then, sonication was performed for 30 min at high power and was subsequently centrifuged at 3000 and 4000 rpm (just for LFE) for 10 min, to eliminate larger flakes  and after drying the ferromagnetic graphene particles were collected. Here, the droplets were rotated and evaporated base on the Leidenfrost effect. In this method, the source of the magnetic field and temperature were Heater-stirrer and the source of pressure was the pressure inside the droplets. In fact, all parameters were simultaneously applied to graphene flakes55. Apart from strain, temperature and the magnetic field are significant parameters to manipulating the electronic and magnetic properties of graphene which is studied here. In all the methods, ferromagnetic graphene powders (FGPs) was created  and negligible impurities such as Ni, Co, Fe, and Mn, the weak ferromagnetic behavior is observed in pristine graphite  and the results are shown in Fig. 45. Because the temperature and pressure are applied in all directions, all flakes can be felt the same conditions in Oven- and Autoclave-method. The details of the hysteresis loop were summarized in Supplementary Table Similar to LFE-method, the slope created in the VSM diagram of Heater-method in high magnetic fields, related to the non-uniformity of particles. In Heater-method, the non-uniform conditions are felt by flakes because of gradual removal of the solution, non-uniform of the magnetic field, and non-uniform heat flow. In LFE-method, the slope created is related to the different behavior of droplets56. In Fig. Magnetic force microscopy (MFM) is used to observe the magnetic domains. This technique is similar to atomic force microscopy (AFM), based on cantilever oscillations. The surface of materials is probed by the magnetic tip in different distances. Each line is scanned twice, in one of them, the tip moves near the surface to collect the topography data and in another one the tip moves far from the surface to collect magnetic data60.Raman and IR spectrum complement each other and show an excellent map of active vibrational modes. By means of these techniques, a lot of information is obtained about the electronic systems and the lattice vibrational modes of graphene at Brillouin Zone (BZ). Raman spectroscopy is widely considered as a key diagnostic tool for symmetric vibrational modes, whereas IR is used for recognizing the asymmetric modes. Although most vibrational modes are active, some of the modes are silent. Generally, in Raman spectroscopy, the laser light is used to excitation and Raman scattering is detected. In addition, the electronic band structures can involve in the resonance process such as double resonance (DR) and triple resonance (TR) Raman process. For example, the phonon modes far from the center of BZ are activated by the DR Raman process. Whereas, at Γ point the phonon is usually activated by first-order Raman process57. Raman spectra of all FGPs and graphite are shown in Fig. Lattice structure, the lattice vibrations, the electronic systems, and their changes can be investigated by Raman spectroscopy. Therefore, each parameter that affects the electronic systems and phonon modes of graphene can be detected and probed by Raman spectroscopy6h, which is belonged to one-layer graphene and graphite, at the Γ point. The irreducible representation of them are ΓGraphene = A2u + B2g + E1u + E2g and ΓGraphite = 2(A2u + B2g + E1u + E2g). Three optical modes in graphene are E2g (Raman active) and B2g (silent). In graphite, nine optical modes exist containing three IR active, five Raman active, and one silent (B2g) modes. IR active modes are a doubly degenerate E1u appear in ~ 1588 cm−1, generated from asymmetric in-plane vibrational mode and one asymmetric out-of-plane mode, A2u, located in ~ 868 cm−161. In Supplementary Fig. 1u and A2u modes are observed in most samples. Also, these two peaks are too weak in graphite and LFE-sample. For graphite and LFE-sample, E1u peak is located at ~ 1525 cm−1 and in the other samples broadening and redshift of E1u peak is observed.Interpretation of the vibrational mode is based on the structural symmetry and group theory. The highest symmetry is D3d and D3h at Γ point, respectively. Also, the symmetry information along the Γ–K direction is different and their phonons are activated by DR Raman process. For example, the symmetry of monolayer and NLG (even) at M point is D2h and C2h, respectively62. Generally, changes in the electronic system and the lattice structure can create new modes, and even the combination and splitting of some modes can appear the complex modes in the Raman spectrum.By increasing the number of layers, the symmetry is reduced and subsequently, the modes are changed. For example, N-layer graphene (NLG) symmetry with the even and odd layers is D−1. These doubly degenerate, E2g, is called G mode and its position is sensitive to external perturbations, such as defects, doping, strain, and temperature. The two other peaks appear in ~ 2670 and 1200 cm−1, called 2D and D, respectively57.Basically, three characteristic peaks exist in graphene Raman spectra. One of the main peaks in Raman spectra is generated from symmetric in-plane vibrational mode, located in ~ 1580 cm2g (~ 128 cm−1) and E2g (~ 43.5 cm−1) modes, respectively. As another example in AB-2LG, C and LB modes are located at ~ 31 cm−1 (Eg) and ~ 90 cm−1 (A1g), respectively. In addition, C mode in AB-3LG and ABC-3LG are located at ~ 33 cm−1 and ~ 19 cm−1, respectively. Generally, interlayer vibrations depend on twisting, stacking order, and the number of layer68. In Fig. −1, however, many modes are observed in Fig. Ultralow-frequency modes are directly related to interlayer vibrations. In graphite, the out-of-plane and in-plane interlayer vibrations, layer-breathing (LB) and shear (C) modes, are related with Bnd E2g ~ 3.5 cm−1 nd E2g ~ 3.5 cm−1 D/IG is used to estimate the amount of defects in graphene flakes57. Only edge defects can be created in the samples, related to the sonication process in the dispersion of graphene. Because of using the same dispersion method in all samples, it is expected that the edge defects are the same and ID/IG are equal  can interact. Therefore, the position and the shape of the G peak are changed and led to broadening and splitting72. Broadening of G mode in Heater-method can be related to the new modes such as E2  is observed under stress. In addition, the G mode are sensitive to temperature80.Changing the interatomic distance in crystal lattice under stain and stress lead to manipulating electronic systems. Recently, the strain effect on electronic systems is considered and remarkable results such as shifting the Dirac cones, opening band gap, shifting and splitting Raman modes, and inducing strong pseudo-magnetic field is reported. As an example, in graphene, shifting the vibrational frequency and splitting the G mode (G−1) is activated by the resonance process. The component of the 2D peak dependent on electronic bands and laser wavelengths. Here, the laser wavelength is the same in all measurements. Due to several electronic bands are in multilayer graphene, the many 2D components can be expected. Because of these features, the 2D peak is used to determine the number of layers. In addition, the stacking order also can be strongly effected on electronic bands and change the spectral profile of the 2D peak. Therefore, the complexity of 2D LFE-sample and Autoclave-sample compared to other samples can be related to more manipulation of electronic systems . Ethanol solution is prepared by pure ethanol (Merck) and deionized water. After dispersion, elimination of larger flakes is occurred by centrifuge (Hettich EBA20). To prepared ferromagnetic graphene Heater-stirrer  and oven (Memmert) are used. For investigating magnetization of graphene, Vibrating Sample Magnetometer  is used. Natural defects of graphite and manipulation of electronic structure are investigated by Raman spectroscopy (HORRIBA). UV–Vis (mini 1240) and FTIR (8400S) spectrophotometer analyzes are performed by SHIMADZU instruments for investigation graphene concentration and vibrational modes, respectively. Magnetic field sensor  is used to measure magnetic fields of Heater-stirrer.Supplementary Information.


The activity of this new material was evaluated in situ and in vivo. The performance of the synthesized material was measured in different saline solutions  as an electrolyte. The results showed that the new material exhibits very good performance (157 F/g at 5 mV/s and 1.0 M NaCl compared to 19.5 F/g for pure graphene oxide). In the desalination test, which was performed in batch mode, the salt adsorption capacity and the efficiency of salt removal were 9.2 mg/g and 98%, respectively. To check the stability, the desalination test was repeated several times, and no change in the performance was observed. The results provide evidence that the newly synthesized material is a potential electrode material for CDI water desalination with satisfactory salt removal ability.In this study, N-doped and TiO Capacitive deionization (CDI) is a promising new technology compared to other traditional desalination methodologies, such as thermal or nonthermal distillation, due to its low energy consumption, low capital and processing costs, easy operation and maintenance processes, and environmentally friendly characteristics, with no hazardous chemicals used for electrode regeneration. Furthermore, CDI does not require high pressures, has low equipment and operational costs, employs low voltages and low power (low energy cost), is safe and involves no expendables. Small CDI units can be used in remote locations and run by solar panels, and some of the energy can be recovered by utilizing stored energy. CDI can achieve arsenic removal to below drinking water standards. CDI is strongly recommended as an environmentally friendly and economical technique for removing salt ions from saline water. The main concept of CDI is to use the electrosorption/desorption processes of an electric double layer capacitor as an electrochemical water purification method. By applying an external electrical potential , salt ions in the feed water will be attracted by the oppositely charged electrodes. Reversing the potential leads to ejection of the desorbed ions and regeneration of the electrodes for a continuous desalination process3.Most existing desalination technologies require a great deal of energy. Even nuclear power has proven too expensive for desalination and is the most expensive water supply option. Thus, energy conservation has prompted researchers to develop new water desalination technologies5. The most suitable materials that can be used as electrodes are carbon-based materials, including aerogels7, activated carbon (AC)8, mesoporous carbon (OMC)10, carbon nanofibers (CFs)11, carbon nanotubes (CNTs)12, and graphene13, which are the best candidates for CDI electrodes. Moreover, other high-performance carbon electrodes have been developed15.Researchers are in a race to develop CDI technology, and at the heart of this research are the electrodes. Effective electrodes should have high electrical conductivity, a high specific surface area with adjusted and well-controlled pore sizes, and excellent electrolyte wettability. The electrode materials should have a potential at zero charge, which is the potential at which anions and cations are adsorbed onto a surface . Many innovative electrode materials have been registered in patents16. Other electrode materials have also been investigated. Ryoo et al. used modified activated carbon cloth as an electrode to enhance the capacitive deionization performance. However, this was only very fundamental research17.The ability of an electrode to absorb ions basically depends on the surface properties of the electrode material, such as the electrical conductivity, surface area, and pore size and distribution. Therefore, carbonaceous mixtures, such as carbon nanotube and nanofiber (CNT–CNF) composite films, have also been proposed as electrosorption electrode materials21. Unfortunately, the known high hydrophobicity of graphene harms its performance in aqueous media due to the low contact angle, which adds additional resistance. However, the oxygenated groups on the surface of graphene oxide (GrO) make this material more hydrophilic than graphene. On the other hand, the electrical conductivity of GrO, compared to that of graphene, is negatively affected by these active groups. In fact, GrO’s electrical conductivity depends on the carbon/oxygen ratio in the sample. During the oxidation step, the sp2 carbons are replaced by sp3 carbons with oxygen functionalities, and this process will create a band gap by pulling the bands apart. Consequently, graphene oxide can behave as an insulator when graphene is fully oxidized. However, the partially oxidized GrO behaves as a semiconductor due to the appearance of sp3 and sp2 regions22, and the electrons can pass through the sp2 regions via Klein tunneling22. Insertion of a transition metal oxide and nonmetals within the GrO layers could play a crucial role in enhancing electron transfer23.Among the reported nanostructure carbonaceous materials, graphene has drawn the most attention due to its very large surface area and excellent electrical conductivity2 and N-doped graphene oxides have been developed as efficient nonprecious electrodes for capacitive deionization. It is expected that this fabrication process will meet many CDI electrode requirements.In this work, a fast and green synthesis route for controlling the morphological structure is presented. TiO4 from Cica-reagent, Japan) and urea hydrazine were used without any further purification.Commercial graphite powder (<20 μm) purchased from Sigma Aldrich was utilized as a graphene precursor. Titanium chloride solution , 0.1 g urea (as a source of nitrogen) and 150 micro liter hydrazine were added. All these mixtures were subjected to closed reflux system at 150 °C for 5 h. Then filtration and drying at 60 °C under vacuum for 1 day.The graphene oxide (GrO) was synthesized via a modified Hummer’s methodX-ray diffraction was conducted to analyse the phase and crystallinity using Rigaku X-ray diffractometer  with Cu Kα (λ = 1.54056 Å). 2θ angles from 10° to 80° has been used. A spectrometer (JY H800UV) equipped with an optical microscope was used to collect Raman spectra at room temperature. Scanning electron microscope , field-emission scanning electron microscope  and Transmission electron microscope  operated at 200 kV equipped with EDX analysis has been used to investigate the surface morphology of the produced samples.27. Three-electrode system were used: counter electrode from platinum wire, Ag/AgCl, reference electrode and the prepared materials as the working electrode. VersaStat4 potentiostat device was used to control the whole system. The specific capacity was simply calculated by integrating the area under the CV curve to determine the average value according to the following equation29.CDI is a solt seperation process that can be simply described as adsorption/desorption processes occurring on the electrode surface by means of electrostatic force. The salt ions are adsorbed on the charged electrode and form an electric double layer (EDL). As polarity reversed, the regeneration process take place and the ions are desorbed from the electrode surface and released back to the solution. The electrode is the main part of CDI unit. the salt adsorption capacity (SAC) of the CDI electrode material strongly depends on the properties including high electrical conductivity, large capacitance, high wettability and electrochemical stability. The salt adsorption capacity was analyzed by cyclic voltammetry cell simulate the CDI flow cell using the proposed materials. Cyclic voltammetry measurement was done by using different NaCl concentrations  after adjusting the sweep potential range from −0.4 to 0.6 V vs Ag/AgCl in the electrochemical cell. This range was selected according to that reported for graphene and graphene doped materials2 is one of the most widely used semiconductors with supercapacitor electrodes that exhibit excellent performance and a wide band gap and are placed in contact with a redox electrolyte. The purpose of using TiO2 and N-doped GrO is to improve the electrochemical characteristics, as TiO2 intercalates between the graphene oxide nanosheets.The Hummers approach was adopted to synthesize single- or few-layered chemically exfoliated graphene oxide (GrO), followed by postprocessing with thermal treatment or chemical reduction to achieve a better reductive degree of graphene, also known as reduced graphene oxide (rGO). Moreover, GrO with enough oxygen functional groups, small particles, and massive exposed edge sites offers a promising matrix for surface modification, especially for heteroatom doping. TiO2 and N-doped graphene oxide nanostructure can be easily controlled, as shown in Fig. 2 and N-doped GrO semisphere with a diameter of nearly 10 µm. This SEM image confirms that the sample contains spherical TiO2 nanoparticles. The right image shows a magnified part of the image on the left. TiO2 and N-doped GrO is in the form of layers with a thickness on the nanoscale. These introduced forms of TiO2 and N-doped GrO separating layers are very effective for use in capacitive deionization with highly improved salt adsorption capacity; consequently, an optimum CDI electrode can be obtained.The morphology of the TiO2 and N-doped GrO material. Ultrathin wrinkled graphene oxide NPs with systematic distribution can also be observed. The image indicates that TiO2 intercalated into the flexible and ultrathin wrinkled graphene oxide sheets with a few layers to form a needle-like structure. Decoration of the prepared graphene oxide by small particles of TiO2 could be proved by TEM images , (004), (200) and (204) crystal planes, respectively, indicate the formation of the anatase phase of TiO2.Figure 30. This technique can efficiently and easily prove the formation of graphene oxide, as it can distinguish GrO from the utilized graphite precursor. For instance, in the case of pristine graphite, the obtained Raman spectrum shows a prominent G peak at 1581 cm−1, which corresponds to the first-order scattering of the E2g mode31. However, the G band is broadened and shifted to 1594 cm−1 in the case of graphene oxide. Moreover, because of extensive oxidation, the D band, which appears at 1363 cm−1, becomes dominant, indicating that the in-plane sp2 domains decrease. Finally, the D/G intensity ratio in the case of graphene oxide approaches unity, while that of graphite is very small31. Figure 2 nanoparticles and doping with nitrogen. It can be easily concluded from these spectrum corresponding to pristine GO that the chemical procedure utilized was successfully applied to prepare graphene oxide. Raman spectroscopy can also easily distinguish the two compounds. For the reduced graphene oxide, the intensity of the D band is higher than that of the G band. Furthermore, the D/G ratio is very high, especially in the case of single (or few)-layered graphene. However, in our case, as shown in the figure, no change in the ratio can be observed after decoration and doping. Actually, the presence of functional groups on the surface of graphene oxide makes it preferable in capacitive deionization applications due to the enhanced ability for ion adsorption compared to graphene.The rotational, vibrational and other low-frequency modes of compounds can be easily investigated by Raman spectroscopy32. This result confirms the doping of the prepared composite by nitrogen. Decoration of the prepared graphene oxide by small particles of TiO2 could be proven by XRD analysis  analysis is a powerful technique to investigate surface elements and their electronic structure. Moreover, some elements cannot be detected by XRD, such as nonmetals and even noncrystalline metals. However, XPS is a powerful analytical technique that can detect all elements except hydrogen and helium. Figure sis Fig. . As showCyclic voltammetry (CV) is a good electrochemical technique to investigate the surface activity of functional materials.2 and N-doped GrO electrode at different scan rates. The analyses were conducted using different NaCl solution concentrations  at 25 °C in an applied voltage window of −0.4~0.6 V (vs. Ag/AgCl reference electrode). As shown in the figure, smooth ellipse-shaped cycles were obtained at all the assigned scan rates for all concentrations. This finding indicates, due to the absence of any redox peaks, that the surface is very stable and free of any functional groups. Moreover, an increase in the cycle width indicates an increase in the amount of adsorbed ions, which can be understood as an improvement in the formation of the electric double layer. Actually, the high surface area due to the exfoliated GrO sheets increased the number of absorbed ions. Additionally, no chemical reactions are involved, allowing the ultracapacitor to be rapidly charged and discharged  thousands of times, which is highly beneficial in CDI technology.Figure 2 only as a dopant did not distinctly enhance the electrochemical absorption of the ions, especially at a content close to that used in this study25. To show the advantage of the utilized codoping strategy (TiO2 and nitrogen), Fig. 2 and N-doped GrO in the presence of 1.0 M NaCl solution at a scan rate of 5 mV/s. As shown in the figure, a very considerable enhancement in ion adsorption for the introduced material was obtained compared to that of pristine graphene oxide. Moreover, the observed tail  in the case of GrO can be assigned to the oxygen evolution reaction. In other words, pristine GrO possesses electrocatalytic activity toward the water reduction reaction to produce oxygen gas. Even though this activity is very small, it is undesirable in CDI technology. In addition to the improvement in ion adsorption, the proposed doping process annihilates the electrocatalytic activity.Notably, our previous study concluded that utilizing TiO2 and N-doped GrO and pristine GrO, respectively, which translates to an 8-fold increase in the specific capacitance upon doping of graphene oxide.Numerically, the observed cycles in the above figures can be translated into the specific capacitance values by applying Eq. 37:, V (L) is the total volume of the solution, and M (g) represents the mass of the electrodes. Figure Practically, the real performance of any proposed electrode for the CDI unit is estimated by investigation of its ion adsorption and durability. Figure 2 nanoparticles and doping by nitrogen atoms. Incorporation of the metal oxide and doping by nitrogen distinctly improve the specific capacitance of the graphene oxide, resulting in an 8-fold increase in the specific capacitance. As an electrode in the capacitive deionization unit, the modified graphene oxide shows high salt removal efficiency and satisfactory salt adsorption capacity, with high stability for both parameters. Overall, this study opens a new avenue for titanium oxide nanoparticles and nitrogen as effective dopants to improve the characteristics of carbonaceous materials to be exploited as efficient electrodes in capacitive deionization units.The Hummers method is a good chemical procedure for the preparation of graphene oxide nanosheets through a chemical route. Hydrothermal treatment of the prepared graphene oxide in the presence of titanium chloride and urea results in decoration of the carbonaceous sheets by TiO


Tremendous progress has been advanced by research into graphene and its derivatives with great benefits toward low-cost, portable, and real-time tactile sensors/electronic skin.The review presented herein direct future efforts aimed at high-quality graphene-based tactile sensors and their implications for the wider scientific community.The paper also are informative regarding some basic and crucial issues regarding graphene and its derivatives, such as charge-transport principles, doping/trapping behaviors, correlations between structure/morphology and properties/functions. Skin is the largest organ of the human body and can perceive and respond to complex environmental stimulations. Recently, the development of electronic skin (E-skin) for the mimicry of the human sensory system has drawn great attention due to its potential applications in wearable human health monitoring and care systems, advanced robotics, artificial intelligence, and human–machine interfaces. Tactile sense is one of the most important senses of human skin that has attracted special attention. The ability to obtain unique functions using diverse assembly processible methods has rapidly advanced the use of graphene, the most celebrated two-dimensional material, in electronic tactile sensing devices. With a special emphasis on the works achieved since 2016, this review begins with the assembly and modification of graphene materials and then critically and comprehensively summarizes the most advanced material assembly methods, device construction technologies and signal characterization approaches in pressure and strain detection based on graphene and its derivative materials. This review emphasizes on: (1) the underlying working principles of these types of sensors and the unique roles and advantages of graphene materials; (2) state-of-the-art protocols recently developed for high-performance tactile sensing, including representative examples; and (3) perspectives and current challenges for graphene-based tactile sensors in E-skin applications. A summary of these cutting-edge developments intends to provide readers with a deep understanding of the future design of high-quality tactile sensing devices and paves a path for their future commercial applications in the field of E-skin. A tactile sensor is a kind of device that simulates the tactile sense of human skin and can detect and analyze the strength, position and time sequence of an external mechanical force with micron-level resolution by micro/nano processing technology and intelligent data analysis. As one of the most important sensor components in electronic skin (E-skin), tactile sensors have become a popular international research area due to their potential applications in wearable human health monitoring and care systems, advanced robotics, artificial intelligence, and human–machine interfaces. Among all kinds of tactile sensors, flexible, low-cost, conformal, portable and wearable real-time-monitoring functional electronic devices based on graphene and its derivatives should be generally concerned as the next generation of sensing devices for E-skin applications –5. On onWith a special emphasis on the state-of-the-art works published in 2016, these latest developments use the most advanced methods of material assembly, device construction and signal characterization and represent the forefront of graphene-based tactile sensors, laying the foundation and identifying the direction for future commercial applications. The main contents of this review provide a general synopsis on the functional supramolecular nanoassemblies of graphene and its derivatives with respect to progress during the tactile sensing era for E-skin applications. Some historically significant seminal works achieved before 2016, which are of paramount importance in shaping this field, are also highlighted to provide a foundation. For other interesting yet earlier studies, we encourage the interested readers to consult other excellent reports. This review is organized as follows: First, we briefly introduce the related concepts and preparation methods of graphene and its derivatives for tactile sensors. Then, with an emphasis on the impactful protocols of how to improve the performance of this kind of sensor, the unique roles and advantages of the employed graphene materials are discussed and highlighted by addressing representative paradigms. Finally, the current perspective and challenges of graphene sensors are outlined. We hope that the discussions will be beneficial to future investigations aimed at high-quality graphene-based tactile sensors.sp2-hybridized crystal structure, where each carbon atom has three equivalent valence orbitals  together in a plane forming a triangle and a pz orbital perpendicular to the basal plane, which arranges the carbon atoms in a honeycomb lattice. The hybridization of one s and two p orbitals leads to the formation of covalent σ-bonds with other neighboring carbons, and the pz orbitals overlap each other to form delocalized π-bonds; the abundant delocalized electrons are responsible for the extraordinary electronic properties of graphene. The concentration of the electrons and holes of graphene can up to 1013 cm−2, and the mobility can reach 200,000 cm2 V−1 s−1 [Since Geim and Novoselov discovered graphene in 2004, it has received tremendous attention as an ideal material to construct electronic devices due to its unique physical properties . As is k V−1 s−1 –20. Thisπ–π stacking [π-conjugated molecules to realize a responsiveness to various stimuli, confirming graphene materials to be excellent scaffolds for various sensors [Additionally, graphene has a perfect 2D structure, which offers abundant active sites on the basal plane to react with functional groups by means of conjugation reactions or the absorption of various functional moieties via hydrophobic interactions, dipole–dipole interactions, or stacking . This chstacking –24. Part sensors –30.Moreover, graphene materials also possess excellent transmittance and mechanical properties with light transmittances of up to 97.7%, fracture strains of up to 25% and a Young’s modulus of ≈ 1.1 TPa, which provide graphene a significant opportunity for the construction of flexible and stretchable electronic devices used in tactile detection , 32.Most importantly, many studies have discussed the physical properties of graphene and its sample preparation routes –35. For C) of a parallel plate capacitor can be defined as C = ε0εA/d, where ε0, ε, d, and A are the vacuum dielectric constant, the relative permittivity of the elastomer dielectric, the distance, and the overlapped area between the two parallel plates, respectively. With excellent electrical properties, mechanical flexibility and optical transmittance, graphene has become one of the most promising materials for electrodes in tactile piezocapacitive sensors.In recent years, great progress has been made in fabricating pressure sensors based on different sensing mechanisms, including capacitive , 43, traFor example, to obtain a tunable-sensitivity flexible pressure sensor, a classic method was employed by Luo and coworkers, wherein graphene was used as the electrodes, and polydimethylsiloxane (PDMS) pyramids with different spacings were used as the dielectric layer . By a th2 wafer by wet etching. (3) On another substrate, a piece of Si/SiO2 wafer was patterned by deep reactive ion etching to form an array of circular or hexagonal holes of a given diameter, periodicity and depth, arranged in various patterns such as a hexagonally packed lattice. (4) The graphene-polymer membrane was aligned with the patterned SiO2 surface using a tape-supported transfer process. It was found that sensors covering an area of just 1 mm2 showed reproducible pressure transduction under static and dynamic loading up to pressures of 250 kPa. The measured capacitance change in response to pressure was in good agreement with calculations.In the above configurations, the tunable sensitivity was obtained by changing the space between the pyramids or the morphology of graphene; indeed, the tunability of the suspended membrane area and the dielectric gap were the most important factors to the sensing performance Fig. b. Althoue-beam evaporation, which are generally tedious to work and crosstalk between adjacent cells is inevitable. These drawbacks can be overcome simply by replacing the dielectric layer. Nylon netting composed of polyethylene terephthalate (PET) is a flexible, low-cost insulating polymer with a regular microporous structure and excellent mechanical properties, and was first selected and sandwiched between graphene films by He et al. [The microstructure dielectric layer of the abovementioned devices to some extent can overcome the slow response and relaxation times caused by the high viscoelasticity of PDMS, leading to a substantially higher sensitivity and faster response/relaxation time. However, the construction of such capacitive sensors often requires intricate processes such as traditional lithography and e et al.  as the d−1), rapid response (≈ 70 ms), and high stability over 2500 cycles of loading/unloading. The authors also demonstrated a pixelated sensor array for pressure mapping without any significant crosstalk between adjacent cells, as shown in Fig. An air gap between the surrounding spacers in each tactile cell is another effective method to reduce the difficulty in capacitive tactile sensor preparation. Pyo et al.  presente2 and 8 × 8 array (64 channels) was fabricated by Kang and coworkers [From the above examples we can see that most of the capacitive tactile sensors have mainly focused on pressure or strain sensors that transduce physical touch into electronic signals, which cannot fulfill the demands of E-skin applications. Indeed, in addition to a position-sensing capability through contact, a 3D-sensing capability for the recognition of 3D shapes and the distance of approaching objects before contact occurs is significantly important both in wearable electronics applications and in the robotics field . Furtheroworkers . As disp4HCO3 in a PDMS sponge, Kou and coworkers achieved a composite. When the sponge was sandwiched between two electrodes, a flexible wireless pressure sensor with a high sensitivity, wide operating rage, rapid response time, low detection limit, and good stability was obtained [Graphene and its derivatives are versatile and sometimes can be used as a good dielectric material for capacitive pressure sensors. By adjusting the proportion of graphene and NHobtained . Ho et aobtained  developeobtained . By utilobtained . With a As seen from the above examples, both graphene layer and graphene foam can be used to construct high-performance capacitive tactile sensors. Graphene layers with good morphology, high crystallinity and uniform thickness are usually obtained through CVD. The graphene electrodes of capacitive tactile sensors obtained in this way not only present excellent electrical properties but also control the transparency through altering the growth process. Furthermore, the electrodes can also conform to substrates of different surface morphologies, thus, realizing the patterning of electrodes and ultimately improving the tactile sensitivity of devices. What is more interesting is that the CVD method can achieve large-area graphene layers, making the realization of integrated of tactile sensor components easier and laying a solid foundation for tactile sensors with good spatial resolution. However, the high energy consumption, high cost and high pollution stemming from the production process pose difficulties to realizing industrial production. In contrast, graphene foams are usually obtained through GO, which can be prepared in large quantities by solution methods at room temperature. Furthermore, GO has different electrical properties due to the varying degrees of reduction, which enables it to be used as both electrodes and the insulating layer of capacitance tactile sensors. When the graphene foam, which is porous and flexible, acts as the dielectric layer, the distance between the two parallel plates is easily adjusted under the action of external forces, thus, greatly improving the sensing performance of capacitive tactile sensors. However, even with a strong reduction, the oxygen-containing functional groups do not completely disappear, which greatly reduces the crystallinity and conductivity of RGO, and results in the material not being a good electrode for capacitive tactile sensors. Therefore, due to the graphene layer and the graphene foam having respective advantages and disadvantages for constructing capacitive tactile sensors, suitable forms of graphene and its derivatives with different morphologies should be chosen according to the actual practical applications.R = ρL/A, where ρ is the resistivity, L is the length, and A is the average cross-sectional area. When sensors are in operation, various related parameters are used to evaluate their qualities. Among them, the most fundamental parameter is the gauge factor (GF), which reflects the sensitivity to external physical action. The GF is defined as GF = (∆R/R)/ε, where ΔR/R is the normalized resistance and ε is the mechanical strain/pressure. A higher GF means a higher sensitivity [Although capacitive tactile sensors based on versatile graphene materials forge ahead on wearable electronic devices, it has been widely accepted that high sensitivity, high resolution, and mass production can hardly be realized at the same time, and the constructed devices are still far from practical uses. Owning to the excellent electrical property of graphene and its nanoscale flexibility, minor stress deformations could lead to a dramatic change in resistance . Therefositivity . To achiAs the high aspect ratio of 1D architecture favors the rapid capture and release of external stimuli, increasing effort has been focused on fabricating pressure sensors based on 1D active material. However, the innate 2D structure of graphene makes obtaining 1D microscopic structure difficult. Thus, we need to search for other tools to help. As reported by Nakamura and coworkers, using a nickel wire as a template, 1D hollow tubing CVD graphene fibers (TGFs) could be obtained, coated with PDMS, and used as the active material for resistance-type strain sensors, as shown in Fig. Electrostatic spinning is a special fiber manufacturing process in which a polymer solution or melt is sprayed in a strong electric field. By taking advantage of this commonly used method, composite nanofibers of carbon nanotubes and graphene were fabricated by Lee et al.  as showc, whereiGO and RGO contain abundant oxygen functional groups on their basal plane and edges, which make their self-assembly into 1D fiber architectures via solution processes possible. Fu and coworkers prepared a kind of conductive glass fibers (GFs) fabric by dip-coating GO on the surfaces of GFs, followed with an HI reducing process . Taking With the increasing demand for high-conductivity films, 2D graphene films have attracted significant attention due to their transparency and flexibility for wide-ranging application in optoelectronics, light-emitting diodes, solar cells, and sensors . For tac3/Ar plasma treatment of graphene, changed its morphology, structure, chemical composition, and electrical properties. Due to the tunneling behavior originating at localized defects, the graphene structure doped with nitrogen atoms exhibited a significant increase in sensitivity by one order of magnitude compared to that of the unmodified graphene sheet, as shown in Fig. −1) was obtained, substantially higher than that of tactile sensor devices with relatively flat graphene electrodes. More interestingly, as the complete separation of the two graphene layers occurred when the sensor was not subjected to any pressure, such a device could be used as an on/off and energy-saving device.In addition to directly using CVD graphene as an active material in tactile sensors, the modification and microstructure of graphene can further improve the sensing performance of devices. From the point of view of material modification, Haniff et al.  found thAlthough CVD graphene-based tactile sensors show the high sensitivity and reliability needed for sensor devices, their low yield, high production costs, and complex processes hinder their development toward practical applications . Fortunaπ–π interface interactions between graphene sheets, those 3D materials with an ultralight density and flexibility also possess a high conductivity and mechanical strength. In addition, their scalable production makes for an attractive choice for practical implementation [Active materials based on a 3D porous structure are the most common well-shaped and self-supported graphene hierarchical nanostructures used in graphene-based tactile sensors. Based on the large stacking interfaces and the entation . Recentlentation –78, varientation –81, cellentation , multilaentation , all kinentation –86, and entation –89.A typical example using commercially available PU and PVC sponges as templates was reported by Zhang et al., wherein graphene-wrapped sponges were obtained by soaking sponges in a hydroquinone/GO mixed solution and then vacuum annealing under certain conditions . The as-As is known, the majority of sponges provided on the market are PU sponges. The production process for this kind of chemical product is neither environmentally friendly nor conducive to human health, so tactile sensors based on PU sponges are difficult to realize for practical applications of E-skin , 83. To In addition to natural fabrics, paper is another kind of environmentally friendly substrate with a 3D hierarchical nanostructure and good elasticity that also has potential to be a good alternative to improve the performance of pressure sensors. A typical paradigm was reported by Tao et al. , who mixAlthough the 3D graphene structures prepared through the abovementioned templates can significantly improve the sensing performance of tactile sensors, the opacity of sponges, fabrics and tissue paper hinders the construction of transparent E-skin , 92. PDMBeyond PDMS, other flexible porous polymers, such as porous inverse opal acetylcellulose (IOAC) films, thermoplastic polyurethane electrospun fibrous mats, poly, polyester textiles, and polyaniline (PANI) can also be used as templates to construct 3D graphene structures –97. The 2) and Ecoflex with such a GPN. It was found that the conformal nanostructure of MoS2 on the GPN surface could produce improved resistance variations against external strain and pressure. As a result, the MoS2/GPN/Ecoflex sensor exhibited noticeably improved sensitivity over that of previously reported GPN/PDMS sensors in a pressure test. Copper foil, as the most commonly used metal for the preparation of graphene by CVD, has also been used to fabricate porous graphene 3D structures [Flexible and transparent polymers to some extent can solve the problem of sensor transparency. Nevertheless, all of the porous structure cannot effectively remain in sensors after the polymer has infiltrated into the as-prepared graphene foam, and the pores can achieve enhanced sensing performances , 98. To ructures , 84.Fig.−1, an ultralow detection limit of approximately 0.1 Pa, and a superior separation of 0.2-pascal-scale difference.Generally, the size and distribution of the pores in the foam, as well as the thickness of the pore walls, are essential to the sensitivity of graphene foam-based pressure sensors . Therefoπ–π interactions. Then, the formed emulsions were immediately immersed into liquid nitrogen for 10 min to keep their macroporous structure. (3) The aerogels were obtained after lyophilization and a thermal treatment. Thereafter, the authors used polyethylene glycol sorbitol monooleate (Tween 80), instead of SDS, as a sparkling agent, and an automatic egg beater, instead of a blender, to obtain a sparkling graphene block (SGB) with bubbled cavities maintained well, as illustrated in Fig. −1), making this material a promising candidate for applications in actuators, elastic conductors, strain/pressure sensors, and wearable devices, as shown in Fig. 2, GaN, and CdS, to the graphene aerogel 3D structure could enhance the tactile sensing properties, where the piezoresistive response was considerably higher than that of the bare aerogel [To fabricate graphene aerogels with ultralight, superelastic, and excellent mechanical and multifunctional properties, surfactants and crosslinkers are often employed in the synthesis system –102. Qu’ aerogel , 105.FigFrom the abovementioned 3D graphene-based tactile sensing paradigms for E-skin applications, we can see that most of the developed strategies focused on high sensitivity, while sensors capable of combing high sensitivities and broad dynamic ranges have barely been proposed. This inequality is because such materials are prone to saturation responses when attempting to obtain measurements involving high pressures . By meanAs we all know, many well-adapted hierarchical structures have been developed through natural selection and are critical for the survival of organisms. For example, to climb a vertical wall, the feet of the gecko have developed a kind of special hierarchical structure so that maximized contact area and intermolecular interactions could be realized , the sel−1 over a wide linear range of 0-2.6 kPa. As shown in Fig. By using a bioinspired hierarchical structure based on the surfaces of organs and consisting of PDMS covered with monolayer graphene Fig. a, Bae anExcept for human epidermis, the epidermal ridges on the skin of the human fingertip, which serve to amplify subtle external stimulations, can also inspire us to design highly sensitive fingertip skin-like pressure sensors. As shown in Fig. In addition to human organs, microstructures from animal and plant organs also provide interesting ideas for the preparation of active materials for graphene-based tactile sensors. A more obtrusive example is the Shar-Pei dog, and the higher dimensional patterns of Shar-Pei skin can sustain large in-plane stretching and still provide tactile perceptions so that wrinkle-crumple RGO electrodes with a high stretchability and strain-insensitive resistance profiles were fabricated by means of sequential deformation processes, as shown in Fig. With the rapid development of materials science, micronano materials with various morphologies and functions have been designed and synthesized. To advance the applications of E-skin, these materials can be integrated with versatile graphene materials in various ways to achieve effects in tactile sensors exemplifying that the combination can be greater than the sum of the individuals .ZnO, as a common inorganic semiconductor possessing a large bandgap and exciton binding energy, an inherently high transparency and excellent luminescence at room temperature, has become a celebrated material widely used in liquid crystal displays, thin-film transistors, light-emitting diodes and other electronic products, particularly tactile sensors , 87. SunIn addition to ZnO, other inorganic materials can also be employed to construct functional composites to enhance the sensing performance of tactile sensors. For example, Ma et al.  synthesiAs an important member of the material family, polymers present many excellent properties, such as light weight, mechanical flexibility, and their generally good solution processability, as well as a good compatibility with large-area and flexible solid supports; all of these characteristics cannot be matched by other materials and endow polymers with the ability to manufacture various sensing devices . FurtherPoly(vinylidene fluoridetrifluoroethylene) (P(VDF-TrFE)), as a natural elastic conductive building block, is usually functionalized with graphene materials and has widely been used in the piezoresistive sensors , 127. Fo−1 at 3–1000 Pa and a high stability for 20,000 loading–unloading cycles could also be obtained [The abovementioned graphene/polymer composites were fabricated by simply mixing graphene and polymers together, hardly yielding well-defined composite materials and negatively affecting the performance of pressure-sensing devices based on such materials. To solve this problem, Lin and coworkers prepared a highly flexible self-healing conductive polymer composite consisting of graphene, poly(acrylic acid) and amorphous calcium carbonate by a biomineralization-inspired process . Strain obtained . Furtherobtained . The GF π-conjugated graphene fragments, GO, as a novel cousin of graphene, can be considered as a 2D surfactant for use as a dispersing agent or to generate Pickering emulsions [−1 and could detect subtle pulse beats and monitor various human movement behaviors in real time , metal carbides and nitrides (MXenes), and montmorillonite (MMT), have recently become a focus for advanced electronics [Another example related to reducing the efforts and cost of the fabrication techniques for the FET-based tactile sensor configuration was reported by Sahatiya and coworkers, wherein 2D graphene/MoSthe gate . Owing tctronics . From anctronics . Althougctronics . Howeverctronics . HoweverAlthough FET-based tactile sensors are generally very sensitive, easily integrated, realize real-time detection, the touch point of the abovementioned FET pressure sensors are mostly located in the gate or channel region . As a re−1, a limit of detection < 1 kPa, and a response time of ≈ 30 ms (Fig. In addition to piezopotential MIM capacitors, the induced triboelectric potential can also be able to couple with FETs for modulating the carrier transport in semiconductor channels and helping to obtain high-performance devices . For exa ms Fig. b. Meng eRecently, targeting high-performance graphene-based tactile sensors, great progress has been achieved mainly in terms of the sophisticatedly designed morphologies of graphene and its derivatives, the working principles aiming at providing a fundamental knowledge of the sensing processes, state-of-the-art protocols targeting high-performance sensing, and the development of synergy with other materials. Indeed, the tremendous advancements accumulated to date have brought tactile sensors a significant step closer to the potential applications of flexible and wearable E-skins, such as health monitoring devices, artificial intelligence, and human–machine interfaces. Continuous efforts to further improve the overall qualities of this kind of sensor, including sensitivity, detection range, pattern recognition and spatial resolution of external forces, response time, stability and reproducibility, limit of detection, capability of digital and intelligent readouts, real-time workability, etc., remain strongly desired. To achieve this improvement, the following aspects will continue to be the major subjects of this field in our opinion.First, the synthesis, assembly and modification of high-performance graphene materials are the basis for the fabrication of high-performance graphene-based tactile sensors. As we mentioned above, graphene and its derivative materials were usually used as conductive electrodes or sensitive materials. As electrodes, graphene has a variety of intrinsic excellent physical properties, such as a high electrical conductivity, transparency, and flexibility, which lay a good foundation for building high-performance wearable devices. However, on one hand, it is difficult to industrialize the production of large-area, high-quality materials via current production technology. On the other hand, ultrathin 2D structures are easily damaged during scratching by external forces, and the reuse of devices can only be realized through the application of complex packaging technologies. Therefore, exploring the preparation process of graphene electrodes remains the basis for commercial applications of graphene-based tactile sensors. From the point of view of sensitive materials, graphene materials are easy to assemble; are easy to modify; are easy to combine with other materials, enabling them to have diverse morphologies and properties; and can be used to improve the performance of tactile sensors. However, as a zero-bandgap semiconductor, the on/off ratio of graphene is relatively low, and the current is difficult to modulate. Although there are many ways to open the bandgap in graphene, these remain in the laboratory. Therefore, scientists should do their best to explore simpler and more effective ways to realize the commercial applications of graphene as an active material.Second, the launch of emerging sensing mechanisms in terms of molecular engineering, supramolecular assembly, and their combination with other protocols will undoubtedly be among the most important ways to construct new types of high-performance tensile sensors. For example, a self-powered sensation matrix could be constructed by sandwiching piezoelectric polymer materials between two graphene electrodes , 150. AcAs we have highlighted, numerous sophisticated strategies have been proposed to achieve high-performance tactile sensors based on graphene materials. However, most of the abovementioned works address only one or a few aspects of a sensor’s quality, which is still far from mimicking human skin. Clearly, a mechanically flexible and fully integrated sensor array for multiplexed monitoring of an individual’s activities, without interrupting or limiting the user’s motions, is still a formidable challenge that cannot be realized by a single capacitive-style, piezoresistive-style or FET-style array. The combination of a couple of the state-of-the-art strategies might be among the most feasible ways to address this topic to some extent.Finally, multifunctional tactile sensing devices capable of digital and intelligent readouts are important issues required by modern E-skin applications. Therefore, the construction of high-performance tactile sensing device arrays along with effective pattern recognition algorithms is particularly important. We believe that with the joint efforts of scientists in chemistry, physics, material science, micronano processing, computer science and other disciplines, the construction of high-performance graphene-based tactile sensing systems for potential commercial uses will soon become a reality.


Tolypocladium inflatum. The suggested nomenclature was proved feasible by naming introns present in mitogenomes of 16 fungi of different phyla, including both basal and higher fungal lineages although minor adjustment of the nomenclature is needed to fit certain special conditions. The nomenclature also had the potential to name plant/protist/animal mitochondrial introns. We hope future studies follow the proposed nomenclature to ensure direct comparison across different studies.Fungal mitochondrial genes are often invaded by group I or II introns, which represent an ideal marker for understanding fungal evolution. A standard nomenclature of mitochondrial introns is needed to avoid confusion when comparing different fungal mitogenomes. Currently, there has been a standard nomenclature for introns present in rRNA genes, but there is a lack of a standard nomenclature for introns present in protein-coding genes. In this study, we propose a new nomenclature system for introns in fungal mitochondrial protein-coding genes based on (1) three-letter abbreviation of host scientific name, (2) host gene name, (3), one capital letter P (for group I introns), S (for group II introns), or U (for introns with unknown types), and (4) intron insertion site in the host gene according to the cyclosporin-producing fungus The online version of this article (10.1186/s43008-019-0015-5) contains supplementary material, which is available to authorized users. Fungi constitute a huge group of highly diverse organisms, with 2.2–3.8 million estimated species and 144,000 currently known species on Earth    as the reference mitogenome. The 25,328-bp mitogenome of T. inflatum contains all the 15 protein-coding genes typically found in fungal mitogenomes, and there is no intron in any of these protein-coding genes , which is fast when aligning long sequences containing many introns and can always generate satisfactory alignment according to our experience. The default setting of MAFFT works well in most cases. If exon-intron boundaries are not correctly identified (probably due to the interference of intron sequences or presence of short exons) under the default settings, one may consider adjusting the alignment parameters  and/or importing additional sequences to align from a species closely related the test species. In addition, it is always advisable to refer to known annotation results and/or characteristic nucleotides at splice sites of group I/II introns  from Candida glabrata cox1 at position 240. Other examples are included in Table We propose a new nomenclature system for introns in fungal mitochondrial protein-coding genes based on 1) three-letter abbreviation of host scientific name, 2) host gene name, (3) one capital letter P , S , or U (for introns with unknown types), and (4) intron insertion site in the host gene according to  three-le host gennad5 in Candida pseudojiufengensis (Cpse.nad5U717) and Candida psychrophila (Cpsy.nad5P717)   Table . The intfungi from different phyla, including both basal and higher fungal lineages . From the intron insertion site numbers, one can also easily understand the phase of an intron, which is phase 0 when an intron inserts between two codons , and phase 1 or 2 when an intron inserts within a codon . These introns are often found at highly conserved regions  also contain group I or II introns in their mitochondrial genes  that are absent in fungal mitogenomes . Insertion site of group I introns are shown in red, group II introns in green, and introns with undetermined intron types in shade. (DOCX 21 kb)Sequences of protein-coding genes of Additional file 2:cob, cox1, cox2, nad1, and nad5 of different fungal taxa plus few non-fungal taxa were aligned by MAFFT, and visualization of the aligned sequences was performed using ESPript 3.0 (Robert and Gouet Intron insertion sites for 22 common introns. Exon sequences of


P = 2.66 × 10–4). Moreover, applying enterotyping on our samples stratified microbiomes into A, B, and C dermotypes. Dermotype C included overrepresentation of Pseudomonas, Staphylococcus and Stenotrophomonas and was markedly associated with delayed healing of RID. Strikingly, coexistence of diabetes mellitus and RID was remarkably correlated with a significant overrepresentation of Klebsiella or Pseudomonas and Staphylococcus. Metabolic abilities of skin microbiome could support their potential roles in the pathogenesis of RID. Cutaneous microbiome profiling at the early stages of RID could be indicative of prospective clinical outcomes and maybe a helpful guide for personalized therapy.Radiotherapy-induced dermatitis (RID) is an inflammatory cutaneous disorder that is acquired as an adverse effect of undergoing radiotherapy. Skin microbiome dysbiosis has been linked to the outcomes of several dermatological diseases. To explore the skin microbiota of RID and deduce their underlying impact on the outcome of RID, cutaneous microbiomes of 78 RID patients and 20 healthy subjects were characterized by sequencing V1-V3 regions of 16S rRNA gene. In total, a significantly apparent reduction in bacterial diversity was detected in microbiomes of RID in comparison to controls. Overall, the raised Proteobacteria/ Firmicutes ratio was significantly linked to delayed recovery or tendency toward the permanence of RID (Kruskal Wallis:  Recently, Egypt recorded an exponentially increased rate of cancer patients. Radiotherapy is substantially considered as a key element of treatment in breast cancer and nearly is applied to > 70% of cancer patients5.Worldwide, there is a raised incidence of cancer6. Overall, cutaneous dermatitis negatively affects individuals’ health and quality of life. Pathophysiology of cutaneous dermatitis was linked to a multitude of predisposing factors that could cause a disturbance in proinflammatory and profibrotic cytokines8. These factors include genetic and epigenetic predisposition in addition to arterial insufficiency, diabetes mellitus, chronic osteomyelitis, radiotherapy-induced dermatitis (RID), rheumatoid arthritis, skin tumors, trauma and venous ulcers10. Distinctly, the vast majority of cancer patients experience skin reactions upon radiation exposure. Several risk factors are assumed to directly trigger or influence the incidence and severity of RID. These risk factors may be intrinsic including age, ethnicity, gender, malnutrition, location and stage of the tumor, and concomitant diseases such as systemic inflammation and diabetes mellitus. While extrinsic factors include the kind and total dose of treatment, volume of treated area and radiotherapy technique11, in addition to bacterial infections12.Although the rapid advances in radiation oncology improved the effectiveness of cancer treatment, skin reactions, which are defined as radiation dermatitis, remain the most common adverse effect of radiation toxicity14.The anatomical and physiological structures of human skin represent the protective shield of the human body. The human skin is influenced by the surroundings and is the site where billions of microbial communities collectively constitute a unique ecosystem that constantly modulates host immunity and metabolism. The normal functions of human skin basically require integral collaborations between epidermal barriers, skin immunity and microbial inhabitants. Both the composition and structure of the microbial ecosystems associated with the human skin are influenced by several factors which include host-related such as age and gender, in addition to external stimuli as weather, lifestyle, and use of medicated preparationsDespite the skin microbiomes have been intensely investigated over the past years for their implication in several dermatological disorders, their roles in the pathogenesis and prognosis of RID demand comprehensive investigations. We aimed to provide new insight into the potential impact of skin microbiome on the prognosis of RID.The present study was approved by the ethics committee of the Faculty of Pharmacy, Port Said University, Egypt (Reference no. A-6-2019). This study was carried out following the principles of the Declaration of Helsinki. Recruited patients in this study provided informed consent at sampling time.16. The inclusion criterion was mainly based on patients with newly diagnosed and grade 2 confirmed RID. The exclusion criteria were: (1) systemic antibiotic intake; (2) topical application of corticosteroids and antibiotics. Besides, healthy subjects were involved in the current study. All recruited patients provided informed consent prior to sampling.This study was performed for over 16 months (between June 2019 and September 2020). Participants were prospectively enrolled at outpatient clinics of dermatology department, Al Mabarrah Health Insurance Hospital, Zagazig, Egypt. The prognosis of RID was identified by follow-up of patients for 12 months. Diagnosis and grading of RID were carried out according to CDC Cutaneous Radiation Injury: Fact Sheet for Physicians17. The collected swabs were used for genomic bacterial DNA extraction using DNeasy PowerSoil Kit Catalog No. 12888  with previously reported modifications18. V1-V3 hypervariable regions of 16S rRNA gene were amplified using the following primers (underlined bases define adaptors): Forward Primer 5′ AATGATACGGCGACCACCGAGATCTACACATCGTACGTATGGTAATTC AATTACCGCGGCT GCTGG; Reverse Primer 5′ CAAGCAGAAGACGGCATACGAGATAACTCTCGAG TCAGTCA GCCGAGTTTGATCMTGGCTCAG19. Then, PCR amplicons were sequenced by Illumina MiSeq at IGA Technology Services .Skin swabs were collected from patients by rubbing the site of RID with a premoistened swab (0.15 M NaCl with 0.1%Tween 20). Also, antecubital fossa, represents the moist sites, was rubbed by swab for collecting specimens from healthy subjects20, which included: trimming, filtering and denoising of row reads as previously described 21. Finally, high-resolution RSVs were generated by removing the potential chimeric sequences.The analysis and classification of 16S rRNA reads were mainly based on ribosomal sequence variants (RSV). QIIME2 platform with DADA2 plugin was used for sequence preprocessing23.RDP’s naive Bayesian classifier was applied on representative RSVs for taxonomy assignment against SILVA SSU Ref NR dataset v.132 at 99% sequence similarity24. The core genus in our dataset was defined as the genus that was detected in 80% of all samples, while the core genus for each study group was defined as the genus that was present in all samples of this group.Bacterial diversity was estimated using QIIME2 on the entire dataset without subsampling. Alpha diversity was determined using two approaches; regarding richness (number of observed species and Chao1 index) and evenness of communities (Shannon diversity index). The statistical significance of pairwise comparisons was assessed using unpaired Wilcoxon rank sum test, while multiple group comparisons were estimated using Kruskal–Wallis rank sum test and all P values were corrected using false discovery rate method (FDR)25. Similarity distances between cutaneous microbiomes were visualized by principal coordinates analysis (PCoA) based on weighted UniFrac distance matrix. Permutational Multivariate Analysis of Variance  was applied to define the significance of weighted UniFrac distance-based clustering of samples26.Phylogenetic relatedness of bacterial communities was identified using both unweighted and weighted UniFrac matrices27.To determine the dermotypes of cutaneous microbiomes, a widely used enterotyping pipeline was applied to all genera in our dataset. Clustering of samples and the optimum number of clusters were achieved using partitioning around medoids (PAM) algorithm, Jensen–Shannon divergence (JSD) and Calinski-Harabasz (CH) index28 against Greengenes database (V 13.8)29 at a 97% identity using closed-reference script for OTU picking in QIIME, v 1.9.129. Functional potential of cutaneous microbiomes was predicted using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) for mapping the Kyoto Encyclopedia of Genes and Genome (KEGG) Orthology (KO) Database at level 332. LEfSe was applied to the metagenomic data to define the taxon or metabolic pathway (logarithmic LDA scores > 3.0 and α = 0.05) that were differentially abundant among healthy subjects and patients with RID (https://huttenhower.sph.harvard.edu/lefse/)33.For predicting the functional profile of cutaneous microbiota, representative RSVs were employed for further taxonomic classification using UCLUSTP ≤ 0. 01) using R package, Hamsic34. R packages; Phyloseq and ggpubr, were applied for calculation and plotting of diversity indices (https://rpkgs.datanovia.com/ggpubr/)36, while ggplot2 was used for graphical illustrations of results 37.Correlations between either communities’ members or structure of microbiota and patient’s clinical data were defined by Spearman correlation coefficient  and biosamples (SAMN168624511: SAMN16862528).16S rRNA row reads of this study have been deposited in NCBI bioproject under accession number PRJNA665254 (Ninety-eight subjects were included in this study (20 controls and 78 patients with RID). Patients’ data regarding clinical characteristics, demography and outcome were listed in Table In total, 11,276,243 row sequences  were inputted in Qiime2 for merging, quality checking, removal of low quality reads  and potential chimeric sequences  that were resulted in 9,283,731high quality sequences that were used for downstream analyses.P = 2.58 × 10–7: Spearman; r = -0.68, P = 0.001) . In contrast, microbiomes of chronic ulcers were significantly predominated by Proteobacteria and low abundance of Firmicutes .16S rRNA reads were taxonomically assigned to 19 phyla, 58 classes, 109 orders, 237 families, 658 genera and 4643 OTUs. In total, cutaneous microbiomes associated to RID showed a remarkable representation of certain bacterial taxa at different taxonomic levels that was in contrast to healthy subjects  . Cutaneous microbiomes of RID that turned to chronic ulcer had significantly reduced bacterial diversity in comparison to either healthy individuals or rapidly healed RID .Identification of bacterial diversity associated with RID was performed using both evenness and richness indices. Skin microbiomes of healthy subjects were significantly more diverse than those with RID (Wilcoxon test: –5) Fig. . SurprisBoth structure and composition of bacterial communities significantly drove the classification of cutaneous microbiomes to health-state based clustering Fig. a. PCoA bEnterobacteriaceae or Staphylococcaceae were the most predominant families in several RID specimens, while genus level analysis was provided a clear separation of genera belonging to the previous families .Studying the structure of RID microbiomes at the family level unable to provide a deep understanding of the potential causative agent of RID. For instance, either ies Fig. a. In genKlebsiella, Pseudomonas, Cutibacterium, Corynebacterium, Bacillus and uncultured Enterobacteriaceae were detected in all samples with variable abundance . Additionally, the predominance of these species was accompanied by a slightly enriched representation of uncultured Enterobacteriaceae species, Klebsiella varicola, Stenotrophomonas maltophilia, Rothia dentocariosa and Acinetobacter johnsonii. Nevertheless, unclassified sequences represented about 21.83% of cutaneous microbiomes of controls, while in RID microbiomes they were constituted 1.69%. Strikingly, K. pneumoniae was negatively correlated to P. aeruginosa in all study groups . Also, the strongest correlations were detected between P. aeruginosa, S. aureus and S. maltophilia.Species level analysis of RSVs revealed the significant enrichment of The collective metabolic functional profile of RID microbiome was significantly associated with the overrepresentation of metabolic pathways that could be attributed to impairments in epidermal integrity, alteration in pH and supporting microbial survival such as lipid metabolism, histidine metabolism, enhanced bacterial invasion of epithelial cells, signaling molecules of bacterial toxins, hedgehog signaling pathway, nitrogen metabolism and peptidases . Diabetes mellitus is a metabolic disorder that is significantly linked to developing chronic wounds due to the impairments in wound healing phases that provides favorable conditions for either opportunistic residents or pathogenic bacteria50. Also, colonization of wounds with certain microorganisms was strongly correlated to the persistence of wound via organism induced body changes that include epidermal barrier integrity, alteration in pH and dysregulation of the immune system53. Overall, developing chronic ulcer in diabetic patients with RID could potentially depend on the colonizing microorganisms through intensifying the epidermal disturbances produced by diabetes mellitus.One of the interesting findings is the significant association between diabetes mellitus and developing a chronic ulcer in RID patients (Spearman: r = 0.86; Klebsiella genus was inversely correlated to other predominant genera in all datasets, Pseudomonas, Staphylococcus and Stenotrophomonas have exhibited a significant coexistence especially in prolonged RID and chronic ulcers (Supplemental Table S. aureus and P. aeruginosa on the clinical outcome of cystic fibrosis is controversial, the comparisons with mono-infection of S. aureus or P. aeruginosa showed that the pair coexisting was significantly associated with prolonged hospitalization and exacerbation of the disease54. Also, the bidirectional interplay between this pair was manifested in the inhibition of phagocytosis, in addition to the promotion of staphylococcal biofilm formation by P. aeruginosa55. Additionally, Stenotrophomonas maltophilia was detected in our samples with significant prevalence especially in RID patients. S. maltophilia has emerged as an opportunistic pathogen with global concerns, due to its significant association with the high crude mortality in pneumonia and bacteremia of immunosuppressed patients56. S. maltophilia was also reported to cause wound infections, soft tissue infections, bone and joint infections, and cellulitis58. Interestingly, in our study, the coexistence of S. maltophilia and P. aeruginosa was strongly correlated to develop chronic ulcers in RID. This observation was in agreement with the previously reported cooperative pathogenicity of this pair in cystic fibrosis59.The main interesting finding of the present study is the collective correlations between community members. While Staphylococcus genus was defined as a core genus in healthy and diseased groups. Although S. aureus was significantly enriched in RID microbiomes, it was overrepresented in the rapidly healed RID. These findings were matched with the previous study which reported that the S. aureus infection was conjugated with limited exacerbations and a short duration of hospitalization54.As expected in the cutaneous microbiome, K. pneumoniae showed fluctuated representation in our specimens. In microbiomes of RID, K. pneumoniae was significantly overrepresented especially in samples that turned to a chronic ulcer, and its abundance was strongly linked to diabetes mellitus. As it is known that K. pneumoniae have been implicated in liver abscess, meningitis and psoas abscess60. Furthermore, K. pneumoniae may harbor diverse virulence characteristics including capsules, multidrug resistance and virulence factors that enhance invasion and promote epidermal deteriorations62. The coexistence of these characteristics and diabetes mellitus could boost epidermal damage, aggravate the inflammatory processes and retard the repair of the epidermal barrier.Notably, The limitations of this study are the slightly small sample size of each group, dependence on unculturable approach, lack of microbiome characterization at different sampling time and isolation of causative agents in order to define their virulence characteristics including antimicrobial resistance patterns.Klebsiella or coexistence of Pseudomonas, Staphylococcus and Stenotrophomonas potentially prompt the chronic ulcer formation in RID patients. Cutaneous microbiome profiling at the early stages of RID could be indicative of the prospective clinical outcomes and might be a helpful guide for personalized therapy and management of RID. Finally, shifts in the structure and prevalence of cutaneous microbiota that accompany the RID could support the candidate role of microbial dysbiosis in the pathogenesis of RID.We have studied the microbiome of patients with RID in order to accentuate the underlying bacterial structure and to provide new insight about the potential role of microbiota as an influential factor in pathogenesis and clinical outcome of RID as well. A notable positive correlation has existed between bacterial diversity and recovery rates of RID. The overwhelming predominance of Supplementary Information.