The introduction of graphene will certainly uncover new advanced materials, and many more future technologies will become realistic in the forthcoming years.The present review article includes more recent publications about the biomedical application and cellular interaction of graphene. It is also updated with modern approaches such as use of graphene inks for 3D printing application.Moreover, the importance of protein corona in modulating the cellular interaction, which was overlooked in previous review publications, is also included in this article.The possible biological outcomes and toxicity when graphene is exposed to living organisms at the cellular and organ level are explained. sp2 hybridized carbon framework of one atom thickness, is reputed as the strongest material to date. It has marked its impact in manifold applications including electronics, sensors, composites, and catalysis. Current state-of-the-art graphene research revolves around its biomedical applications. The two-dimensional (2D) planar structure of graphene provides a large surface area for loading drugs/biomolecules and the possibility of conjugating fluorescent dyes for bioimaging. The high near-infrared absorbance makes graphene ideal for photothermal therapy. Henceforth, graphene turns out to be a reliable multifunctional material for use in diagnosis and treatment. It exhibits antibacterial property by directly interacting with the cell membrane. Potential application of graphene as a scaffold for the attachment and proliferation of stem cells and neuronal cells is captivating in a tissue regeneration scenario. Fabrication of 2D graphene into a 3D structure is made possible with the help of 3D printing, a revolutionary technology having promising applications in tissue and organ engineering. However, apart from its advantageous application scope, use of graphene raises toxicity concerns. Several reports have confirmed the potential toxicity of graphene and its derivatives, and the inconsistency may be due to the lack of standardized consensus protocols. The present review focuses on the hidden facts of graphene and its biomedical application, with special emphasis on drug delivery, biosensing, bioimaging, antibacterial, tissue engineering, and 3D printing applications.Graphene,  Graphene, the basic structure for all graphitic materials, is a two-dimensional (2D) sheet of carbon atoms arranged in a honeycomb lattice structure. The outstanding properties of graphene including its mechanical strength, electrical conductivity, thermal conductivity, and large surface area highlight its widespread applications. However, in reality, the hydrophobic nature of graphene impedes its use in the biomedical field . GraphenAmong the graphene family materials, GO, a derivative of graphene with oxygen-containing functional groups, is widely recommended for biomedical use as it is readily soluble in water (hydrophilic). GO possesses carboxyl, epoxyl, and hydroxyl functional groups distributed throughout the basal plane and its edges. The stronger hydrogen bond interaction between the oxygen molecule of the epoxide groups of GO and water molecules maintains its stacked structure .sp2 conjugation of the lattice, making GO an insulator [Graphite oxide (GtO), another form of graphene, differs from GO in its structure, but, chemically, the two are similar. GtO is a highly stacked structure with oxide functionalities, whereas GO has a wide spacing between the layers because of water intercalation . The strnsulator . The elensulator , hydroqunsulator , ascorbinsulator , sodium nsulator , and strnsulator , 11 has nsulator  because Despite the tremendous increase in the number of literature studies on graphene synthesis, the large-scale industrial production of graphene is still difficult to achieve because of the various synthetic methods adopted, which vary with their application. Graphene is obtained from GO by removing oxygen-containing functional groups via simple chemical or thermal reduction. Basically, graphene synthesis is categorized into two types: a top-down and a bottom-up approach. The top-down approach uses chemical ablation, electrochemical oxidation, or plasma treatment to cut down larger graphene sheets into smaller pieces. In contrast, the bottom-up method involves building up larger graphene sheets from simple carbon precursors.Graphite, an allotrope of carbon, is a naturally available chemical having numerous defects in its structure. It is the precursor material for the synthesis of different forms of graphene. Mechanical exfoliation of graphene from pyrolytic graphite using Scotch tape was developed by Geim and Novoselov . The adv4 with concentrated H2SO4 gives explosive Mn2O7 (manganese heptoxide), which oxidizes graphite [Chemical exfoliation of graphite remains a simple, efficient, and cost-effective method for producing hydrophilic GO Fig. . The prographite .Fig. 1Prsp2-hybridized carbon sheets into a graphitic sp2 domain surrounded by oxidized sp3 domains and several defects [Oxidation of the defect sites of graphite can be initiated using different oxidizing agents such as nitric acid, potassium chlorate , and pot defects . The oxi defects .2), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and N,N′-dicyclohexyl-carbodiimide, followed by the addition of amines or hydroxyl-forming covalent amide or ester bonds. In addition, polymers can also be grafted on the carboxylic end of GO to make it dispersible in water and solvents. Functionalization of the epoxy groups involves the ring-opening reaction [π–π stacking and van der Waals interaction. Preparation of stable dispersions of graphene remains an unsolved problem because of its hydrophobic nature. This can be achieved by sonicating graphene suspension for several hours and also by using surfactants or polymers. Details of the effect of different solvents and surfactants and the importance of sonication to obtain a stable aqueous dispersion of GO were well thoroughly investigated by Khan et al. [N,N-dimethylformamide (DMF), ethylene glycol, acetone, pyridine, 2-propanol, methanol, ethanol, and dimethyl sulfoxide (DMSO), and was sonicated for 400 h along with the addition of sodium cholate. The treated GO samples were stable in water, DMF, ethylene glycol, and pyridine, and remained as homogenous dispersion even after 3 weeks without any aggregation. Similarly, Paredes et al. [N-methylpyrrolidone (NMP), pyridine, THF, dichloromethane, o-xylene, and n-hexane) following 1 h of bath sonication. Immediately after sonication, the GtO remained dispersed in almost all the solvents. However, long-term stability (3 weeks) was achieved only in water, ethylene glycol, DMF, NMP, and THF. Both these studies identified suitable solvents to improve GO dispersibility for further application. Simple magnetic stirring and a heating method were also demonstrated for the synthesis of aqueous dispersion of single-layer GO [GO contains chemically reactive functional groups such as carboxylic acid on the edges and hydroxyl and epoxy groups on the basal plane. These oxygen-containing functional groups are modified to formulate biocompatible graphene for clinical use. The carboxylic acid groups are activated by various chemicals such as thionyl chloride  or chemical (reducing agents) reduction conditions results in the formation of RGO. The chemically reduced graphene sheet undergoes incomplete reduction, leaving behind a few oxygen-containing functional groups. Chemically reduced graphene looks similar to pristine graphene in terms of electrical, thermal, and mechanical properties. Commonly followed hydrazine-based reduction increases the presence of ohydride , hydroquohydride , gaseousohydride , strong ohydride , 11, andohydride  are also2 gas, which creates enough pressure to separate the stacked structures [GO is also treated at higher temperatures to exfoliate its stacked structure. This physical method of reduction extrudes COructures . Howeverructures .Electrochemical reduction of GO was developed to avoid the use of harmful reductants such as hydrazine. In this method, GO films are deposited on the surface of various substrates; after placing electrodes on the opposite end of the film, voltammetry is run . The exaA green route for graphene synthesis under alkaline conditions was reported . The metExfoliation and reduction of sulfuric acid-intercalated GtO at > 100 °C resulted in single-layer graphene sheets of 1.07 µm mean diameter following dispersion in DMF . When grMicrowave-assisted reduction, ion bombardment, hydrazine-free aqueous route, ultraviolet irradiation, thermal reduction, chemical reduction, electrochemical reduction, and solvothermal reduction have been reported in literature . In therShear mixer has been demonstrated to exfoliate graphite into individual graphene nanosheets. The combined effect of the rotor and the stator generates shearing, impacting, pressing, turbulence, and cavitation. The mechanical rotation of the rotor creates a shear force that peels off thin GO nanosheets from GtO particles by cleaving the inter-plane bonds. Another force, the impact force, is created owing to the mechanical impact against the rotor, which removes nanosheets from the edges and also induces in-plane fractures . A high-Biomedical application of graphene is a new fascinating area that is beyond imagination. The overwhelming properties of GO that support its clinical use are the amphiphilicity, surface functionality, fluorescence quenching ability, and surface-enhanced Raman scattering property. The hydrophobic nature, large surface area, ripples, and grain boundaries on defective sites of graphene are important factors when considering them for biomedical use. The first-ever use of GO as a nanocarrier for drug delivery reported by Sun et al.  paved th2 domain afford them high loading capacity, high solubility, and biocompatibility. Multimodal GO with multiple functions can be produced by conjugating polymers, proteins, and biomolecules via simple physisorption or chemical conjugation. GO acts as an efficient nanocarrier for delivering water-insoluble anticancer drugs. The water-insoluble anticancer drug SN38 was successfully loaded onto amine-terminated PEG-grafted GO by noncovalent adsorption and was targeted against cancer cells [π electron on the graphene surface helps in loading of aromatic anticancer drugs through π–π stacking or hydrophobic interaction. Selective killing of cancer cells was achieved by loading doxorubicin (DOX) onto antibody-conjugated PEGylated nano-GO (NGO) sheets. The quinone portion of DOX binds to GO via π–π interactions, whereas hydrogen bond is formed between the amino/hydroxyl groups of DOX and the hydroxyl/carboxyl groups in GO. It was noticed that the drug loading and release kinetics depends on the pH. Maximum drug loading capacity was observed at neutral pH, whereas more than 70% of the drug was released at an acidic pH of 2 [GO with its oxygen-containing functional groups (COOH and OH) has been reported as an effective carrier for drug or gene delivery Fig. a. Despiter cells . The del pH of 2 . Under a pH of 2 .Fig. 2DrGO also improves the solubility and bioavailability of camptothecin (CPT), a quinoline alkaloid that kills cancer cells by inhibiting the DNA enzyme topoisomerase I. Since cancer cells are known to express high amounts of folate receptors, controlled and targeted delivery of multiple drugs is made feasible by conjugating folic acid onto graphene sheets for effective killing of cancer cells. De Sousa et al.  utilizedTwo “off–on” switches of a photoresponsive drug release system made of GO and mesoporous silica nanoparticles (MSNs) were developed . Here, tGene therapy is a grafting technique to successfully treat various genetic disorders. Successful gene therapy is achieved by developing a vector that protects the DNA from endonuclease and possesses high transfection efficiency. Molecular beacons and aptamers can be delivered inside cells with the help of GO for specific detection of biomolecules. It is well known that cationic polymers such as polyethyleneimine (PEI) induce a proton sponge effect on endocytosis and promote endosomal escape for efficient gene delivery. For the facilitation of gene delivery, GO functionalized with positively charged cationic PEI was transfected with plasmid DNA . The plaGene silencing in cells can be achieved by delivering siRNA using PEI-GO Fig. b. For exGraphene acts as a perfect platform for binding of protein molecules, and it also protects proteins from proteolysis. Hence, graphene-based materials can be used as a nanocarrier for the intracellular delivery of therapeutic proteins. Bone morphogenetic protein (BMP) is a well-studied osteoinductive protein for inducing bone regeneration. Local delivery of BMP to induce osteogenesis for bone regeneration is highly appreciable in clinical treatment. GO-coated Ti implant was developed as a vehicle for the delivery of BMP at the site of interest . Sustain−2) of pristine GO provides effective PTT efficiency in vivo [Photothermal therapy (PTT) is recently considered as a minimally invasive and highly efficient method for cancer treatment. PTT involves the conversion of light energy into heat energy by certain light-absorbing agents under irradiation. When the excited molecules come to a ground state, they release energy in the form of heat that causes thermal ablation of cancer. Nanomaterials with NIR absorbance are highly appreciable for PTT as they avoid interference from biological tissues. Compared to that of the commonly employed PPT agents such as gold nanoparticles, the lower power density  and low-power NIR irradiation  was noticed in a 4T1 tumor mice model. Complete destruction of the tumor in 1 day was achieved, with no tumor regrowth observed for another 40 days. GO decorated with iron oxide and gold nanoparticles was fabricated to obtain a multifunctional nanocomposite with strong superparamagnetism and enhanced NIR absorbance. The biocompatibility of the nanocomposite was further increased by surface functionalization with PEG. This multifunctional nanocomposite showed remarkable photothermal ablation of cancer with decreased toxicity. Magnetic resonance (MR) and X-ray dual-modal imaging were made possible owing to the presence of iron oxide and gold nanoparticles [2 and H2O. The instant formation and collapse of microbubbles damage the cancer cells.GO shows higher absorbance in the NIR region, a property that is utilized for photothermal destruction of tumor cells (hyperthermia). PEGylated nanographene sheets showed high tumor uptake efficiency when injected into a mouse tumor model . It alsoarticles . It was articles . IrradiaCombined chemo- and photothermal therapy is an effective way to treat cancer compared to monotherapy. The high drug loading capacity and NIR absorbance property of graphene can be utilized for the synergistic treatment. To achieve this, the authors in  developeπ–π stacking, showed effective killing of MGC803 cancer cells under irradiation [π–π interaction. This complex can be utilized to treat tumors under hypoxic conditions as well as deep solid tumors such as the glioblastoma multiform tumor [π–π stacking. This was further modified with aptamer sgc8, which specifically binds to the protein expressed by CCRF-CEM cancer cells. These nanoconjugates are effectively endocytosed by the cells, and upon laser irradiation, they induce the formation of heat and singlet oxygen  is a novel treatment for cancer as it combines photosensitizer-mediated targeted killing. Upon light activation, the photosensitizer molecule gets excited and induces the formation of reactive oxygen species (ROS) that cause irreversible damage to cancer cells. Chlorine 6, a photosensitizer that is loaded onto folic acid-conjugated GO via hydrophobic and adiation . Porphyrrm tumor . PEG-GO rm tumor . Ultrasorm tumor . Aptamerrm tumor . ICG, a hyrin IX , and NGOhyrin IX  for combEffective delivery of photosensitizers for PDT along with enhanced fluorescence imaging was achieved by Yan et al. . In theiπ–π stacking. Upon irradiation, the fluorescence of PPa is quenched owing to the energy transfer from PPa to GO through the fluorescence resonance energy transfer (FRET) mechanism. PPa-NGO-mAb enters into the cell and accumulates in the mitochondria. While entering the mitochondria, PPa-NGO-mAb comes into contact with the lipid mitochondrial membrane where π–π interaction is broken, resulting in fluorescent regain of PPa. This phototoxicity on/off system based on a graphene material is a novel carrier for subcellular targeting and attacking of αvβ3-expressing tumor cells via the production of singlet oxygen  was effectively conjugated onto the surface of polyvinylpyrrolidone (PVP)-functionalized RGO (nanocarbon) for effective PDT . CoatingGraphene has attractive application in the development of electrochemical sensors and biosensors owing to its excellent electrical conductivity, large surface area, and high electron transfer potential. Graphene-based field effect transistor biosensors have been developed to detect biomolecules such as nucleic acids, proteins, and growth factors, which monitor the changes in an electrical signal. Developing graphene-based biosensors relies on the fluorescent quenching property of graphene. The detection of nucleic acid is done by tagging GO with fluorescent-labeled ssDNA. GO quenches the fluorescence of ssDNA. This ssDNA forms a double helical structure when it comes into contact with the target complementary sequence. The formation of a double helix displaces GO from an ssDNA strand, resulting in fluorescence recovery Fig. a.Fig. 4GFRET biosensors, FET biosensors, and biosensors for DNA detection  have beeEscherichia coli (E. coli) [E. coli antibodies and was passivated using Tween 20. This graphene biosensor detects E. coli with high specificity and selectivity. The conductance of the graphene sensor increases with the increase in the number of E. coli attached on the graphene sheets. In addition, graphene is also used for the sensitive detection of biomarkers for early detection of life-threatening diseases  . CVD-groses Fig. c. In the present . Use of π–π stacking. The adsorption of the peptide to GO quenches the fluorescence emission from the dye molecule. Competitive binding of LPS induces fluorescence recovery, releasing dye-labeled peptide from the GO sheets. This method is a rapid, selective, and sensitive method for the detection of LPS/endotoxin in aqueous solution at room temperature. The detection limit of the sensor was found to be 130 pM.Graphene-based biosensors have also been developed for the detection of lipopolysaccharides (LPSs). The most commonly employed enzymatic assay for LPS detection is the limulus amebocyte lysate (LAL) assay. However, the LAL assay has certain pitfalls: it is highly susceptible to changes in temperature and pH and requires monotonous sample preparation and controlled experimental conditions. Several efforts have been taken to develop alternate methods for the detection of endotoxin using synthetic sensors . However, these sensors are expensive, are not robust, and are less sensitive to the detection of endotoxin at the picomolar regime. A GO-based fluorescence turn-on biosensor was developed for the detection of endotoxins . Tetrame3O4 magnetic beads were hybridized to a complementary DNA 1 probe. Incubation of these magnetic beads in LPS solution promotes the binding of LPS to LBA aptamer, concomitantly releasing DNA 1. The released DNA 1 together with the help of an assistant probe binds to the capture probe and unwinds its hair pin structure, forming a Y-shaped junction structure. This Y-shaped capture probe is then cleaved by exonucleases, releasing both the DNA 1 and the assistant probe. The released probes are now available for binding to new capture probes and the cycle continues. The continuous action of rehybridization, nicking, and release creates a large number of cleaved capture probes. The cleaved capture probes bind with a DNA 2 nanocomposite. DNA 2 nanocomposite is made of DNA 2-labeled AuNPs-Tb-Gra. The large surface area of graphene increases the immobilization of toluidine blue (Tb) and enhances the electrochemical signal. Both graphene and gold nanoparticles (AuNPs) amplify the signal, providing the detection of LPS at the femtogram level  [Recently, magnetocatalytic graphene quantum dot (GQD)-based Janus micromotors were synthesized for the detection of endotoxin released from 0111:B4) . These msp2 and oxidized domain. Annealing induces phase transformation in GO that promotes oxygen diffusion, resulting in the formation of nanosized (1-1.5 nm) graphitic domains responsible for the blue photoluminescence. This procedure permits oxygen diffusion only and does not remove oxygen from the graphitic structure; hence, subsequent conjugation of drug molecules is also possible. Jin et al. [Since GO possesses strong absorbance and fluorescence property in the NIR region, it is used as an imaging tool. Fluorescence can be induced in GO by manipulating the synthesis conditions such as the pH, rate of reduction, and size. The presence of functional groups on the sides of the planar graphene can be conjugated with fluorescent dyes for bioimaging. Simultaneous imaging and drug delivery using GO were recently reported by Cheng et al. . Under mn et al.  stated tized 1-1. nm graphn et al. .Fig. 5Bi3O4 nanoparticles were immobilized on GO nanosheets and were used as a contrast agent in MRI, which not only exhibited enhanced MRI signal but also were biocompatible [GO protects DNA from hydrolysis by DNAse I owing to the steric hindrance preventing the binding of DNAse I. Hence, graphene can be used for the delivery of aptamer probes for in vivo imaging of biomolecules. In a study , GO nanompatible .E. coli are less sensitive to graphene than Gram-positive bacteria such as Staphylococcus aureus [Both GO and RGO are known to possess antibacterial activity against a wide range of bacteria. It was found that Gram-negative bacteria such as s aureus . The pres aureus  proposed−1, whereas for Gram-positive bacteria, MIC was found to be 4–8 µg mL−1 [l-lysine, chitosan, lactoferrin, and polyvinyl-N-carbazole) have also been developed to provide antibacterial surface for biomedical applications. Recently, Zarafu et al. [Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (S. aureus) bacteria. These functionalized GO hybrids exhibited improved inhibitory activity against bacteria compared to amines alone.RGO nanowalls were found to be more toxic toward bacteria than GO because of better charge transfer with the bacterial cells and of the presence of sharp edges of RGO . The sha µg mL−1 . Graphenu et al.  function2O2) photolysis. Direct photolysis samples were more effective in inhibiting the growth of the bacteria E. coli K12 than the indirect photolyzed samples. These differences could be attributed to the size of GO after photolysis. Indirect photolysis that involves addition of H2O2 resulted in the formation of smaller GO sheets than those formed using direct photolysis. The larger GO from direct photolysis can effectively interact, wrap the cells, and induce membrane deformation. In addition, it also possesses greater oxidation capacity toward GSH, depleting the cellular antioxidant level. Another study demonstrated the use of GO as a reservoir for loading of antimicrobial peptide (G(IIKK)4I-NH2) and for its sustained release [4I-NH2 enhances its binding on the negative charge surface of GO. Upon binding, G(IIKK)4I-NH2 monomers undergo structural transition to form an α-helix secondary structure. Layer-by-layer assembly of a GO-G(IIKK)4I-NH2 nanocomposite showed similar MIC values to those obtained from free G(IIKK)4I-NH2. Sustained release of antimicrobial peptide from this nanocomposite demonstrates an effective approach for surface coating of devices to achieve long-term antibacterial activity.The antibacterial activity of GO photolyzed under simulated sunlight was studied recently by Hou et al. . To indu release . The posIn contrast to the above findings, enhanced bacterial growth was observed on the GO surface . The bacπ–π interaction), whereas GO does not affect adipogenesis owing to electrostatic binding of insulin. However, graphene enhances osteogenesis by pre-concentrating osteogenic factors [Successful tissue engineering depends on the biocompatible substratum that offers cells to attach, grow, and proliferate. Stem cells are one of the most promising candidates for tissue regeneration because of their differentiation into cells of specific lineage. Recently, graphene has been put on the spotlight as a reliable scaffold for the attachment and proliferation of stem cells, especially MSCs and neuronal cells. Several cell lines were cultured on the surface of graphene-coated substratum, e.g., osteoblasts , NIH-3T3 factors .Fig. 6GrAnother important property of graphene is its electrical conductivity. This property enables graphene to modulate neural stem cell activity. The findings suggested that graphene differentiates human neural stem cells into neurons Fig. a. NeuralThe unique mechanical and physical properties of graphene and its ability to induce differentiation of stem cells unlock opportunities in dental applications. High mechanical strength, durability, and biocompatibility are the prerequisite for any dental materials. Fabrication of graphene-based materials into dental composites will increase the mechanical properties such as the compressive strength and the compressive modulus . GO-induβ cells. Altogether, the above findings show the promising use of graphene at a safe dose for stem cell therapy and regenerative medicine.Induced pluripotent stem cells (iPSCs) are another source of cells that have major potential in tissue engineering and regenerative medicine. Both graphene and GO were shown to induce spontaneous differentiation of iPSCs into cells of ectodermal and mesodermal lineages . However−1) and high tensile strength (< 1 MPa), are mechanically resilient, possess the ability to withstand strain (> 80%), and are highly bioactive [Three-dimensional (3D) printing is a revolutionary technology, having promising application in tissue and organ engineering. Fabrication of 2D graphene into a 3D structure is made possible with the help of 3D printing. Three-dimensional printing is an efficient technology for enabling the direct production of 3D bulk objects. In this technology, polymer, ceramics, or metals can be heated and deposited layer by layer under computer control to build 3D monoliths that are designed using software associated with the printer. The development of 2D and 3D printing graphene-based ink has helped scientists and engineers develop modern devices, sensors, and constructs for tissue engineering. Three-dimensional graphene printed materials possess high electrical conductivity  . The 3D In vivo studies in mice showed that a subcutaneously implanted 3DG scaffold had no severe immune response or fibrous capsule formation. Pores of the scaffold were covered with extracellular matrix structure collagen. Vascularization and tissue integration were noticed after 30 days of implantation. Macrophages appeared to surround the implanted site, which probably indicates the removal of graphene sheets. Absence of pathological lesions in organs  indicates that the graphene sheets either were cleared away from the system or remained in close proximity to the implanted site.Similarly, GO was incorporated into a nanocomposite based on polyurethane/poly lactic acid, which increases the mechanical strength and thermal property of 3D nanocomposites. It was found that the mechanical strength depends on the printing orientation. It was also revealed that the nanocomposite provides good biocompatibility with NIH3T3 cells, promising their potential use as a biomaterial scaffold in tissue engineering . Multi-cWhen a nanoparticle is injected into the body, it enters the circulatory system immediately. The biomolecules present in the blood stream, especially the protein, are quickly adsorbed onto the nanoparticles and form a complex called “corona.” The formation of protein corona alters the physicochemical properties of nanoparticles and modulates the way they interact with the biological system and defines the biological fate of the nanomaterial. Generally, protein corona formed on the surface of nanoparticles is categorized into two layers: “hard” and “soft” corona Fig. a. Hard cπ–π stacking interaction between the graphene sp2 carbon atoms and the aromatic rings of amino acids facilitates the adsorption of aromatic residues, whereas the van der Waals interaction between the side chain (guanidinium group) of arginine and the graphene drives the adsorption of basic amino acid residues. During the adsorption process, water molecules are displaced in the interactive region and are squeezed out. The other side of the protein is fully solvated by water. Adsorption also induces large conformational changes in the structure of the protein; hence, the amino acids present in the interior of native proteins are now exposed to the graphene surface. Both GO and RGO have higher protein adsorption capacity than that of single-walled carbon nanotubes (SWCNTs) owing to their flat exposed surfaces. Between the two, RGO adsorbs less protein owing to the lower availability of oxygen groups. Understanding of adsorption kinetics is important to determine the saturation limits, structural and conformational changes, and difference in biological responses. In general, binding of proteins onto the surface of graphene increases with time, and once it reaches the equilibrium, no further adsorption occurs or the protein may fall off from the surface of the nanomaterials. The binding affinity of four proteins  on the surface of pristine graphene, GO, and RGO was studied by Chong et al. [π–π stacking.Molecular dynamic simulation studies showed that basic amino acid residues (arginine and lysine) and aromatic residues  play a key role in binding the protein onto the surface of graphene sheets . The strg et al. . Measure−1) was pre-incubated with fetal bovine serum (FBS) before exposing it to cells [Binding of proteins from serum not only increases the size of the nanoparticles but also attenuates the cytotoxicity induced by graphene. The high surface-to-volume ratio of graphene helps in the adsorption of large amounts of proteins. The defects present on the planar surface of graphene provide an additional binding site for proteins. About 90% cell viability was obtained when GO  Fig. c. All otIt is well known that the complex interaction between graphene and the biological system induces numerous responses inside the cells. Several reports are currently available on the toxicity induced by pristine graphene and graphene derivatives , 146. ThIt is a fact that nanomaterials are often contaminated with endotoxin during synthesis, and handling that may cause septic shock in patients following administration. Endotoxins are lipopolysaccharides derived from the outer membrane of Gram-negative bacteria. LPS binds to pattern recognition receptors and activates immune cells to secrete pro-inflammatory mediators such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, mediating an inflammatory response. Endotoxin detection is performed using rabbit pyrogen test and LAL assay. Endotoxin detection is difficult in carbon nanomaterials including graphene owing to their interference with LAL assay, which may lead to flawed results. Recently, a TNF-α expression test was developed to detect LPS in graphene samples at noncytotoxic doses . This teBim and Bax (pro-apoptotic factors) activation induces mitochondrial permeabilization, and these factors are released into the cytosol, which eventually activates several cascades of caspase enzymes, ultimately resulting in cell death , IL-10, IL-12, TNF-α, monocyte chemotactic protein-1, and interferon-γ are believed to be released upon GO exposure. Graphene activates the TLR and NF-κB signaling pathways, resulting in an inflammatory response Fig. a. GO is The in vitro hemocompatibility of GO and graphene sheets was studied in isolated RBC membranes using hemolysis assay . The res−1), no obvious cytotoxicity was noticed for GQDs. However, at a concentration of 100 µg mL−1, only 50% of the cells were found to be alive. Even after surface functionalization, significant toxicity of GQDs has been reported by a few studies. Like other graphene materials, GQDs also provoke cytotoxicity, increased ROS formation, and genotoxicity in mammalian cells. They can induce DNA damage in cells without affecting their viability by increasing the expression of p53, Rad 51, and OGG1. Although GQDs are mainly distributed in the cytoplasm, with no direct contact with the nucleus, they induce DNA damage indirectly through ROS generation [GQDs, which have a chemical structure similar to that of graphene but smaller in size, are generally considered to be nontoxic. At a lower concentration (< 50 µg mLneration . They haneration . Biologineration . p21 is 14C-labeled few-layer graphene (FLG) showed that around 47% of the FLG remains in the lungs even 4 weeks post-intratracheal instillation [Several in vivo reports are available on graphene material toxicity. However, the results are inconsistent because of the difference in the route of exposure and the characteristics of the graphene synthesized. Quantitative measurement of illation . The preRecently, the biodistribution and toxicokinetics of PRGO in mice following intraperitoneal and intravenous administration were examined . It was −1 body weight) 1 and 30 days before cohabitation. No change in mating behavior was noticed in the female mice. Moreover, less-deformed fetuses were seen in mice injected with RGO at the early stage of gestation (~ 6 days). However, when RGO was injected at a later stage of gestation (~ 20 days), most females died at high dose, whereas the surviving females had abortions. This study [The effect of RGO on female reproductive ability and offspring health was recently studied . In theiis study  also expThe 2D graphene structure offers several advantages over conventional nanoparticles owing to its unique physicochemical characteristics. The successful application of graphene materials will pave a new way for building a nanoplatform in biomedical research. However, the use of graphene in the biomedical field is still in its nascent stage, with numerous challenges to be overcome. The toxicity induced by graphene nanomaterials in a biological system causes significant safety concerns pertaining to the use of the nanoparticles. Several measures to reduce their toxicity such as the use of biocompatible polymers and a green route for synthesis have already been reported in the literature. Desirable characteristics can be obtained by manipulating the synthesis under controlled conditions. More attention should be paid to understand the nano-bio interaction of graphene materials in a living system. A simple cytotoxicity test is not a valid screening criterion to describe any material as being biocompatible and suitable for clinical translation. Graphene is included in the list of hazardous materials by the European Scientific Committee on Emerging and Newly Identified Health Risks. There are still many gaps to be filled, considering the toxic potential of graphene. All physicochemical parameters including the size, shape, agglomeration, layer thickness, lateral dimension, and atomic composition should be considered while evaluating the toxicity. From a biological perspective, the effect of concentration, duration, route of exposure, and presence of impurities should be thoroughly investigated. The contradictory outcomes, difference in synthesis methods, and lack of reproducibility hinder the use of graphene in real-world applications. Since there are no regulatory guidelines available for testing the toxicity of nanomaterials, a standard protocol must be established to avoid such conflicts. In conclusion, graphene promises an exciting nanoplatform for biomedical applications, yet there are many questions that need to be addressed. It is recommended that graphene derivatives should undergo extensive safety evaluations or validations before considering them safe for biomedical applications or clinical use.


Being the first successfully prepared two-dimensional material, graphene has attracted extensive attention from researchers due to its excellent properties and extremely wide range of applications. In particular, graphene and its derivatives have displayed several ideal properties, including broadband light absorption, ability to quench fluorescence, excellent biocompatibility, and strong polarization-dependent effects, thus emerging as one of the most popular platforms for optical sensors. Graphene and its derivatives-based optical sensors have numerous advantages, such as high sensitivity, low-cost, fast response time, and small dimensions. In this review, recent developments in graphene and its derivatives-based optical sensors are summarized, covering aspects related to fluorescence, graphene-based substrates for surface-enhanced Raman scattering (SERS), optical fiber biological sensors, and other kinds of graphene-based optical sensors. Various sensing applications, such as single-cell detection, cancer diagnosis, protein, and DNA sensing, are introduced and discussed systematically. Finally, a summary and roadmap of current and future trends are presented in order to provide a prospect for the development of graphene and its derivatives-based optical sensors. Furthermore, two photon imaging with GO as a fluorescent label has been widely used in the fields of cancer cell imaging and food safety  biological imaging via two-photon excitation spectroscopy thanks to the giant red-edge effect . In 2014d safety . In 2016d safety . In addid safety . The NCs2+, a hairpin structure is formed. In this structure, the charge transfer process between GO and mercury ions causes the fluorescence of GO to be quenched. The present optical sensor shows a limit of detection as low as 0.92 nM and excellent selectivity over various metal ions. The fluorescence sensing based on charge transfer has been applied to the detection of more types of pollutants in environment and agriculture . In 2018, Zeng et al. successfully synthesized silver nanospheres coated with graphene oxide (Ag@NGO) (−4 to 10−9 M). In additional, SERS based on a reliable graphene-based substrate has been successfully used to discriminate various environment pollutants, including organic pollutants, heavy metal ions, pathogens, and antibiotics. Compared with traditional SERS substrates, graphene has proper modification and improved biocompatibility, which also makes graphene-based SERS substrates widely used in biosensing, drug delivery, and bioimaging fields. However, graphene-based substrates also need to address many difficulties and challenges. First of all, the stability of graphene-based substrates needs to be improved, especially when various biochemical reactions occur on the surface of graphene. In addition, non-specific adsorption on the graphene surface will also cause serious disturbances. Therefore, it is urgent to develop graphene based substrates with high stability and high specificity.Undoubtedly, graphene has been widely employed in the design of SERS substrates. Graphene has overcome some of the limitations of the SERS substrates since it endows the SERS substrates with better stability, sensitivity, reproducibility, and biocompatibility. More importantly, high-performance graphene based substrates have great application potential in numerous fields. Various biomarkers and biomolecules, such as DNA, nucleosides, proteins, bacteria, and fungi have been successfully detected through the employment of multiple graphene-based substrates . In addiubstrate . They ha(Ag@NGO) . The hyb(Ag@NGO) . Then thOptical fiber sensors have received world-wide attention due to their high sensitivity, small size, good anti-electromagnetism disturbance ability and other potential advantages. As the first prepared two-dimensional material, graphene has been used extensively in the design of optical fiber sensors due to its unique optical properties . In this3 gas sensor based on graphene/microfiber hybrid waveguide (GMHW)  . During cladding . It was achieved .The interaction between incident beam and graphene mainly includes interband and intraband transitions. In the far infrared and THz bands, the electronic response consists mainly of intraband transition (free carrier response), which can be well described by the Drude model. Instead, in the near infrared and visible range, the absorption of graphene consists mainly of interband transition, which is wavelength independent. Since the absorption of single layer graphene is only 2.3%, a variety of structures have been designed to enhance the absorption of graphene .2/Si substrate.In 2013, Ye et al. discovered that graphene exhibits strong polarization dependent optical absorption under total internal reflection . Compare−8) and sensitivity (4.3 × 107 mV/RIU), as well as an extensive dynamic range. s- and p-polarized beams by a polarization beam splitter. The difference in intensity of the two modes is measured by a balanced detector. The researchers regarded cancer cells and normal cells as microspheres with different refractive indices. When cancer cells and normal cells pass through the surface of graphene, the refractive index changes induced are different, thus enabling the detection of cancer cells. In addition, the polarized absorption optical properties of graphene have also been used in sensor design. In 2014, Xing et al. used the polarization dependent optical absorption of graphene in combination with microfluidic technology to achieve high sensitivity detection of cancer cells . They obs- and p-polarized beams by a polarization beam splitter. The detector measures the difference between s- and p-polarization caused by the modulated pump beam. By exploring the effect of the polarization angle of the pump beam on photothermal signal, Gao et al. explored the photothermal anisotropy of BP and ReSe2. Furthermore, the photothermal anisotropies of BP/ReSe2 heterostructures were also explored. The photothermal contrasts of samples were observed to change with different stacking angles indicating that the photothermal anisotropy of heterostructures is dependent on the stacking angle. These findings provide new prospects for designing novel optical devices based on two-dimensional anisotropic materials. In addition, the PTD technique has been used for identification of the crystalline orientation of anisotropic two-dimensional materials on a transparent substrate. Compared with traditional crystal orientation determination methods, the PTD overcomes typical challenges associated with transparent substrates, including insulating and rough surfaces, and enables the unambiguous identification of crystalline orientation. In 2018, Gao et al. used the polarization dependent absorption of two-dimensional materials and a modulated pump beam to measure their photothermal signal . PhotothIn summary, the recent progress in graphene and its derivatives-based optical sensors have been reviewed, covering aspects related to fluorescence sensing, graphene-based substrates for surface-enhanced Raman scattering, optical fiber biological sensors, and other kinds of graphene based optical sensors. The last few years have witnessed a dramatic increase in research effort on graphene-based optical sensors, both at the fundamental level and from a technological point of view. Graphene and its derivatives-based optical sensors have several advantages, including fast response, high sensitivity, and high flexibility, and are widely used in cancer cell imaging, DNA sensing and protein detection. Although tremendous efforts have been devoted to this research field over the past several years, there still undeniably remain several significant issues that are required to be addressed and explored. The challenges involved include not only the strategies to synthesize graphene and its derivatives but also the integration of sensors into practical applications. In the synthesis process, a major challenge is to synthesize high-quality graphene, and its properties can be tuned through effective chemical methods. Another challenge is to achieve a low-cost, environmentally friendly preparation method for graphene and its derivatives. For the practical application of optical sensors based on graphene and its derivatives, the primary issue is to develop high-sensitivity and high-specificity sensors, while another important objective is to enable miniaturization for integration into wearable devices. Overcoming the above challenges will not only boost graphene and its derivatives toward applications in optical sensing technology, but will also greatly advantage peoples’ life.X-GG and L-XC conceived the idea and designed the manuscript. X-GG wrote the paper. W-SJ, X-KL, and FX participated in the collection and sorting of relevant materials. All authors discussed the results and commented on the manuscript.This work was supported by the Natural Science Foundation ofChina (Grant 62005189), Natural Science Foundation of Shanxi Province (201901D211071), the open foundation of Key Laboratory of Weak Light Nonlinear Photonics, Ministry of Education (OS20-2), the Science and Technology Research Project of Henan Province (182102311130), the Key Scientific Research Items of Henan Province Colleges and Universities (20A416006), the Doctoral Research Grant Program of Xinxiang Medical University (XYBSKYZZ201719), the Natural Science Foundation of Henan Province (Grant No. 182300410107).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Xenopus tropicalis. These stable intronic sequence RNAs (sisRNAs) persist through the onset of zygotic transcription when synchronous cell division has ended, and the developing embryo consists of approximately 8000 cells. Despite their abundance, the sequence properties and biological function of sisRNAs are just beginning to be understood.Stable introns and intronic fragments make up the largest population of RNA in the oocyte nucleus of the frog X. tropicalis oocyte nucleus using published high-throughput RNA sequencing data. Our analysis revealed that sisRNAs, have an average length of ~ 360 nt, are widely expressed from genes with multiple introns, and are derived from specific regions of introns that are GC and TG rich, while CpG poor. They are enriched in introns at both ends of transcripts but preferentially at the 3′ end. The consensus binding sites of specific transcription factors such as Stat3 are enriched in sisRNAs, suggesting an association between sisRNAs and transcription factors involved in early development. Evolutionary conservation analysis of sisRNA sequences in seven vertebrate genomes indicates that sisRNAs are as conserved as other parts of introns, but much less conserved than exons.To characterize this population of non-coding RNA, we identified all of the sisRNAs in the In total, our results indicate sisRNAs are selected intron regions with distinct properties and may play a role in gene expression regulation. RNA is one of the three major macromolecules essential for living organisms and consists of three major types: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). Whereas mRNAs code for proteins , tRNAs aXenopus tropicalis [Drosophila melanogaster [Drosophila [In higher eukaryotes, the majority of protein-coding genes have one or more non-coding introns interspersed within the coding sequence that are spliced from the primary transcript . The splopicalis  and lateopicalis . Althougopicalis . These sopicalis , 10. Thunogaster , 18–22. osophila , 22–24.X. tropicalis genome using the published high-throughput sequencing data of RNA from the GV [To further characterize sisRNAs, we identified all sisRNAs in the m the GV . We thenXenopus tropicalis [X. tropicalis genome  from the GV of the frog opicalis  was usedopicalis . We idenome Fig. . Similarome Fig. . Togetheome Fig. a, which ome Fig. . Taken tTo determine the number of genes with sisRNAs, we first divided the genes of each dataset into 10 groups according to the number of introns in a gene. Genes without introns were excluded from further analysis. From the 9448 RefSeq genes, 93.8% (8864) have multiple exons while only 6.2% (583) of the genes have a single exon . For example, genes with a single intron have an average of 0.60 sisRNAs, genes with 5 introns have an average of 2.06 sisRNAs, and genes with 10 or more introns have an average of 5.06 sisRNAs  than the genome (40.07%). The GC content in sisRNAs alone  is higher than that of the introns  , CpG density and CA|TG density are widely used and important parameters to analyze sequences characteristics. Thus, we calculated these parameters for each intron with/without sisRNAs and compared these results with sisRNAs alone. The total GC content of the introns with sisRNAs 40.59%) is higher , middle introns (M) and last − 3′ end introns (E). The results indicate that last − 3′ end introns are very similar to the middle intron, while the first − 5′ end introns have different compositions and are slightly longer  in sisRNAs to study the potential functions of sisRNAs. We searched for enriched DNA motifs among the 935 position weight matrices (PWMs) collected from the TRANSFAC databases  in sisRNlis Fig. c. NF-κB lis Fig. a. These To further investigate the role of sisRNAs, we asked whether the genes with sisRNAs share any function. GO (Gene Ontology) enrichment analysis  of the 5X. tropicalis. As expected, the boundary of exon and intron has the highest conservation score  enrichment analysis for the genes host sisRNAs in each cell type , after the 12th cell division. The transcription rate must be extremely high in the oocyte and the transcripts must be very stable to allow a sufficient amount of RNA to be deposited in the cytoplasm such that the RNA:DNA ratio is not significantly depleted as the cells divide before MBT [Xenopus oocyte and early embryonic development.The biological function of sisRNAs in the fore MBT , 37. In fore MBT . HoweverXenopus, numerous circular intronic sequences have been identified in cultured human cell lines (Hela and H9) [Recently, 9000 sisRNAs have been found in the cytoplasm with about half of these confirmed as lariat molecules . These s and H9)  implying and H9) .Xenopus tropicalis oocytes [X. tropicalis and X. laevis [We showed that consensus TFBS of Stat3 is the most enriched motif in the introns from which sisRNAs are generated, and observed that Stat3 is highly expressed in  oocytes . A recen. laevis . It has . laevis . The sig. laevis . Stat3 b. laevis –43. Anal. laevis . In tota. laevis . A recen. laevis . Another. laevis , which mXenopus tropicalis [X. tropicalis genome, and as expected, TG is enriched. Interestingly, we observed higher GC content in sisRNAs, which suggested that the base composition of sisRNAs is not merely the consequence of deamination of 5mC, there must be other mechanisms playing a role in the evolutionary origin of sisRNAs. A recent study showed that exons of lncRNA loci also have a high GC content due to purifying selection [DNA methylation at the 5′ position of cytosine (5mC), primarily in CpG context, is observed in nearly every vertebrate examined, including opicalis . 5mC canopicalis . Deaminaopicalis , and as opicalis . The unmopicalis  tend to opicalis . As a reelection , thus itX. tropicalis sisRNA sequences in six other vertebrate genomes, including zebrafish, chicken, opossum, rat, mouse and human. Our results indicated that sisRNAs are not conserved: sisRNAs are as conserved as other parts of introns, but much less conserved than the exons. Cytoplasmic sisRNAs have been identified recently in X. tropicalis oocytes and these sisRNAs are only derived from a relatively small number of specific introns [We also assessed the evolutionary conservation of the  introns , it is wXenopus and Drosophila, implying that sisRNAs might be widely expressed in many other species [Thus far sisRNAs are identified in human, mouse, chicken, zebrafish,  species , 22. ConIn conclusion, our results suggest that sisRNAs are not transcribed from random part of introns but specific regions with distinct properties. The sisRNAs are GC rich while CpG poor, and preferentially enriched in the 3′ end of the mRNA transcript. Specific TFBSs involved in gene regulation are enriched in the regions from which sisRNAs arise, suggesting an association with specific proteins, such as Stat3, and further experiments are required to investigate this association. With more and more sisRNA data available in different species, the potential biological functions of sisRNAs would hopefully to be revealed soon.X. laevis XTC cells were obtained from [Xenopus tropicalis were downloaded from the University of California Santa Cruz Genome Bioinformatics website (http://genome.ucsc.edu/) [X. tropicalis genome assembly was xenTro2 (assembly version 4.1). Exons, introns, 5’UTRs, and 3’UTRs for refSeq genes and Ensembl genes were determined using UCSC annotations. UCSC genome browser screen shots were generated using custom tracks of the UCSC web site (https://genome.ucsc.edu/).The GV and cytoplasmic RNA-seq data were obtained from the Gall lab . sisRNA ned from . All thened from . Brieflyned from ; human Rned from . The datsc.edu/) . The refThe Model-Based Analysis of ChIP-seq algorithm (MACS)  was usedb with length of L in a certain gene g, we denote b (1:L). For each position i in b let the variable x at position i (xi) be 1 in case of an overlap with a sisRNA and 0 if it does not overlap. The density of sisRNAs in each bin for a certain joined intron transcript is calculated as: j (joined intron transcript), the set of sisRNA densities is: Gj = {Denj1, Denj2, …, Denj100}. Then the average sisRNAs density for a given bin b for the whole gene set  is calculated as: To calculate the sisRNA density in introns, we first concatenated all the introns for each gene. Then we divided each joined intron transcript into 100 bins. For a bin X. tropicalis genome, we searched the whole genome for each motif. In each scaffold, motifs were searched using MAST in MEME suite [M with the length L we denote M (ystart:yend) to record the positions where the motif starts and ends: y1:y1+L-1, y2: y2+L-1 ... yN:yN+L-1, N being the total number of motifs in the genome. For each position yi: yi+L-1, if it overlapped with the examined regions, sisRNAs or introns, xi=1, otherwise xi=0. For whole sisRNAs or introns, the observed occurrences (OCCobs) and expected occurrences (OCCexp) of the motif are calculated as: N is the total number of motifs in the whole genome, Lr is the total number of base pairs in the examined regions (sisRNAs or introns), and Lg is the total number of base pairs for the whole X. tropicalis genome. The enrichment score E for motif M was calculated as follows: OCCobs is the observed occurrences, and OCCexp is the expected occurrence of motif M in the examined regions (sisRNAs or introns).To determine the enriched motifs in sisRNAs and introns, we calculated an enrichment score for each motif. To avoid the bias of sampling from the ME suite  with theME suite  in whichhttp://david.abcc.ncifcrf.gov/) [P-values < 0.01 were considered as significantly enriched.Gene Ontology (GO) analysis was performed using DAVID  , 58. Go http://genome.ucsc.edu/). The seven genomes and assemblies are: zebrafish (danRer4), X. tropicalis (xenTro2), chicken , opossum (monDom4), rat (rn4), mouse (mm8) and human (hg18). PhastCons scores in each sisRNA or intron were extracted for each nucleotide for ±150 bps relative to the 5′ end, midpoint, and 3′ end respectively. Values were averaged for all sisRNAs or introns.Base by base PhastCons conservation scores based on an alignment and a model of neutral evolution among the seven vertebrate genomes  were dowAdditional file 1: Supplementary Figure 1. Longer introns have more sisRNAs. (A) Boxplot of lengths of introns grouped by the number of bearing sisRNAs: 0 (green), 1 (orange), 2 (purple), 3 (red) and > 3 (blue). (B) Scatter plot of sisRNA numbers versus length of introns. (C) Scatter plot of sisRNA numbers versus total length of introns per gene. (D) Scatter plot of sisRNA numbers versus normalized intron number per Mbps per gene. Supplementary Figure 2. sisRNAs are specific regions of the introns. Boxplots of the comparisons of GC% (A), CpG density (B), CA|TG density (C), and length (D) of the 1st introns (green), middle introns (orange), and last introns (purple) of genes with more than 3 introns, to sisRNAs identified by refSeq (red) and Ensembl (blue) genes. Supplementary Figure 3. The gene ontology (GO) analysis for the genes hosting cytoplasmic sisRNAs across species. Significant enriched GO terms for genes hosting cytoplasmic sisRNAs in human red blood cells (A), human Hela cells (B), mouse red blood cells (C), mouse 3 T3 cells (D), chicken DF1 cells (E), and Xenopus laevis XTC cells (F).


Negative Poisson's ratio (NPR), also known as “auxetic”, is a highly desired property in a wide range of future industry applications. By employing molecular dynamics (MD) simulation, metal matrix nanocomposites reinforced by graphene sheets are studied in this paper. In the simulation, single crystal copper with crystal orientation [1 1 0] is selected as the matrix and an embedded-atom method (EAM) potential is used to describe the interaction of copper atoms. An aligned graphene sheet is selected as reinforcement, and a hybrid potential, namely, the Erhart-Albe potential, is used for the interaction between a pair of carbon atoms. The interaction between the carbon atom and copper atom is approximated by the Lennard-Jones (L-J) potential. The simulation results showed that both graphene and copper matrix possess in-plane NPRs. The temperature-dependent mechanical properties of graphene/copper nanocomposites with in-plane NPRs are obtained for the first time. Graphene is an ideal reinforcement agent for high-performance composites due to its superb mechanical and physicochemical properties –6. RecenNegative Poisson's ratio (NPR), also known as “auxetic”, is a phenomenon associated with a material that experiences transverse expansion when a tensile load is applied in the longitudinal direction. On the other hand, if the material is under a compressive load, it contracts transversely. The NPR materials are a novel class of materials, which have advantages that conventional materials may not possess . MateriaIn most polycrystalline metal materials, the value of Poisson's ratio is in the vicinity of 1/3. However, it is different for metal materials with single crystals where NPR may exist in certain crystallographic directions . Early ro method , moleculo method , 45, molo method , first po method , and memo method .In this study, the classical MD method using the LAMMPS package  is emploFi(ρ) is the embedding energy of atom i which is a function of the atomic electron density ρ, ϕ is the pair potential interaction, and α and β are the element types of atoms I and J. In the case of this study, α and β are the same. The lattice constant and the atomic weight of the FCC copper are selected as 3.615 Å and 63.55, respectively.Copper is an ideal substrate that supports growing graphene layers. In this study, we create a MD model of single crystal copper along the [1 1 0], In the simulation, the tensile and shear behaviors controlled by strain are applied to this MD model to study its in-plane and out-of-plane mechanical properties. The temperature varying from 300 K to 1000 K is also considered in the simulation to observe the effect of temperature on the mechanical properties of the single crystal copper.VR and VΛ are the pairwise attractive and repulsive contributions, respectively, fC is the cutoff function, b is the bond order, and r is the distance between the nearest two carbon atoms. The detailed definitions of these physical quantities can be found in the literature  direction. The variation of the stretching rigidities, the shear rigidity, the in-plane Poisson's ratios, and the thermal expansion coefficients of the graphene from 300 K to 1000 K is shown in −4 nN and 1 million steps are taken for the balance of the whole system. The center deflection of graphene is calculated as the average value among transverse displacements of the nearest carbon atoms to the center point. In our study, the strain load of 10−6/fs is applied on the MD model and the averaged lattice parameters for the time step are converged to within 0.0001 nm. The strain load 10−6/fs with unit fs−1 means that we apply about 2 × 10−5 nm as an incremental displacement per femtosecond on the MD model of graphene. The equilibrium state depends on the difference of lattice parameters within a time step. If the difference is lower than 0.0001 nm, the whole model can be regarded to be in an equilibrium state. The graphene is an atomic-scale two-dimensional hexagonal lattice made by carbon atoms. In a dimensionally fixed graphene, the geometric center point must be in a hexagon cell. In our computation, the transverse displacements of the six carbon atoms of the hexagon cell in the center area of graphene are averaged to obtain the deflection of the graphene for each time point. Applying Equation VG=wE11 is slightly larger than E22 and they are both improved as the volume fraction of graphene is increased. We also notice that the rising trend of the moduli becomes slower when the graphene volume is higher than 7%. The out-of-plane moduli, E33, G13, and G23, with different VG are depicted in E33 is only improved by 7% with 13% volume fraction of graphene when compared with that of the pure copper. Unlike the in-plane shear modulus, the out-of-plane shear moduli, G13 and G23, are actually weakened by adding graphene reinforcement. We observe that the out-of-plane shear properties are degraded by as much as half when the composite contains only 5% volume fraction of graphene. The detailed moduli of the composites with different volume fractions of graphene under various temperatures are presented in ν12 and ν21) of the composite are between those of graphene and copper but closer to the graphene NPRs. With increase in temperature, the NPRs of the composite are decreased and the NPRs can reach about -0.09 at 1000 K. The effect of graphene volume fraction on ν12 of the composite is shown in α11 and α22 for all three materials being very close, only their α11 is compared in In order to study the auxetic behavior of the graphene-reinforced copper composites, the NPRs of the composite with 9% graphene volume fraction and its component materials are all shown in VG is 5%.This study not only presents novel MD results for MMCs reinforced by graphene sheets but also provides a new approach for the design of auxetic materials and structures. The graphene sheet and single crystal copper both showing in-plane auxeticity are selected as reinforcements and matrix, and their detailed material properties are obtained through MD simulations. To determine the effective material properties of graphene-reinforced metal matrix composites (GRMMCs), the MD simulations are carried out under different graphene volume fractions and temperature ranges from 300 K to 1000 K. The results showed that the in-plane NPRs of GRMMCs are decreased as the temperature rises or the graphene volume fraction increases and the maximum NPRs may reach -0.11 at 1000 K when the volume fraction of graphene


Furthermore, we observed no significant changes in anticorrosive behaviour of the graphene coated copper samples stored in ambient environment for more than one year.Due to the excellent chemical inertness, graphene can be used as an anti-corrosive coating to protect metal surfaces. Here, we report the growth of graphene by using a chemical vapour deposition (CVD) process with ethanol as a carbon source. Surface and structural characterisations of CVD grown films suggest the formation of double-layer graphene. Electrochemical impedance spectroscopy has been used to study the anticorrosion behaviour of the CVD grown graphene layer. The observed corrosion rate of 8.08 × 10 Corrosion is a naturally occurring phenomenon, which describes the tendency of a material to react with other chemicals resulting in deterioration of stability . A feasiGraphene is a hexagonal two-dimension atomically thin film of carbon. It exhibits many unique properties, including outstanding mechanical properties ,16,17,184) have been used in these papers because it provides stable carbon flow [To improve the coating quality, gas precursors such as methane  as the substrate of graphene coating. All of the substrates were originally sonicated in acetone solution (100%) for 5 min to remove organic residues. After this, the ethanol solution (100%) and deionized water were used to wash samples three times to remove the residual solution followed by heat treatment at 900 °C for 20 min to transform polycrystalline copper into single crystalline Cu(111). The polycrystalline copper substrate after the heat treatment is shown in 2 three times to remove any residual air for growth process. Our experimental conditions allow a heating rate of 18 °C/s and cooling rate of 7 °C/s. To optimize the growth parameters for graphene growth, a set of temperature and growth times were selected  is shown in shown in , and eacCVD grown films on the copper surface were thoroughly characterised. Scanning electron microscopy (F.E.I. Nova NanoSEM 230 and NanoSEM 450) was employed to investigate the surface morphology, and Raman spectra were recorded on a Renishaw inVia with 532 nm excitation laser to confirm the crystallinity and quality of graphene. Considering the possible surface inhomogeneity, at least three different spots on the surface were investigated to determine the characteristics of CVD grown films. The presented spectra are representative curves with baseline corrected.Potentiodynamic polarization and electrochemical impedance spectroscopy (EIS) study was conducted using a Versatile Multi-potentiostat VSP300 with EC-lab software to process the electrochemical data. A glass corrosion cell with the three-electrodes system was used for the electrochemical measurements with a platinum wire as a counter electrode, Ag/AgCl electrode as a reference electrode, and NaCl solution (3.5%) as an electrolyte at room temperature of 23 ± 1 °C. In our measurements, the open circuit potential was monitored for the first 30 min to ensure that the system is operating in high stability. The scan rate of potentiodynamic polarization curves was chosen at 0.5 mV/s in the range −250 mV to 250 mV. EIS tests were conducted by using a sinusoidal potential wave with an amplitude of 10 mV at corrosion potential and the frequencies measuring impedance response between 1 MHz and 10 MHz. All the above tests were repeated at least three times to ensure the reproducibility of the results.−1) and G peak (~1580 cm−1) as well as a very low intensity of the 2D peak (around 2700 cm−1) suggesting the formation of graphitic structure [D/IG) is close to 1 ; value of about 0.3 [In the second set of experiments, when the temperature was ramped up to 800 °C, the coverage of carbon area increased which is possibly due to the formation of graphene  on graphene growth has been analysed at 850 °C. Carbon flakes grown at this temperature 2 min were in the shape of small black dots or lines . Raman sThe above study suggests that a temperature of 850 °C and growth time of 6 min is the optimized condition to get good quality graphene films with high coverage. These samples were chosen for anti-corrosion test using an electrochemical method. EW) of copper is required, which can be calculated as below:Consequently, potentiodynamic polarization was applied for the quantitative determination of corrosion rates. From CR) can be determined by this equation [The calculation for the corrosion rate CR2, and ρ the density of copper substrate is 8.91 g/cm3, and the corrosion rate constant K = 3272 mm/(A cm year) [corrI) of bare substrates and graphene-coated samples is 1.58 × 10−3 mA and 6.51 × 10−5 mA, respectively.Here, the atomic weight of Cu is 64 g/mol and the valency is +2. The operative area of each sample is 3 cmcm year) ,43. From−13 m/s. The graphene covered sample mitigates the corrosion, with an average CR of 8.08 × 10−14 m/s, which is 24 times lower than the copper corrosion rate. This anti-corrosion effect is higher than the values reported previously [After the calculation, the average CR of the bare copper substrate is 1.97 × 10eviously ,34, and eviously ,45,46,47Z’) and the imaginary impedance (Z’’) [Another electrochemical experiment applied to test the anti-corrosion ability of coating is EIS Nyquist plots are shown in ce (Z’’) .(3)Z2=ZZ’’ tends to be zero, the absolute value of impedance Z is equal to that of Z’ which can be approximated to the resistance of sample surface. Consequently, the diameter of the semicircle in the Nyquist plot can represent the ability of electron transfer in the graphene coating or the surface of a bare copper substrate [According to the above equation, when the ubstrate . It is eubstrate ,34.Z’ [In addition, we analysed the data in the form of bode plots which provides the relationship between impedance Z and frequency ω. In the whole process of ω decreasing, the Z of coated samples exceed that of bare substrates. When the ω is sufficiently small (the y-intercept) and Z mainly results from Z’ ,40, the Moreover, to study the stability of anticorrosive behaviour, we conducted the corrosion test experiments on samples after 15 months of the first test. The samples were stored in an ambient environment. The potentiodynamic polarization results are shown in In this work, graphene films were grown by evaporating ethanol in a CVD system, and we found that the films can serve as anti-corrosive layers. Graphene films grown at a temperature of 850 °C for 5–6 min show excellent anti-corrosion property over significant surface coverage. Our study shows that good quality graphene films can be prepared using readily available ethanol as a carbon source in a CVD process. The potentiodynamic polarization and EIS also separately confirmed that, compared with bare Cu substrates, lower corrosion rate and higher impedance can be achieved by graphene coating.


Directed self-assembly of block copolymers (BCPs) enables nanofabrication at sub-10 nm dimensions, beyond the resolution of conventional lithography. However, directing the position, orientation, and long-range lateral order of BCP domains to produce technologically-useful patterns is a challenge. Here, we present a promising approach to direct assembly using spatial boundaries between planar, low-resolution regions on a surface with different composition. Pairs of boundaries are formed at the edges of isolated stripes on a background substrate. Vertical lamellae nucleate at and are pinned by chemical contrast at each stripe/substrate boundary, align parallel to boundaries, selectively propagate from boundaries into stripe interiors , and register to wide stripes to multiply the feature density. Ordered BCP line arrays with half-pitch of 6.4 nm are demonstrated on stripes >80 nm wide. Boundary-directed epitaxy provides an attractive path towards assembling, creating, and lithographically defining materials on sub-10 nm scales. Directing the position, orientation, and long-range lateral order of block copolymer domains to produce technologically-useful, sublithographic patterns is a challenge. Here, the authors present a promising approach to overcome the challenge by directing assembly using spatial boundaries between planar, low-resolution regions on a surface with different composition. Directed assembly of BCP domains oriented vertically to the substrate is particularly important because vertical domains can be utilized to produce nanostructures for logic gates6, data storage7, electrical contacts8, optical devices9, sensors10, and separation membranes12, and to provide a template for materials synthesis15.Directed self-assembly of block copolymers (BCPs) provides a scalable route for rationally fabricating large-area patterns with dense nanoscale features at precise locations on a surface16 and chemoepitaxy19—have been used to direct the assembly of vertical BCP domains into organized, useful patterns. Graphoepitaxy uses topographic features, such as trenches, whereas chemoepitaxy uses periodic chemical patterns to direct assembly of BCPs with controlled domain position, orientation, and lateral order. In both techniques, assembly of vertical lamellae can be controlled by tailoring template dimensions23 as well as interfacial interactions between the template surfaces and polymer blocks24, and feature density can be multiplied compared to the template dimensions to enhance resolution beyond the limits of conventional lithography26.Predominantly, two techniques—graphoepitaxy29) have been extensive4, directed assembly via these methods faces several challenges. For example, graphoepitaxy is sensitive to underfilling and overfilling of BCP in the trenches31, and requires fabrication of topographic structures that interfere with subsequent processing if the structures cannot be removed32. On the other hand, chemoepitaxy requires the formation of narrow guiding features that have widths of 0.5–1.5 times the BCP domain spacing (L0), where a width of 0.5L0 is ideal23. Therefore, to achieve BCP assemblies with sub-10 nm domains via chemoepitaxy, it is advantageous to use sub-10 nm guiding features; however, such features are difficult to fabricate. Moreover, at sub-10 nm scales, line-edge and surface roughness of the guiding features can become significant compared to L0, disrupting BCP registration33. While alternative chemical patterns with larger guiding features up to 3.5L0 in width have been used to direct assembly of vertical lamellae, the lamellae are not spatially registered and complex geometries  cannot be directed to assemble37.While the achievements of graphoepitaxy and chemoepitaxy .4 at 910 °C in a flow of H2 and Ar38. This bottom-up synthesis yields graphene stripes with tunable width from virtually 0 to 100s of nanometers by adjusting the growth time, with high-aspect ratio due to the anisotropic nature of growth, with single-atom thickness of 3 Å due to a self-limiting growth mechanism, and with atomically sharp edges due to faceting of the graphene crystals (with edge roughness <5 Å over edge lengths >10 nm)40. Furthermore, the surfaces of graphene and Ge are relatively pristine and uncontaminated  is used to produce graphene stripes that are polydisperse in width (0–100 nm) to study the effect of stripe width on assembly. Second, CVD employing graphene seeds that are lithographically positioned on Ge(001) is used to produce regular arrays of graphene stripes with controlled placement, unidirectional alignment, and reduced polydispersity43. Ultimately, it should also be possible to grow continuous films of graphene and then arbitrarily pattern graphene into stripes via lithography.To fabricate the chemical pattern, graphene stripes are synthesized directly on Ge001) wafers via CVD by decomposing CH wafers v44 with L0 of 16.8 nm and PPC-b-PS-b-PPC tri-BCPs45 with L0 of 12.8 and 14.5 nm -block-polystyrene (PPC-b-PS) di-BCPL0, thereby multiplying the feature density compared to stripe width L0. In fact, self-aligned vertical lamellae of PPC-b-PS-b-PPC (L0 of 12.8) assemble on stripes of every possible width spanning 15–100 nm   on relatively unoxidized Ge001) surfaces . In contrast, here, assembly is driven by chemical contrast achieved at graphene stripe edges, without the need for high-resolution chemical features.While graphene has been studied as a template for directing BCP assemblyL0 of 12.8 nm).The kinetics of boundary-directed epitaxy show that vertical BCP lamellae first nucleate at the edges of each stripe, but only after a mass transfer event in which the BCP partially flows off each stripe and onto the substrate, thereby inducing spatial variation in film thickness. These phenomena are demonstrated in Figs. L0, discontinuous dot-like domains form in the stripe interiors .The MC simulations indicate boundary-directed epitaxy is relatively robust with respect to the chemical contrast at the stripe/substrate boundaries, the surface tension of the BCP blocks, and the initial BCP film thickness (Supplementary Note L0 of 25 nm) on graphene stripes on Ge. In this case, the graphene stripes are still preferential to PS, but Ge is neutral48, resulting in assembly of small disordered grains of vertical lamellae on Ge and horizontal lamellae on graphene  , Ge(110) , Ge(001) with 6° miscut towards Ge[110] , and Ge(001) with 9° miscut towards Ge[110]  substrates are loaded into a horizontal furnace with a quartz tube inner diameter of 34 mm. The system is evacuated to <10−2 torr and then refilled to atmospheric pressure with a flow of Ar and H2. After annealing the substrates at 910 °C, CH4 is introduced to begin graphene growth. The furnace is slid away from the growth zone to terminate growth while maintaining the same atmosphere used during graphene synthesis.Graphene stripes are deposited directly on Ge wafers via CVD45. During polymerization, telechelic PS homopolymers with hydroxyl end group(s) serve as chain-transfer agents (CTAs). After deprotonation, the end-capped hydroxyl groups initiate the alternating incorporation of propylene oxide (PO) and CO2 in the presence of the most common Salen cobalt complex, producing PPC/PS BCPs. After removal of a small amount of PPC homopolymer, PPC-b-PS or PPC-b-PS-b-PPC with nearly uniform molecular weight and narrow molecular weight distribution is obtained. The reactions are conducted at room temperature using a PO:CTA:cobalt molar ratio of 2000:20:1 and 2.0 MPa of CO2. The volume ratio of PPC to PS in the BCP products is controlled by tuning the reaction time. After growth of graphene stripes on Ge via CVD, a BCP film with thickness of ~L0 is spin-coated onto the template surface. The BCP film is thermally annealed on a hotplate in an inert environment  to microsegregate the BCP domains. Boundary-directed epitaxy is achieved by thermal annealing, without relatively complex solvent annealing58 or topcoats59.PPC-b-PS di-BCP and PPC-b-PS-b-PPC tri-BCPs are synthesized via in-situ chain-transfer polymerization3/2 peaks are acquired using analyzer pass energy of 50 eV (resolution of 0.1 eV) and collection time of 50 ms. XPS peaks are fit to a Voigt function (Lorentzian:Gaussian ratio of 3:7) using the Powell fitting algorithm, after subtracting a Shirley-like background. Static contact angles are measured using an optical contact angle system (DataPhysics OCA 15). Deionized, distilled H2O droplets (volume of 7 µL) are dispensed onto the surfaces and H2O contact angles are measured immediately after droplet formation.Samples are imaged with SEM (Zeiss LEO 1530) using an in-lens detector. Surface morphology is characterized via AFM (Veeco MultiMode SPM) in tapping mode. Characterization of surface composition is performed using XPS (Thermo K-Alpha) with spot size of 400 μm and monochromatic Al Kα radiation (1486.7 eV). Spectra of the C1s and Ge2p61 (see Supplementary Note N. The system Hamiltonian has three contributions: (1) bonded interactions between beads, (2) non-bonded interactions between beads, and (3) interactions of beads with the template, and the adjustable thermodynamic parameters describing the interaction terms are (1) incompatibility between blocks, xN, (2) incompressibility of the B and A blocks, kBN and kAN, respectively, and (3) interaction strength between the stripe and B, the stripe and A, the substrate and B, as well as the substrate and A, λB-stripeN, λA-stripeN, λB-substrateN, and λA-substrateN, respectively. Negative and positive λ values imply attraction and repulsion, respectively. The non-bonded interaction Hamiltonian is expressed as a functional expansion in terms of number densities up to the third order64, which allows the coexistence of dense and sparse (or vacuum) regions to enable prediction of phenomena at the non-flat free surface at the top of the BCP film.Simulations are conducted using a coarse-grained bead-spring model that is derived from the theoretically informed coarse-grained (TICG) method6 MC cycles. Each MC cycle consists of M bead displacement steps, where M is the total number of beads in the system. The lateral dimensions of the simulation box are 17.5Re × 7.5Re × 3.75Re (L0 is ~1.25Re), where Re is the mean-squared end-to-end distance for an isolated non-interacting chain. The template, which consists of an isolated chemical stripe on a background surface, is below the BCP film. The BCP film has an initial thickness of 1.25Re and an initial chain density of 128Re−3. The system is annealed at a given set of adjustable parameters. The values of xN, kBN, kAN, λB-stripeN, λA-stripeN, λB-substrateN, and λA-substrateN used in Fig. The model described above is evolved using MC simulations (see Supplementary Note Supplementary Information


Cyberlindnera jadinii is widely used as a source of single-cell protein and is known for its ability to synthesize a great variety of valuable compounds for the food and pharmaceutical industries. Its capacity to produce compounds such as food additives, supplements, and organic acids, among other fine chemicals, has turned it into an attractive microorganism in the biotechnology field. In this review, we performed a robust phylogenetic analysis using the core proteome of C. jadinii and other fungal species, from Asco- to Basidiomycota, to elucidate the evolutionary roots of this species. In addition, we report the evolution of this species nomenclature over-time and the existence of a teleomorph (C. jadinii) and anamorph state (Candida utilis) and summarize the current nomenclature of most common strains. Finally, we highlight relevant traits of its physiology, the solute membrane transporters so far characterized, as well as the molecular tools currently available for its genomic manipulation. The emerging applications of this yeast reinforce its potential in the white biotechnology sector. Nonetheless, it is necessary to expand the knowledge on its metabolism, regulatory networks, and transport mechanisms, as well as to develop more robust genetic manipulation systems and synthetic biology tools to promote the full exploitation of C. jadinii. Saccharomyces cerevisiae is by far the most relevant industrial yeast species, Cyberlindnera jadinii is an example of the so-called non-Saccharomyces yeasts . . C. jadi carrier ,114.Km of 1.43 mM, being competitively inhibited by molybdate, selenate, thiosulfate, chromate, and sulfite [The saturable and unidirectional sulfate transporter D is pH-, sulfite ,116. TheC. jadinii cells [The proton-symporter for nitrate E is reprii cells .Km of 1.0 µM of NH4+ [C. jadinii [Km = 410 µM), a high (Km = 23 µM) and low-affinity (Km = 495 µM) transporter for L-methionine and a high (Km = 5.6 µM) and a low affinity (Km = 530 µM) for L-leucine were also described [An ammonia carrier F, reveal of NH4+ . Several jadinii ,122. A pescribed .C. albicans Pdr1 homolog CjCdr1 in the S. cerevisiae ADΔ strain conferred resistance to geneticin (75 μg), micafungin (40 μg), and nystatin (500 μg). These two proteins present similar substrate specificity, although CjCdr1 is more resistant to Rhodamine 6G [C. jadinii aquaglyceroporin CjFps1 (S. cerevisiae Fps1. The heterologous expression of CjFPS1 in a glycerol-consuming S. cerevisiae wild-type strain (CBS 6412-13A) promoted cellular growth improvement on glycerol as the sole carbon source [Thirty ABC transporters H from diamine 6G . The C. n CjFps1 I shares n source .C. jadinii, a proton-symporter and a facilitated diffusion  [The low- and the high-affinity systems for D-xylose transport K with Km 2.6 mM) . The hig 2.6 mM) .C. jadinii (Km 0.06 mM), pyruvate (Km 0.03 mM), propionate (Km 0.05 mM), and acetate (Km 0.1 mM) (Km 4.0 ± 0.5 µM), succinate (Km 0.03 ± 0.01 mM), fumarate, oxaloacetate, and α-ketoglutarate [Km of 0.056 mM for citrate and is competitively inhibited by isocitric acid, while aconitic, tricarballylic, trimesic, and hemimellitic acids did not affect citrate uptake [Vmax 0.516 nmol of malic acid s−1 per mg (dry wt. of cells) and Km 1.529 ± 0.024 mM malic acid, (b) Vmax 0.585 nmol of succinic acid s−1 per mg (dry wt. of cells) and Km 1.789 ± 0.089 succinic acid, and (c) Vmax of 1.14 nmol of s−1 per mg (dry wt. of cells) and Km of 0.59 mM citric acid [ATO1 and six genes homologous to ScJEN1 (unpublished results). In S. cerevisiae, these two genes encode distinct monocarboxylate transporters [Four mediated transport systems for organic acids are described in  jadinii L [29,30, 0.1 mM) L-II is a 0.1 mM) L-III is lutarate ,131. Thee uptake . The folric acid . Recentlsporters ,135 and sporters ,141,142.sporters ,143,144.Cyberlindnera jadinii has been widely explored as a source of single-cell protein, having the ability to produce vitamins , organic acids , and proteins . The capacity to utilize and degrade a great variety of carbon sources and its natural ability to produce significant compounds make C. jadinii an attractive microorganism for industrial applications [C. jadinii is still lagging behind when compared to other non-Saccharomyces yeasts, mainly due to the inexistence of extensive knowledge on its metabolism, regulatory networks, and transport mechanisms. The genomic characterization of several strains is also necessary to reveal the genetic features underlying the existing interspecies variability, particularly between its teleomorph and anamorph state. The potential of this yeast as a therapeutic agent is due to its already known antagonistic effects on human pathogens and utilization as a probiotic agent. Its protein secretion system is another attractive feature for the heterologous expression of soluble proteins. Nonetheless, only with the improvement of advanced genetic manipulation systems and the development of synthetic biology tools will the full exploitation of C. jadinii biotechnological potential be achieved. These and other advances will certainly allow C. jadinii to become a robust microbial cell factory in an expanding era of metabolite bioproduction.As biotechnological applications expand, it becomes necessary to explore novel expression hosts as more efficient and robust microbial cell factories are demanded ,124,125.ications ,8,10. Adications . However