Field-effect transistor (FET)-based biosensors allow label-free detection of biomolecules by measuring their intrinsic charges. The detection limit of these sensors is determined by the Debye screening of the charges from counter ions in solutions. Here, we use FETs with a deformed monolayer graphene channel for the detection of nucleic acids. These devices with even millimeter scale channels show an ultra-high sensitivity detection in buffer and human serum sample down to 600 zM and 20 aM, respectively, which are ∼18 and ∼600 nucleic acid molecules. Computational simulations reveal that the nanoscale deformations can form ‘electrical hot spots’ in the sensing channel which reduce the charge screening at the concave regions. Moreover, the deformed graphene could exhibit a band-gap, allowing an exponential change in the source-drain current from small numbers of charges. Collectively, these phenomena allow for ultrasensitive electronic biomolecular detection in millimeter scale structures. Field effect transistors based on graphene hold promise for sensing applications. Here, the authors report a millimeter-sized transistor based on deformed graphene as a biosensor that can detect nucleic acid molecules having detection limit of ~18 molecules of DNA in physiological buffer solution and ~600 molecules in human serum. These methods also offer considerable promise for forensics7, environmental monitoring8 and global personalized medicine9. In particular, field effect transistor (FET) based detection of nucleic acids has drawn great attention as label-free and highly sensitive biomolecular sensing platform, which can be readily integrated with other electronic components, such as data analyzers and signal transducers. 2D materials, such as graphene, are attractive due to their unique properties, such as ambipolar field effect, high carrier mobility, biocompatibility, mechanical strength, and flexibility10. 2D materials intrinsically exhibit high sensitivity in detection of charged biomolecules due to their ultimate thinness and extremely high surface to volume ratio. Compatibility to the conventional CMOS fabrication process is another potential advantage of using 2D materials, which carbon nanotube, Si-nanowire, nanoparticles do not have. Especially, large area graphene, which is grown through chemical vapor deposition (CVD) method, has been utilized in electrical systems, such as FET device for bio-sensing5 including detection of pH11, microorganisms12, blood sugar12, and more specifically, proteins at concentrations of 10 fM13, and nucleic acids (DNA or RNA) at the 100 fM concentrations14. There are a few reports that showed DNA and RNA detection at aM level, however, had significant level of background noise and lacked robust controls16. Further sensitivity would be highly desirable for detection of very rare molecules, such as micro RNA (miRNA) or cell-free DNA (cfDNA), from unamplified samples17.All-electrical detection of biomolecules and specifically nucleic acids are of great interest for gene-expression investigations18. Unfortunately, PCR is susceptible to interference by the inhibitory factors in biological samples, therefore not suitable to analyze miRNA directly from blood or serum samples19. Moreover, the result can be misinterpreted by bias and artifact due to the amplification efficiency of different sequences20. Therefore, there is an urgent need to develop amplification- and purification- free method to directly detect miRNA from biological samples such as serum.It is important to detect DNA/RNA, such as miRNA circulating in serum or plasma with high ionic strength. Such detection could enable liquid biopsy, which can replace invasive tumor-tissue biopsies in many diagnostic applications. The existing approaches to monitor cancer-related miRNA is based on the polymerase chain reaction (PCR)22. Outside the Debye length, which is <1 nm in physiological solutions, the charges are electrically screened. An increase in the Debye length can result in reduced screening effect and allow for a more sensitive electrical detection of charged biomolecules. While methods have been proposed to overcome this intrinsic limitation of FET biosensors22, these have focused on detecting biomolecules which are larger than the Debye length itself. None of the reports has tried to overcome the concentration limit of detection by modulating the Debye screening. Moreover, none of the works have modulated Debye screening in clinical solution such as serum or plasma22.One of the major hurdles to lower the detection limit of FET-based biosensor is shielding of the molecule charge by the counter ions in solution (termed Debye shielding)23. Thus, we hypothesized that if the surface of the sensing channel can be curved or bent at the micro- and nano-meter scale, the Debye length could be modulated resulting in higher sensitivity. Previous works have shown that 3-dimensional “crumpled” graphene can be created by deformations at the micro- and nano-scale on pre-strained thermoplastics by relieving the stress and inducing buckle delamination of the graphene24. This approach can be used to engineer curving and bending of 2D materials and thin films. Several applications have been investigated using the mechanically-tunable crumpled graphene such as stretchable photosensors25, nanoplasmonic sensor26 and strain gauges27, and the in-plane strain can change the electronic properties of graphene, opening a bandgap with a 1% stretch29. Moreover, crested 2D materials FETs recently showed large increase in the mobility as compared to standard devices30. It has also been separately shown that flat 2D semiconducting material such as MoS2 can be 10 times more sensitive than flat semi-metallic 2D graphene for biosensing applications31. Hence, we hypothesize that nanoscale bending of 2D graphene in 3-dimensions could result in high sensitivity due to modulation of the Debye length (or volume), and possibly due to strain induced band-gap opening in the graphene channels. Such deformed graphene (curved or bent 2D) layers have not yet been used for biosensor applications.Computational reports have also predicted that the curved morphology of sensing materials can affect the Debye length (or volume), which can increase in concave regions of a nanowire sensor32. The process does not require electron beam lithography to fabricate the nano-confined devices. The realization of the bent and crumpled graphene is achieved by macroscopic manipulation of the 2D layer and the resulting “nano”-sized features exhibit the superior sensing performance, while allowing facile fabrication and reproducibility. We demonstrate detection of 22-mer single and double stranded molecules by adsorption and hybridization experiments, respectively. We also showed that the target miRNA (let-7b) and a DNA probe hybridization was detectable as low as 600 molecules in 50 µL of buffer and undiluted human serum directly without amplification. The performance of the sensor was further enhanced using peptide nucleic acid (PNA) probe, which showed 600 zM of LOD, ~18 molecules in an hour of incubation time. We show via molecular dynamics simulations the formation of electrical ‘hot spots’ at the nanoscale crumpled graphene regions where the Debye length can increase and also result in a local high potential due to the charge of DNA/RNA. Furthermore, the bending of the graphene monolayer at these hot spots can result in opening of a bandgap and provide for an exponential increase in the conductivity in vicinity of the biomolecules. These effects combined can allow for a measurable current change in millimeter scale channels even with 600 zM concentration of the target molecules.Here, we report the use of these deformed and bent (crumpled) graphene FET-based electrical biosensors for ultra-sensitive detection of DNA/RNA molecules down to 600 zM of limit of detection (LOD) on millimeter scale structures. To the best of our knowledge, this is the highest sensitivity reported so far using any electronic biosensor for detection of DNA. Because of the simple fabrication process, the presented approach has several benefits over structures such as nanoribbons or nanopores24. To produce the crumpled FET device, the graphene on polystyrene substrate was annealed at 110 °C for 4 h. This annealing process induces the shrinkage of the underlying pre-strained thermoplastic substrate which results in buckling of graphene24. The optical image of the crumpled graphene device is in Supplementary Fig. The scheme for the graphene FET DNA sensing is illustrated in Fig. 24. AFM derived topography data  and atomic force microscope (AFM). Disorganized herringbone-like structures were observed Fig.  and the 34. The conductance of the graphene channel was modulated by liquid-gate potential applied to the solution reservoir. The accumulated ions modulate the conductance of the graphene channel by either p- or n-doping effects because of the ambipolar characteristics of graphene. The ambipolar transport characteristics of the FETs are illustrated in Fig. Id vs. Vgs curves, the Dirac points shifted as Vds changed. Dirac points were positioned between Vgd of 0 and 0.5 V, which is mainly related to the work function difference of Ag/AgCl gate electrode and the graphene. The measurements were repeated over time and confirmed that the Dirac voltage values were stable in PBS buffer solution before using the devices for measurements  , we measured a clear Dirac point shift, but about half of the earlier measurements in PBS. We measured shifts at 20 aM compared to the negative control tests as shown in Fig. n) of graphene and therefore a shift in Dirac point (ΔVD) given by refs. 46CT is the total gate capacitance, and Δn is directly proportional to the charge density of the unscreened DNA molecules 45. The counter-ion screening of the DNA molecules lowers the net charge of adsorbed DNA and affects the detection sensitivity. As shown in Fig. To investigate the phenomena underlying the experimental results and the effect of ionic screening of DNA molecules, we studied the electrostatics and charge distribution of DNA and ions near flat and deformed graphene surfaces using molecular dynamics (MD) simulations (see the methods section for the simulation details). The presence of the unscreened charges (acting as dopants) carried by DNA molecules near the surface of the graphene produces long-range electrostatic potential leading to a change in the charge carrier density  and σ is the graphene surface charge density. As shown in Fig. n) leading to a larger Dirac point shift. In the flat, convex and across configurations, because of weaker confinement, mobile ions are less restricted and freely present in the vicinity of the DNA molecule, screening its charge to a larger extent  in MD simulations with a diameter of 2.45 nm and a length of 8 nm as shown in Fig. 24. As a proof of principle, we created uniaxial crumple and probed the surfaces by AFM. The image in supplementary Fig. n) of the graphene. Such a deep and narrow trench, referred to as an “electrical hot-spot”, could provide very low ionic screening for an adsorbed DNA molecule by exposing most of DNA to the graphene surface. Therefore, The bending used in Fig. n) to explain our measured detection of aM concentrations of DNA. Sarkar et al. showed that the existence of a bandgap in a single-layer MoS2 leads to a higher sensitivity of charge detection compared to its graphene counterpart31. Here, we hypothesize the creation of a band-gap in bent graphene and next calculated the bandgap (Eg) for flat and crumped graphene in the absence and presence of DNA bases using density functional theory (DFT) and GW methods. Typically, GW methods are more accurate for graphene than DFT. Supplementary Table μ) in graphene decreases with increasing bandgap 47. As n is inversely related to μ, the change in n (and the corresponding Dirac point shift) due to bandgap widening upon addition of DNA can be obtained48. To achieve the observed Dirac point shift for aM concentrations used in our experiments, the change in bandgap from 0.4224 eV (crumpled graphene with no DNA) to 1.7641 eV (crumpled graphene with base A) must occur in at least ~10−7% of the area of our graphene sensor  DNA from the surface by a distance of 3 ntIn future, the devices could be miniaturized to micro- or nano-sized sensor in an array format. There are many fabrication and integration process challenges towards this goal. Some of these include; (i) maintaining a high quality crumpled surface if lithography is to be performed after shrinking, (ii) performing lithography first to form smaller flat graphene islands and then performing the heating or local shrinkage to cause crumpling while keep those smaller islands attached to the underlying surface, (iii) integration of silicon FETs at each pixel for row and column addressing to form larger arrays in a silicon substrate, etc.We have demonstrated nucleic acid molecule detection on crumpled graphene FET electrical biosensor with unprecedented sensitivity using DNA and PNA as a probe. DNA adsorption and hybridization tests were demonstrated using cancer-related biomarker miRNA let-7b sequence in buffer and in human serum. The limit of detection was found to be 600 zM for crumpled graphene FET biosensor and 2 pM for flat graphene. We show via experiments and simulations that the nanoscale bending and deformations increases the Debye length in ionic solution to decrease the screening of the DNA/RNA molecules, thus contributing to the dramatic enhancement of sensitivity as compared to flat graphene FET sensors. To explain the results, Molecular Dynamics simulations revealed generation of large electrical potential due to DNA molecules in the nanoscale crevices and deformed regions that exclude ions, compounded by the formation of electrical bandgap in the deformed graphene regions. These attributes coupled with increased molecular residence time due to increased roughness can result in “electrical hotspots” allowing for a change in local conductivity, hence allowing atto-molar detection of DNA in millimeter scale sensors. We demonstrate the applicability of the technology by target molecules detection in undiluted human serum for cancer-related miRNA. This technology can open opportunities for the development of more reliable and efficient diagnostic tools, and electrical point-of-care and implantable biosensors for early detection of biomolecules for various human diseases.2) gas flow continuously. Monolayer graphene growth was initiated when 50 sccm hydrogen and 100 sccm methane (CH4) were introduced to the CVD chamber at annealing temperature of 1030 °C, and the synthesis came to its end when two reaction gases were turned off. CVD furnace was then cooled down with flow of argon (Ar) gas, which completes the monolayer graphene synthesis process. To compare the performance among different source of graphene, graphene was also purchased from Grolltex and Graphenesquare. All the graphene showed the same LOD.Monolayer graphene was grown on a copper foil via chemical vapor deposition (CVD). Before placing into the CVD furnace, copper foil was degreased with acetone and IPA, followed by nitrogen blow drying. The foil was then annealed at elevated temperature for 3 h, while 50 sccm of hydrogen  (PMMA). Undesired graphene formed at the back side of copper foil was removed by oxygen plasma etching. The sample was cut into 1 mm × 15 mm pieces with scissors or a razor blade. PMMA serves as a supporting layer while copper foil was etched by floating on 0.15 M sodium persulfate for about 5 h. The PMMA/graphene was rinsed by moving from the sodium persulfate solution to deionized (DI) water. The PMMA/graphene was then transferred onto a polystyrene substrate. The PMMA layer was removed by soaking in acetic acid for 5 min. The sample was annealed at 110 °C for 2 h to shrink the polystyrene substrate into ¼ of the original size and crumple the graphene. To fabricate transistor, conducting silver paste was used as source and drain electrodes at both ends of the graphene. Silicone rubber (DOWSIL™ 3140 RTV Coating) was used to insulate source and drain electrodes from liquid and construct a solution reservoir.Pyrenebutanoic acid succinimidyl ester (PASE) (20 mM) in dimethyl sulfoxide (DMSO) was treated on the graphene overnight and rinsed with pure DMSO, ethanol and DI water; 50 μM of probe DNA solution was added on PASE-modified graphene for 3 h. The graphene FET with probe DNA functionalization was rinsed with 1× PBS. 200 mM ethanolamine solution was treated for 1 h to saturate the possibly unreacted amino group on PASE and rinsed with ethanol and 1× PBS solution. The volume of all treated chemicals and samples was 50 μL.The target DNA or RNA incubation was conducted by dropping complementary and negative control strands with concentrations that are indicated in the legends in Fig. Vgs) to the 0.1x and 1× PBS buffer solution. For the human serum test, the tests were performed in in serum. In case of DNA absorption, the graphene chips were incubated in PBS overnight because of wettability issue of hydrophobic graphene. For the serum test, the graphene chips were incubated in serum overnight and the blank measurements were repeated till there was no shift only with serum. Then target and NC RNA in serum was treated on the chip. Vg was swept from −0.5 to 1 V and drain–source voltage (Vds) was picked between 0.03 and 0.1 V. Drain–source current (Ids) was measured at an assigned Vds.I–V curves and resistance were measured in a semiconductor parameter analyzer equipped with a probe station. Ag/AgCl electrode was used to apply gate voltage (CT) based on CV is shown in Fig. The capacitance measurements of flat and crumpled graphene on PS substrate were carried out using a CS 350 potentiostat  with three electrodes, including reference, counter, and working electrode. Here, silver chloride (Ag/AgCl), platinum (Pt), and the surface of the graphene channel on PS substrate were used for reference, counter, and working electrode, respectively. Cyclic voltammetry (CV) was chosen for the characterization method, while all three electrodes were immersed in 1× PBS solution for the measurement. Normalized total capacitance by measured graphene area 53. To study the effect of the crumples on the EDL formation near graphene, different simulations with different graphene surface topologies were created  for all the systems. The systems are non-periodic in z direction . The Lennard-Jones (LJ) potential with a cutoff distance of 1.2 nm is used. The long-range electrostatic interactions are calculated using the PPPM54. SPC/E water model is employed. The SHAKE algorithm, which maintains the rigidity of each water molecule, is applied. Sodium chloride salt solution of various concentrations (1.2 and 0.6 M) is considered. The LJ parameters between atoms of graphene and water are modelled by the forcefield developed by Wu et al.55. The CHARMM forcefield56 is used for the DNA strands and ions.Molecular dynamics simulations were performed using the LAMMPS packageted Fig. . Flat an58 with a time constant of 0.1 ps. The production simulations were carried out in NVT ensemble for 10 ns. Trajectories of particles were dumped every picosecond to study the structure of DNA and ions near the graphene sheet.Before starting the equilibrium and production simulations, the energy of each system was minimized for 15,000 steps. The equilibrium simulations were then performed in NPT ensemble for 2 ns at a pressure of 1 atm and a temperature of 300 K. This ensures that the water reaches its equilibrium density. The systems were further equilibrated in NVT ensemble for another 2 ns at 300 K. Temperature was maintained at 300 K by using the Nosè-Hoover thermostat61. The local density approximation (LDA)62 functional of Ceperley-Alder is employed based on the projector augmented wave (PAW) method60. The cutoff energy for the plane wave basis set is 550 eV for all calculations. All ionic positions are optimized by a conjugate gradient method until the forces on each ion are less than 0.01 eV/Å. The size of flat graphene and crumpled graphene model is about 12 Å × 12 Å and 8.9 Å × 12 Å, respectively. A vacuum separation of 40 Å between graphene and its periodic replicas is employed to eliminate the interaction between them. For accurate calculations of the electronic structures, the Brillouin zone is sampled using 18 × 18 × 1 k-point grid. The lattice parameter of flat graphene is computed to be 2.445 Å which is consistent with previous theoretical and experimental results64.The optimized geometry of the adsorbed DNA nucleobases on flat and crumpled graphene are obtained using DFT calculations. All DFT calculations are performed using the Vienna Ab initio simulation package (VASP)65 was performed to obtain the band structures using VASP61. Each system has been initialized using DFT with the LDA66 exchange-correlation functional, energy cutoff of 400 eV, and Gaussian smearing of 0.05 eV. The number of total bands is set to 512 in all structures to ensure a significant number of empty bands is available as required by GW method. The energy cutoff of the response function and the number of frequency grid points are respectively set to 90 eV and 32 to control the high memory demand by the calculation of GW. Gamma-centered k-points are selected to be 6 × 1 × 1 for structures without a DNA base and 4 × 1 × 2 for structures with a DNA base. Finally, the vacuum space is maintained large enough (>20 Å) in the periodic directions to avoid unphysical interactions between the periodic images.Single-shot approximation of GW (G0W0)Supplementary InformationPeer Review File


In idiopathic pulmonary fibrosis (IPF), several factors may have a negative impact on the nutritional status, including an increased respiratory muscles load, release of inflammation mediators, the coexistence of hypoxemia, and physical inactivity. Nutritional abnormalities also have an impact on IPF clinical outcomes. Given the relevance of nutritional status in IPF patients, we sought to focus on some critical issues, highlighting what is known and what should be further learned about these issues. We revised scientific literature published between 1995 and August 2019 by searching on Medline/PubMed and EMBASE databases including observational and interventional studies. We conducted a narrative review on nutritional assessment in IPF, underlining the importance of nutritional evaluation not only in the diagnostic process, but also during follow-up. We also highlighted the need to keep a high level of attention on cardiovascular comorbidities. We also focused on current clinical treatment in IPF with Nintedanib and Pirfenidone and management of gastrointestinal adverse events, such as diarrhea, induced by these antifibrotic drugs. Finally, we concentrated on the importance of pulmonary rehabilitation program, including nutritional assessment, education and behavioral change, and psychological support among its essential components. More attention should be devoted to the assessment of the undernutrition and overnutrition, as well as of muscle strength and physical performance in IPF patients, taking also into account that an adequate clinical management of gastrointestinal complications makes IPF drug treatments more feasible.  In recent years, nutritional status has increasingly gained attention in the evaluation of patients with chronic respiratory diseases since their clinical course is often characterized by progressive weight loss and reduction of muscle mass ,2,3. Idiopathic pulmonary fibrosis (IPF) is a fibrosing interstitial lung disease of unknown etiology characterized by rapid progression and poor prognosis ,5.As shown in Furthermore, given the rapidly progressive nature of the disease, with a median survival time of only two to five years, all these factors often develop and worsen in a short period of time.It is worth noting that not only may the “downward spiral” of the underlying pulmonary disease affect the nutritional status, but also nutritional abnormalities may have an impact on clinical outcomes. For instance, lower body mass index (BMI) and body weight loss have been associated with increased mortality ,11,12. CGiven the relevance of evaluating nutritional status in IPF patients, a topic which remains still largely understudied, we sought to focus on some critical issues, highlighting what is known and what should be further learned about these issues.clinicaltrials.gov databases including observational and interventional studies. Keywords used for each database are reported in We revised scientific literature published between 1995 and August 2019 by searching on Medline/PubMed, EMBASE, and Box 1Nutritional status AND (IPF OR acute exacerbation of IPF), nutritional assessment AND (IPF OR acute exacerbation of IPF), nutrition AND (IPF OR acute exacerbation of IPF), body mass index AND (IPF OR acute exacerbation of IPF), nutritional interventions AND (IPF OR acute exacerbation of IPF), dietary habits AND (IPF OR acute exacerbation of IPF), body composition AND (IPF OR acute exacerbation of IPF), metabolic rate and (IPF OR acute exacerbation of IPF), body weight AND (IPF OR acute exacerbation of IPF), weight loss AND (IPF OR acute exacerbation of IPF), weight gain AND (IPF OR acute exacerbation of IPF), malnutrition AND (IPF OR acute exacerbation of IPF), overweight AND (IPF OR acute exacerbation of IPF), obesity AND (IPF OR acute exacerbation of IPF), cardiovascular comorbidities AND IPF, cardiovascular risk AND IPF, dyslipidemia AND IPF, metabolic syndrome AND IPF, diabetes mellitus AND IPF, gastroesophageal reflux disease AND IPF, nintedanib AND , pirfenidone AND , IPF AND pulmonary rehabilitation.Studies targeting children and conference abstracts were excluded. In addition, given the aim of the present review, we included only studies on IPF excluding those on other interstitial lung diseases (ILDs). Topics and structure of the review were discussed and approved by the expert panel. Reviewing main topics were based on the author’s expert view on the current status of the field under discussion.Specific nutritional counseling and dietary advices for IPF patients are not yet available, with the exception of dietary recommendations to reduce the side effects of antifibrotic treatments (see the following sections). The implementation of a nutrition care process may be useful especially in those patients with IPF who are at risk or already malnourished . AccordiFrom a practical point of view, nutritional status may be assessed by evaluating nutrient intakes, energy expenditure, body composition, laboratory data, and body functions. Overall, only a few studies  have so far evaluated the nutritional status of IPF patients.To the best of our knowledge, there are no consistent results available on nutrient intakes in IPF. Two studies carried out in Japan ,15 showe2 in most studies from Western Countries [2) in those carried out in Japan and South Korea [Main anthropometric data were reported in many papers. Mean BMI was around 26–28 kg/mountries ,19,20,21th Korea ,23,24,25Thus, a high proportion of overweight/obese patients (overnutrition) may be expected ,21. As aThe assessment of health-related physical fitness provides relevant information on body functions by assessing muscle strength, physical performance, and exercise tolerance. There is a small amount of literature on this issue for IPF patients. An inverse association between handgrip strength and age or dyspnea was observed ,28. GaitConsidering that there are no guidelines about the nutritional follow-up in IPF patients, in our opinion, nutritional status should be re-evaluated at regular intervals in all IPF patients with regard to formal diagnosis, weight loss, and nutritional problems. According to the best clinical practice, the periodic evaluation should consider  dietary Previous studies have reported that body weight changes with time have a strong correlation with clinical outcomes in several diseases ,31. BodyFinally, as far as body composition is concerned, changes in muscle mass body composition, i.e., a reduced cross-sectional area of erector-spinae muscles, are associated with a worse prognosis in IPF patients .Acute exacerbation is characterized by acute respiratory worsening, being a severe event in the course of IPF and leading to high mortality . In this.Malnutrition is a highly prevalent consequence of hospitalization, especially in critically ill patients admitted to intensive care units (ICU) . MalnutrNutrient deficiency has been correlated with a prolonged length of ICU/hospital stay and is strongly associated with increased morbidity and mortality among critically ill patients ,37. ConvCardiovascular comorbidities, particularly coronary artery disease and arrhythmias, have been frequently observed in patients with IPF ,44,45. SPrevalence of diabetes mellitus also appears higher than in general population, even after the exclusion of individuals treated with systemic corticosteroid therapy. However, the prognostic significance of such finding is unknown . MetformAs already mentioned, observational studies reporting BMI in patients with IPF showed a presence of overweight or obesity in the majority of patients ,11,18.Although in IPF patients lower BMI and weight loss show a negative impact on disease outcomes more than overweight and obesity (see above), these latter issues may have a major impact in advanced stages as demonstrated by the fact that obesity is associated with an increased mortality risk in patients receiving bilateral lung transplantation and that obese patients may not be eligible for transplant . InteresA recent meta-analysis confirmed that gastroesophageal reflux disease (GERD) and IPF may be associated with each other; however, this association was found in case-control studies with smoking being a possible major confounder . The cauAlthough dietary intervention might play a role in effective treatment and management of GERD  and reflThe two currently available anti-fibrotic drugs, nintedanib and pirfenidone (PFD), have offered a therapeutic chance to IPF patients. However, as reported below, both of them cause adverse events that may interfere with food intake and absorption, potentially leading to weight loss and malnutrition. To the best of our knowledge, the relation between nutrition and anti-fibrotic therapies has not been carefully investigated and needs special attention. There is evidence that a weight loss > 5% occurs in about 20% of IPF patients, even in the absence of anti-fibrotic treatments . MoreoveIn IPF patients, the treatment with Nintedanib was not associated with serious adverse events , but was associated with increased gastrointestinal events, particularly diarrhea (62.4% in treatment group vs. 18.4% in placebo group). These episodes were mild-to-moderate and led to a low 4.4% incidence of discontinuation in the clinical trial . AdditioGastrointestinal adverse events can be managed by reduction to 100 mg twice daily of Nintedanib dose or temporary suspension as needed. For diarrhea control, concomitant usage of loperamide ,73 as weMoreover, Ikeda et al.  observedA retrospective comprehensive analysis of safety outcomes of treatment with pirfenidone (PFD) in 1299 IPF patients from five clinical trials  has shown that overall PFD was well tolerated over a 9.9 years follow-up. Mild to moderate nausea (37.6%), diarrhea (28.1%), dyspepsia (18.4%) and vomiting (15.9%) were the most frequent adverse events . PFD has2) had 418 treatment-related adverse events with nausea, vomiting, and diarrhea being the most common [In real-life studies, anorexia and gastrointestinal disturbance are the most common reasons for cessation of PFD ,81. Ikedt common . In ordeThe benefit of specific dietary recommendations other than those for oral hydration has not been well established in controlled trials.However, a diet can play a role in treating diarrhea avoiding foods that are high in fat, high in fiber, milk and dairy products , spicy foods, alcohol, caffeine-containing products, some fruit juices  . Main rePulmonary rehabilitation (PR) has been widely used in the treatment of chronic respiratory diseases, playing a fundamental role in improving exercise capacity, health-related quality of life, and breathlessness.PR is one of the most effective nonpharmacological treatments, and it is recommended by guidelines for patients with IPF, even if the evidence is still inconclusive.As summarized in in the review performed by Xuequing et al., pulmonary function was evaluated only in three studies, showing a significant positive effect of PR on FVC in IPF patients, with no statistically significant improvement was reported for DLCO .A randomized controlled trial, conducted by Perez-Bogerd et al. showed that exercise capacity, health status, and muscle force improved significantly after PR, maintaining such results after one year . In addiMoreover, Chehere et al. reported a 53% non-responder rate, related to the severity of exercise-induced desaturation and the impairment of DLCO before PR ; such isThe selection of patients for rehabilitation is of paramount importance because specific characteristics may be associated with a greater response to PR. Subjects with a higher FVC, less exercise desaturation, less disability, and more severe breathlessness demonstrated a more remarkable improvement in rehabilitation outcomes .The pulmonary rehabilitation program should consist of a comprehensive intervention of patient-tailored therapies . In partThe other components of PR include education, psychological support, and training in behaviors  that will assist in disease management.Morisset et al. investigated educational needs of IPF patients and provided some topics for educational programs such as disease education, symptoms management, medications use, oxygen therapy, and end-of-life counseling .Psychological support is another component of rehabilitation programs since depression and anxiety are frequent comorbidities in IPF patients. Uncontrolled data suggest that pulmonary rehabilitation might improve psychological health in patients with IPF through improvements in symptoms such as dyspnea, exercise tolerance, and sense of control over the disease .All of these components of PR must work together to achieve optimal outcomes for patients affected by IPF . StudiesTo date, evaluation of nutritional status and physical fitness, the nutrition care process, and physical activity are all largely unexplored issues related to the clinical management of patients with IPF.The increased interest in body composition and in the assessment of nutritional status in COPD over the last few years has led to the establishment of different nutritional risk profiles, based on different patients’ metabolic phenotypes . Such asRecently, more attention has been focused on comorbidities in IPF, especially from a prognostic point of view. A prediction model, named TORVAN, assessed the ability of comorbidities to improve prediction of survival in IPF patients beyond the variables included in the GAP model. The TORVAN model also suggests that GERD, pulmonary hypertension, lung cancer, valvular heart disease, and atrial arrhythmias were the comorbidities with a higher impact on IPF patients survival . There hClinicalTrials.gov Identifier: NCT03770845) and nutritional intervention  in IPF patients are ongoing, and their results are expected in the near future.However, epidemiological studies performing a complete nutritional evaluation  and overnutrition, as well as of low muscle strength (dynapenia) and low physical performance, taking also into account that an adequate clinical management of comorbidities, particularly gastrointestinal complications, makes IPF drug treatments more feasible.To the best of our knowledge, there is a paucity of longitudinal studies which should evaluate the prognostic impact of some comorbid conditions that affect IPF patients, such as obesity and cardiovascular diseases. Moreover, the current lack of nutritional intervention studies does not allow to assess their efficacy in undernourished or over nourished IPF patients.Hence, the clinical guidelines for IPF management ,61 shoulIn light of the above, it seems therefore quite clear that a multidisciplinary approach is needed not only in the diagnostic process, but also in follow-up and advanced phases of the disease—so that various specialists should be involved in multidisciplinary care of IPF, including nutritionists (physicians and dietitians) and rehabilitation specialists in addition to pulmonologists, nurses, and psychologists. Such holistic care might improve IPF patients’ quality of life and at the same time may be also a clear reference point for patients, who often feel alone with their disease .


Drosophila models of TDP-43 proteinopathy, we identified TDP-43 dependent translational alterations in motor neurons impacting the spliceosome, pentose phosphate and oxidative phosphorylation pathways. A subset of the mRNAs with altered ribosome association are also enriched in TDP-43 complexes suggesting that they may be direct targets. Among these, dlp mRNA, which encodes the glypican Dally like protein (Dlp)/GPC6, a wingless (Wg/Wnt) signaling regulator is insolubilized both in flies and patient tissues with TDP-43 pathology. While Dlp/GPC6 forms puncta in the Drosophila neuropil and ALS spinal cords, it is reduced at the neuromuscular synapse in flies suggesting compartment specific effects of TDP-43 proteinopathy. These findings together with genetic interaction data show that Dlp/GPC6 is a novel, physiologically relevant target of TDP-43 proteinopathy.Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative disease in which 97% of patients exhibit cytoplasmic aggregates containing the RNA binding protein TDP-43. Using tagged ribosome affinity purifications in The online version contains supplementary material available at 10.1186/s40478-021-01148-z. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive loss of motor neuron function, culminating in death due to respiratory failure , 79. MutTDP-43 is a nucleo-cytoplasmic shuttling, DNA/RNA binding protein which is predominantly localized to the nucleus where it regulates transcription  and splifutsch and hsc70-4 mRNAs (in axons)  v[+ t1.8] = TRiP.HMS01846}fCRattP40 (VDRC v38377) and y v[+ t1.8] = TRiP.HMS01848}attP40 (VDRC v38379). These lines were crossed with the D42 GAL4 driver  = UAS-dlp.WT}3 (Bloomington Stock #9160) and for RNAi knock-down we used y v[+ t1.8] = TRiP.GLC01658}attP40 (Bloomington Stock #50540). As a control for the RNAi experiments we used y = CaryP}attP40 . CRISPR TDP-43 knock-in models, TDP-43WT and TDP-43G294A in which the endogenous Drosophila TDP-43 (TBPH) had been replaced by human TDP-43 (WT or G294A) were previously described [w[*]; P{w[+ mC] = UAS-fz2-2}16/TM6B, Tb[ +] . Expression of dominant negative Fz2 was achieved with w[*]; P{w[+ mC] = UAS-fz2(ECD)-GPI}3 (Bloomington Stock #44221). For G6PD overexpression, we utilized w1118; UAS-G6PD  v[ sev;P{y[+ t7WT-YFP or TDP-43G298S-YFP were collected and flash frozen in liquid nitrogen. Frozen larvae were homogenized in lysis buffer  and RNAsin Plus 400 units/ml (Fischer Scientific PRN2615). Lysates were centrifuged at 10,000 × g for 10 min and then pre-cleared with magnetic beads  for 1 h at 4 °C. Magnetic beads bound to chicken anti-GFP antibody (Life tech A-11122) were added to the supernatant followed by rotation at 4 ºC for 2 h. Next, beads were washed 3 times with a sugar-rich buffer  and 2 times with a low-density buffer  followed by resuspension in Qiagen RLT buffer  with 1% 2-mercaptoethanol (Sigma Aldrich M6250).100 third instar larvae expressing TDP-43w1118; UAS-RpL10-GFP/CyO, w1118; UAS-TDP-43WT UAS-RpL10-GFP/CyO and w1118; UAS-RpL10-GFP/CyO; UAS-TDP-43G298S/TM6B Hu Tb and the buffers for the immunoprecipitation of RpL10-GFP also included 100 μg/mL cycloheximide.RNA associated with ribosomes was immunoprecipitated using a protocol similar to RIP (see above) except that the genotypes were RNA was isolated from the ventral cord lysates, whole larvae inputs, or immunoprecipitations using a Qiagen RNeasy Mini Kit (Qiagen 74104). For fractionation experiments, dissected ventral nerve cords (VNCs) or neuromuscular junctions (NMJ), RNA was isolated using Trizol (Thermofisher Scientific 15596026). With both isolation methods, RNA was eluted into 20 μl of molecular grade water with quality and quantity determined by a nanodrop spectrophotometer, measuring absorbance at 260/280 nm.Isolated RNA was prepared for RNA sequencing using the SMART-seq v4 Ultra Low Input RNA kit (Takara Bio USA 634888). The Nextera XT DNA Library Prep Kit (Illumina FC-131-1024) was used to tag, clean, and pool the samples with quality assayed by an Agilent 2100 Bioanalyzer. The cDNA libraries for RIP/TRAP were sequenced by the Beijing Genomics Institute using an Illumina HiSeq 4000 and the Illumina Nextera XT Kit (Illumina 15032350) with 100-bp paired end reads. Human fractionation RNA-seq were performed at the Translational Genomics Institute (TGen) using similar protocols.WT and TDP-43G298S complexes, respectively. In TRAP experiments, 9711, 9347, and 9669 genes were detected in RpL10 GFP complexes in the control, TDP-43WT, and TDP-43G298S models, respectively. To identify the “normal” motor neuron translatome we calculated the Log2FoldChange between RpL10 IP and RpL10 VNCs. Next, to identify alterations resulting caused by TDP-43 proteinopathy to the motor neuron translatomes, we subtracted the Log2FoldChange between RpL10 IP and RpL10 VNCs from the Log2FoldChange between TDP-43WT or G298S RpL10 IP and TDP-43WT or G298S RpL10 IP VNCs. Gene ontology (GO) analyses were conducted using David 6.8 [Reads were quality filtered to > Q30 then aligned to the Flybase FB2018_05 genome using MAFFT  with difavid 6.8 , 43. Humavid 6.8  then undavid 6.8 , STRING avid 6.8 , and theg for 30 min. The supernatant became the soluble fraction and the pellet was solubilized in urea buffer , 0.5 mM PMSF, RNAsin Plus 400 units/ml (Fischer Scientific PRN2615), pH 8.5) to generate the urea/insoluble fraction. For human fractionation samples, post-mortem tissue  was homogenized and subjected to the fractionation protocol described above.Fractionations of both overexpression and CRISPR Drosophila models were conducted as previously described . In brieThird instar ventral nerve cords were dissected as previously described . In brieThird instar larval NMJs were dissected as previously described . In brieParaffin-embedded post-mortem spinal cord tissue sections were obtained from the Barrow Neurological Institute ALS Tissue Bank and Target ALS Postmortem Tissue Core. All tissue samples were collected after informed consent from the subjects or by the subjects’ next of kin, complying with all relevant ethical regulations and approved by the appropriate Institutional Review Boards. Clinical neuropathological diagnoses were made by board certified neuropathologists according to consensus criteria. Subject demographics are listed in Additional file dlp mRNA was quantified using Taqman assay (Thermofisher Scientific Dm01798597_m1) using GPDH (Thermofisher Scientific Dm01841185_m1) for normalization. Fold change was calculated using the standard ΔΔCT method [All cDNA was synthesized using Fisher First Strand cDNA synthesis reagents (Thermofisher Scientific K1641). qPCR reactions were conducted in three biological replicates using Taqman Fast Advanced Master Mix (Thermofisher Scientific 4444556) and conducted on either an ABI 7100 or Analytik Jena 844-00504-4 qTOWER qPCR machine. T method .Third instar wandering larvae were placed on a grape juice agar plate. After being given 1 min to acclimate, the larvae were turned on the ventral side and timed until they uprighted themselves and began the first forward motion . Data podlp and Gapdh1 in dissected VNC and NMJs as previously described [FISH was performed using RNAscope (ACD Bio). A custom probe generated by ACD bio for dm-dlp and a control probe dm-Gapdh1 were used to detect escribed . Images For RNA seq analyses, significance was determined using the P adjusted value calculation embedded within Deseq2. All other statistical analyses were performed using GraphPad Prism 7.0 using two-tailed analyses. Normality was evaluated using the Kolmogorov–Smirnov test. All data shown are mean ± standard error of the mean.Drosophila models of TDP-43 proteinopathy that recapitulate key aspects of the disease including locomotor defects, cytoplasmic aggregates and reduced lifespan [Drosophila larvae expressing human TDP-43 protein specifically in motor neurons via the GAL4-UAS system (D42 GAL4 > UAS-TDP-43-YFP) and performed immunoprecipitation experiments to pull down TDP-43 and associated mRNAs (RIP). The mRNAs associated with TDP-43 (WT or mutant G298S) were isolated and subjected to RNA sequencing . Additionally, about 25% of mRNAs enriched with TDP-43 were unique to TDP-43WT or TDP-43G298S  highlight neuronal pathways previously linked to TDP-43 such as NMJ development [futsch [WT, Log2FC = 2.44, Padj = 8.07E−37; TDP-43G298S, Log2FC = 2.68, Padj = 6.86 E−12) and CSNK1E [WT, Log2FC = 0.721, Padj = 3.32E−3; TDP-43G298S, Log2FC = 0.847, Padj = 3.08E−3). Furthermore, we found that of the 834 TDP-43 interacting mRNAs recently identified in murine neurons using TRIBE (targets of RNA-binding proteins identified by editing) [Drosophila RIP data sets   about 1/WT, or TDP-43G298S. Following ribosome immunoprecipitations we isolated total RNA and conducted RNA seq, detecting ~ 9,500 mRNAs . These comparisons identified several genes enriched with or depleted from motor neuron ribosomes in both of the ALS models relative to RpL10-GFP controls (ΔLog2FC > 1 or < −1). Subsequent GO term analyses using David 6.8 [WT, spliceosome components were translationally dysregulated . Interestingly, oxidative phosphorylation genes encoding components of the electron transport chain showed both increased and reduced association with ribosomes , one of 14 genes that exhibited increased translation in a human cell model of TDP-43A315T proteinopathy [Interestingly, genes salient to translation exhibited altered translation Fig. f. In botWT, 37.6% for TDP-43G298S) suggesting that these may be direct targets of translation inhibition. GO term analyses of these TDP-43 enriched, TRAP depleted genes using David 6.8 [WT 27.6%, TDP-43G298S 34.9%) of the genes enriched with ribosomes in the context of TDP-43 proteinopathy were also associated with TDP-43 complexes . This suggests that in addition to specific pathways, a plethora of individual genes might represent salient targets of TDP-43 mediated translational inhibition or activation.To distinguish between translational changes that may be caused directly by TDP-43 versus compensatory alterations, we compared genes altered in their association with ribosomes to those enriched in TDP-43 complexes, in the context of TDP-43 proteinopathy. These analyses show that approximately 1/3 of the genes depleted from ribosomes were also enriched in TDP-43 complexes  and significantly depleted from ribosomes in the context of TDP-43 proteinopathy  family, localized to the plasma membrane via a GPI anchor or secreted in the extracellular matrix. HSPGs consist of a protein core and several heparan sulfate (HS) glycosaminoglycan (GAG) linear polysaccharide chains which exhibit complex sulfation patterns that dictate the ability of the HSPGs to interact with ligands such as wingless [Drosophila, Dlp was shown to function as a co-factor and competitive inhibitor for binding to the Wg/Wnt pathway receptor Frizzled 2 [frizzled 2 mRNA  and ovo/shavenbaby mRNA   and TDP-43G298S  relative to input  gene has been replaced with human TDP-43 [dlp mRNA is a novel TDP-43 candidate target that becomes insolubilized in the context of TDP-43 proteinopathy and are consistent with previous reports of Wg/Wnt alterations in disease.Further substantiating this finding, dlp mRNA enrichment with TDP-43 complexes, insolubilization and decreased association with ribosomes is inhibition of Dlp protein synthesis. To test the impact of TDP-43 proteinopathy on Dlp protein expression we conducted immunofluorescence experiments in the larval neuromuscular system. We examined Dlp expression at the neuromuscular junction (NMJ) and the ventral nerve cord (VNC), which comprises motor neuron cell bodies and compactly packaged neurites, particularly dendrites [WT or TDP-43G298S, and RNAi knock-down of TBPH (TBPHRNAi) are sufficient to deplete Dlp protein within NMJ boutons relative to the w1118 control  and cumulative granule area relative to the w1118 controls. In contrast, TBPHRNAi did not cause significant Dlp granularity phenotypes  we performed RT-qPCR experiments using dissected VNCs and larval NMJ preparations and found no significant changes in dlp mRNA levels between TDP-43 proteinopathy models and controls  to examine dlp using the motor neuron specific driver D42 GAL4 on its own and in the context of TDP-43WT or TDP-43G298S and measured its effect on TDP-43 induced locomotor defects. Using larval turning assays to measure locomotor function we found that although dlp overexpression (dlpOE) on its own resulted in a higher turning time , in the context of either TDP-43WT or TDP-43G298S disease models, it caused a lower turning time, indicating a rescue of locomotor dysfunction , dlp overexpression in this context is not sufficient to mitigate TBPHRNAi induced locomotor dysfunction  enhanced locomotor deficits in both ALS models based on TDP-43 overexpression  or co-overexpressed with TDP-43WT, TDP-43G298S or TBPHRNAi  in Dlp levels at the NMJ compared to w1118 controls . Together with our findings that dlpOE mitigates TDP-43 proteinopathy but not TBPHRNAi induced locomotor defects, these results indicate that Dlp levels at the NMJ are critical in the context of TDP-43 proteinopathy but not TDP-43 loss, and support a cytoplasmic gain of function rather than a nuclear loss of function mechanism. It is surprising that dlpOE in the context of TDP-43WT causes a statistically significant increase in Dlp levels at the NMJ; this suggests a potentially complex regulatory relationship between Dlp and TDP-43 proteinopathy that warrants further investigation.We next asked whether the rescue of TDP-43 induced locomotor dysfunction by NAi Fig. c-n. FirsTo further probe the relationship between TDP-43 induced neurodegeneration, Dlp, and Wg/Wnt signaling we used genetic interaction approaches between TDP-43 and the Frizzled 2 (Fz2) receptor, an established Dlp interactor . These eDrosophila in human patient derived tissues, we evaluated the expression and sub-cellular distribution of the Dlp human ortholog GPC6 [Pvalue = 0.0020, see Fig. Drosophila VNCs overexpressing TDP-43  [TDP-43 proteinopathy is a hallmark of ALS , 78 and  disease , 15. Rec disease . In contDennd4A) . Taken tDrosophila model of TDP-43 proteinopathy that recapitulates key features of the human disease including locomotor dysfunction and reduced lifespan [e.g., translation, mitochondrial function [dlp and the glutathione metabolism pathway. Although glutathione dysregulation has been shown to be deficient both in patient tissue [Here, using a lifespan , 24 we ifunction , 96) andt tissue  and patit tissue , it has t tissue .dlp as a novel target of TDP-43. dlp is translated into a GPI anchored glypican that interacts with the Wg/Wnt pathway receptor Fz2, serving as both a co-factor and competitive inhibitor for substrate binding [WT and TDP-43G298S disease models suggesting that the Wg/Wnt signaling pathway may be compromised by TDP-43 proteinopathy and consistent with published expression studies in patient tissues [i.e., motor neuron) and non-cell autonomous  aspects of Wg/Wnt signaling [dlpOE and dlpRNAi cause locomotor dysfunction suggest that Dlp levels must be exquisitely regulated in motor neurons. Indeed, Dlp overexpression mitigates but does not fully rescue TDP-43 dependent locomotor defects, perhaps due to an inability to restore wild-type Dlp levels. An alternative explanation for this result is that TDP-43 regulates additional mRNA targets.In addition to identifying motor neuron specific alterations to the translatome induced by TDP-43 proteinopathy, we identified  binding . RNAi kn tissues , 35. Altignaling  may be aignaling  and hippWT and TDP-43G298S both associate with dlp mRNA, and correlate with dlp depletion from ribosomes, the severity of some phenotypic alterations was variant dependent. While overexpression of dlp mRNA was sufficient to rescue locomotor dysfunction and Dlp protein levels were altered in the context of both TDP-43 variants, dlp mRNA was insolubilized only in the TDP-43G298S model. Additionally, different pathways were shown to have altered translation between the TDP-43WT and TDP-43G298S models. Differential results have previously been observed between the TDP-43WT and TDP-43G298S models [G298S stability [WT [We show that although TDP-43S models , which atability  and granlity [WT .dlp mRNA from ribosomes using TRAP, an approach that reflects translational changes in the entire motor neuron rather than specific compartments. It is possible that the synapse specific alteration in Dlp levels could also result from transport deficits, trapping NMJ-bound dlp mRNA/Dlp protein in the soma or neurites. Although our qPCR data show no change in steady state dlp mRNA levels, we cannot eliminate the possibility of mRNA and/or protein transport defects leading to the accumulation of Dlp in the VNC and reduction at the NMJ.In both ALS models, analyses of the whole VNC, which contains motor neuron soma and neurites, show that Dlp forms distinct puncta whereas staining at the NMJ revealed a significant reduction in Dlp protein. These findings suggest that TDP-43 dependent translational inhibition may be localized to axons and NMJs, which can explain the relatively low magnitude depletion of RNAi was sufficient to deplete Dlp from the NMJ, but not induce significant neuropil puncta, suggesting that the two phenotypes can occur independently of each other and might result from different mechanisms. Loss of nuclear function versus cytoplasmic gain of function remains an open question, with both mechanisms likely contributing to TDP-43 proteinopathy [RNAi suggests that this phenotype is the result of nuclear TDP-43 function whereas the neuropil puncta are likely the result of toxic gain of function as they are significantly increased only in the context of TDP-43 proteinopathy and not in the context of endogenous TDP-43 loss of function. Furthermore, the fact that restoring Dlp expression at the NMJ is not sufficient to mitigate TBPHRNAi induced locomotor defects while it improves TDP-43WT and TDP-43G298S dependent phenotypes suggests that Dlp levels at the NMJ are critical in the context of TDP-43 proteinopathy and that other factors may be at play in the loss of function caused by TBPHRNAi knock-down. Collectively, our findings suggest a model in which the mislocalization of Dlp found in flies and patient spinal cords results from a combination of translation inhibition and transport defects as previously reported for Futsch/MAP1B [Interestingly, TBPHinopathy , 110. Dlch/MAP1B   howeversmission , furthersmission  and for smission  further dlp as a target of TDP-43 proteinopathy highlights specific strategies including restoration of synaptic specific protein expression, intracellular transport as well as Wg/Wnt and other Dlp linked signaling pathways in ALS.Here we report several novel translational consequences of TDP-43 proteinopathy including effects on the spliceosome, metabolic pathways and the translational machinery itself. Our combined RIP and TRAP approach can distinguish between direct consequences of TDP-43 proteinopathy and compensatory changes in translation, which can inform future therapeutic strategies. Our identification of the glypican Additional file 1. RNAs enriched in TDP-43 complexes, GO term analyses and comparison with mammalian TRIBE targets.Additional file 2. RNAs associated with ribosomes in controls, RNAs with altered ribosome association in TDP-43 proteinopathy and TRAP GO term analyses.Additional file 3. RIP/TRAP overlap in TDP-43 proteinopathy and GO term analyses.Additional file 4. Demographic information for post-mortem patient spinal tissue.Additional file 5. Supplemental information.


This article presents the results of the cross-linking of oxidized flake graphene (GO) using hydrazine at room temperature. Conducting the process at temperatures up to 30 °C allowed to eliminate the phenomenon of thermal GO reduction to its non-oxidized form. In addition, based on the Infrared and Raman spectroscopy as well as X-ray photoelectron spectroscopy (XPS) analysis, the cross-linking ability of GO was observed depending on its size and degree of oxidation. These parameters were associated with selected physicochemical and electrical properties of obtained 3D structures. Three GO flakes sizes were tested in three different oxidation degrees. It was shown that, regardless of the size of GO, it is crucial to achieve a specific oxidation degree threshold which for the conducted tests was a >20% share of oxygen atoms in the whole structure. This value determines the ability to cross-link with hydrazine thanks to which it is possible to synthesize the spatial structure in which the π–π interactions among individual flakes are significantly reduced. This directly translates into the fact that the 3D structure shows an electrical resistance value in the range of 4–103 Ω, depending on the size and oxidation degree of the used material. The explanation of this phenomenon related to the electrical conductivity of 3D structures was confirmed based on the molecular modeling of the chemical structures. Graphene, as the main representative of a growing group of 2D materials, has unique mechanical, optical, and chemical properties not found in any of the currently known 3D materials ,2,3,4,5.Therefore, the purpose of this research was to determine the cross-linking capacity of oxidized graphene powder, depending on the size of the flakes and share of oxygen groups. Synthesis of the spatial material with the quazi-2D properties of graphene will allow to create products based on it, e.g., in the form of water filters, materials capable of reversely storing hydrogen, new lithium-ion battery electrodes or a component of ultra-strong and light composites ,24. In t4 oxidant during synthesis, equaling 25%, 50%, and 100% of oxidant for low, medium, and high oxidation levels, respectively.Graphene oxide (GO) water dispersions were purchased from NANOPOZ company in Poznań, Poland. All samples were synthetized with the modified Hummers method, using the same natural graphite as a precursor. Materials were differentiated by the size of flakes, using grinding and sieving of graphite powder as well as by oxidation level, using different amounts of KMnOThe cross-linking process was performed by introducing to the GO suspension a set amount of hydrazine to obtain a specific concentration. After 120 h, the created structure was left to dry at room temperature.Elemental analysis was performed with a FLASH 2000 analyzer . Before the analysis, water dispersions of graphene oxide were dried for 2 h at 60 °C to prevent thermal reduction while overheating. The analysis was conducted using approximately 2 mg of dry graphene oxide. Two repeatable measurements were done for each material and the average result was presented further.Atomic force microscopy (AFM) measurements were conducted using a Keysight model 5500 microscope . For measurements, the highly oxidized GO dispersions were chosen. Each of them was dissolved in deionized water and smoothly sonicated before analysis. Dispersions were deposited on a freshly cleaved mica surface and dried in ambient conditions for a few hours. Imaging was realized in intermittent-contact mode using All-In-One-Al C cantilevers .The specific surface area and porosity of the cross-linked graphene oxide samples were determined by the BET (Brunauer-Emmett-Teller theory) based on low-temperature (−196 °C) nitrogen adsorption in a Micromeritics ASAP 2020 apparatus .In order to determine the morphology of the obtained test material, the samples were subjected to microscopic examination on a stand equipped with the JEOL JSM-6610LV electron microscope , integrated with the MiniCL-GATAN Cathodoluminescence Imaging System, . The studies were conducted in SEM-EDS mode, with a 25 V acceleration voltage. The analyses were performed using EDS (Energy-Dispersive X-ray Spectroscopy) AZtecEnergy software .Morphology analysis of the produced powders as well as their chemical composition were reinforced using a TEM Talos F200X microscope  from FEI with a maximum value of electron accelerating voltage of 200 kV.The Raman technique was used to identify the structure of the obtained material. The RENISHAW inVia Raman Microscope  was used for the study, which, when activated and stabilized (green laser with a wavelength of 532 nm and a power of 29.3 mW), was calibrated using a silicon sample.−1, range 400–4000 cm−1. The study used a DTGS detector . The beam angle was 80°.The results were supplemented by infrared spectroscopy with the Nicolet iS50 FT-IR . The study was conducted with the following parameters, number of scans 64, resolution 4 cm−6 Pa, where the measurements of the binding energy were according to the Au 4f7/2 core level (84.0 eV). The results of the surface composition were elaborated through CasaXPS software  based on binding energies and peak areas of C 1s, O 1s, Si 2p, N 1s, and Al 2p core levels.Chemical composition analysis was supplemented by X-ray photoelectron spectroscopy (XPS). The XPS of the functionalized graphene oxide powder was obtained using a photoelectron spectrometer  equipped with a hemispherical R3000  high-resolution detecting system and a monochromatized aluminum source Al Kα (E = 1486.6 eV). To compensate for the charge on the surface, a low energy electron flood source FS40A-PS  was used. The wsorking pressure in the chamber during measurements was 5 × 10Electrical resistance was measured by placing a set amount of GO dispersion on a silicon substrate and performing the cross-linking process, after which the system was dried in ambient conditions. Then, a small amount of silver-containing adhesive was placed on the structure so that it penetrated the structure  to form contacts for the electrodes. The distance between each set of contacts was 10 mm.The spatial structure of the cross-linked graphene oxide using hydrazine was modelled using SCIGRESS v.FJ 2.7 software provided by Fujitsu Poland. The created 3D model was optimized in order to achieve the energy minimum in electrostatic interactions and the resulting structural strains.Based on the AFM images, the graphene oxide flake sizes were estimated. Typical AFM topography images are shown in To estimate the graphene oxide oxidation level, the CHNS+O was conducted . PredictThe C/O ratio of obtained graphene oxide materials evolved from 6.58 for the low oxidized large flakes, 2.07–1.74 for medium oxidized ones, and to 1.23–1.16 for highly oxidized flakes. That covers and slightly exceeds the range of typically produced graphene oxide C/O ratio, specified from 4:1 to 2:1 . Such a Based on the analysis of the flakes’ size and composition, the samples are divided into groups with the labeling method presented in −1 and 1600 cm−1 which come from carbon–carbon bonds conjugated with carbon–oxygen bonds.The conducted Fourier-transform infrared spectroscopy (FTIR) tests  prove th−1) and G (approximately 1590 cm−1) as well as weaker signals of G’ (around 2700 cm−1) and D + G (approximately 2925 cm−1). The presence of the D peak is linked to the presence of structural defects, while the G peak is connected to the scattering in the E2g mode [2 bonded carbon. A change in the position of the G peak to the lower values of the Raman shift, even to 1595 cm−1, has also been observed in Reference [The analysis of Raman spectra of the graphene oxide powders is shown in E2g mode  and relaeference , where, D/IG of the analyzed powders were in the range of 0.9 to 1, except for the sample S1GO. In that case, the parameter was 0.5. Similar results for the oxidized graphene powders were also achieved in the research conducted by Kaniyoor and Ramaprabhu [The values of the intensity ratio Imaprabhu ,38. The 2 hybridization. In that case, a shift of the G’ peak towards lower values with the increase of oxygen to carbon share  is observed, as demonstrated by López-Díaz studies [In addition, the analysis of Raman spectra proved that as the average oxidation value of the analyzed graphene flakes increased, there was a shift in both the G and G’ peaks toward lower values of Raman shift , Table 2 studies . This faFor such characterized samples, the process consisted of cross-linking through substitution of hydroxyl and carboxyl groups with hydrazine. For this purpose, eight different water suspensions of graphene oxide were prepared, each with equal concentrations of 4 mg/mL. First, each of the suspensions was sonicated with a frequency of 35 kHz for 5 min to evenly disperse the material. After treating all the samples, the cycle was repeated for another 10 min. Then, 1 mL of cross-linking agent, the hydrazine, was added to each system to start the process, which lasted for 120 h at room temperature (thus avoiding the complete reduction of oxygen groups). After that time, the samples were filtered and dried at room temperature for another 336 h. In order to improve readability, the prefix “C-“ was added to the labeling so as to differentiate between samples before and after the cross-linking process. Examples of changes in macromorphology are shown in 2GO, S3GO, M2GO, M3GO and L2GO were chosen. The choice was based on the samples’ capabilities to cross-link and form 3D structures  [The FTIR spectra of the chosen graphene oxide samples are shown in  (<2 µm) .The results obtained with FTIR spectroscopy are consistent with the XPS spectra analysis, according to which there is an increase of bonded nitrogen coming from the hydrazine (400 eV) with the increase of the oxidation degree of treated graphene oxide . At the D/IG parameter increased for all analyzed samples compared to their state before their cross-linking . This is due to the fact that at room temperature, graphene flakes, through a chemical reaction between oxygen groups and hydrazine, form cross-linked quazi-2D graphene structures. These structures, as a result of transition from sol to gel, with subsequent removal of the aqueous environment, is associated with a reduction in the volume of the cross-linked structures. This fact causes for the produced materials to exhibit relatively high internal stresses. For this reason, information related to, for example, multi-layer graphene, cannot be associated with it, because it is not a real spatial system typical for multi-layer graphene or graphite. In addition, the electron system changes due to the formation of additional chemical bonds. On the other hand, the analyzed structure, due to the cross-linking conditions, may also show local order in which the petals locally arrange in parallel fashion. However, one must be aware that between these flakes there are pillars at the edges and in the defects areas that prevents the flakes from creating physical interactions like in case of graphite. On the other hand, a signal in the Raman spectrum about the creation of two- or three-layered systems may result from the flakes hanging between pillars, the distance of which is large enough that there are local van der Waals interactions.In summary, graphene materials produced as a result of their spatial cross-linking constitute a new subgroup of materials with quazi-2D properties. Due to the fact of their different structural properties, they cannot be analyzed based on Raman spectra in the same way as graphene. The structures show additional chemical bonds, resulting structural stresses and local van der Waals interactions among parallel-oriented flakes.After the complete drying of samples, electrical resistance tests, as well as specific surface examinations were conducted. The obtained results are presented in 3GO powder has been chosen .A correlation can be observed in the case of specific surface values. With a higher degree of oxidation, the specific surface was bigger  which ca1GO, M1GO, and L1GO) were not cross-linked. Their C/O ratio was 4.8; 5.3, and 6.6, respectively.Graphene oxide flakes with low degree of oxidation . In this case, the C/O ratio was 1.2.The most compact structure was achieved for samples with small-sized flakes and their high oxidation degree (C-SThe possibility of cross-linking the graphene oxide flakes, depending on their size and C/O ratio, was confirmed.Based on the conducted research:


A novel class of graphene-based materials incorporated into natural lignocellulosic fiber (NLF) polymer composites is surging since 2011. The present overview is the first attempt to compile achievements regarding this novel class of composites both in terms of technical and scientific researches as well as development of innovative products. A brief description of the graphene nature and its recent isolation from graphite is initially presented together with the processing of its main derivatives. In particular, graphene-based materials, such as nanographene (NG), exfoliated graphene/graphite nanoplatelet (GNP), graphene oxide (GO) and reduced graphene oxide (rGO), as well as other carbon-based nanomaterials, such as carbon nanotube (CNT), are effectively being incorporated into NLF composites. Their disclosed superior mechanical, thermal, electrical, and ballistic properties are discussed in specific publications. Interfacial shear strength of 575 MPa and tensile strength of 379 MPa were attained in 1 wt % GO-jute fiber and 0.75 wt % jute fiber, respectively, epoxy composites. Moreover, a Young’s modulus of 44.4 GPa was reported for 0.75 wt % GO-jute fiber composite. An important point of interest concerning this incorporation is the fact that the amphiphilic character of graphene allows a better way to enhance the interfacial adhesion between hydrophilic NLF and hydrophobic polymer matrix. As indicated in this overview, two basic incorporation strategies have so far been adopted. In the first, NG, GNP, GO, rGO and CNT are used as hybrid filler together with NLF to reinforce polymer composites. The second one starts with GO or rGO as a coating to functionalize molecular bonding with NLF, which is then added into a polymeric matrix. Both strategies are contributing to develop innovative products for energy storage, drug release, biosensor, functional electronic clothes, medical implants, and armor for ballistic protection. As such, this first overview intends to provide a critical assessment of a surging class of composite materials and unveil successful development associated with graphene incorporated NLF polymer composites. Naturally found composite materials like wood, bone, antler, and others, have been used by our ancestors since the beginning of mankind. Artificially fabricated composite materials, with chemically dissimilar phases separated by a distinct and well-connected interface, can be traced back to earlier concretes developed 7 millennia ago . DocumenAmong the polymer composites, those incorporated with fibers have been considered the most important owing to their highest strength and/or stiffness on a weight basis . AccordiToday’s interest for “natural fiber composites” became evident by the exponential growth in publications, starting in the last decade of the 20th century, as revealed in In spite of the aforementioned advantages combined with low density and cost, NLFs exhibit heterogeneity in properties, which may limit their applications. This fluctuation in properties are associated with natural variations on the chemical composition, diameter, crystalline fraction of the cellulose, fiber source type , in addition to the growth stage and cultivation conditions ,34. AnotRecently, research works using graphene incorporated materials suggested a possible emerging solution to the NLFs lower adhesion to polymer matrices ,74,75,76Graphene is a flat monolayer of carbon atoms in a two-dimensional (2D) honeycomb lattice, which is formed by strong sp2-carbon bonds. This is the basic unit for graphitic materials of all other dimensions, such as fullerenes with zero-dimension (0D), nanotubes (1D), and graphite (3D) ,79,80,81The first report about this “lamellar structure of graphite” was in 1859 by Brodie . In the 2∙g−1) [−1∙K−1) [Graphene has exceptional properties such as extensive surface area (2630 m2∙g−1) , high Yo2∙g−1) , high th[−1∙K−1) , and goo[−1∙K−1) . However[−1∙K−1) .The final properties of the graphene-based material depend on the number of graphene layers, average lateral dimension, and atomic carbon/oxygen ratio . Among tThe structure, properties, and the number of layers of the graphene-based materials are closely related to the method chosen for their production . These p3, increased the amount of KMnO4 and performed the oxidation reaction in a 9:1 mixture of H2SO4/H3PO4, instead of only H2SO4, improving the efficiency of the oxidation process and eliminating toxic gas evolution. Chen et al. [3, avoiding the release of toxic nitrogen oxides gases. The GO could remain exfoliated in water and later to be reduced back to graphene. For this reason, this graphene-based material has been successful in several applications, such as electronics, conductive films, electrode materials and composites [The most cost-effective production method is by liquid-phase exfoliation, producing dispersions of graphene ,91. Thisn et al.  proposedmposites ,95,96,97This section presents the research efforts in NLF/polymer composites incorporated with graphene. The use of graphene-incorporated materials in these composites is discussed as well as their related properties. In addition, a brief description of the main techniques to produce these composites is presented.The graphene discovery led to the emergence of new opportunities for the development of novel high-performance and lightweight composites, enabling its application in several fields, from electronics to aerospace. Indeed, a low filler content of this material or its derivatives can significantly improve mechanical, thermal, and physical properties ,105,106.Among the processes shown in In the in situ polymerization, the graphene-incorporated material is mixed with a liquid monomer and an initiator. The reaction is controlled by temperature and time and can also be initiated by radiation ,107. In To improve the fiber’s properties, the coating method is usually applied. It can be done by either immersion into a graphene-based dispersion or spraying the graphene dispersion directly onto the fiber. The first method, the most common, is also known as “dip and dry” and can be followed by a reduction or washing process . The coaDu et al.  investigThe improvement in the mechanical properties of polymeric materials with the addition of graphene-based materials was reviewed by Papageorgiou et al. . They coMany works have reported the effect on the mechanical ,113,115,In The good relation between stiffness and strength of NLF/graphene-based composites can be noted in A great effort is being made to develop natural fiber composites with graphene materials as reinforcement since they exhibit outstanding mechanical properties. In general, the composites are produced using graphene either as a filler, which is mixed directly in the matrix, or coating the fiber, which is used as reinforcement. A potential progress is disclosed in both cases for low volume fractions of graphene-based materials and is presented as follows.Since the rise of graphene in 2004, the application of graphene and its derivatives became a big hit in the materials science field. Researchers from different areas started to investigate the incorporation of graphene (G), graphene oxide (GO) and reduced graphene oxide (rGO) in all kind of materials. Such investigations disclosed the influence on mechanical and electrical as well as thermal and optical properties that could be improved in comparison to “neat” materials. It did not take long to this approach be considered in high performance composite materials ,139. As The first strategy was to use GO as filler in polymer matrix composites associated with some fiber reinforcement. The successful development of these hybrid composite structures lies on the synergy between the matrix and reinforcements as well as in the optimized composition of the constituents. Prasob et al.  investigChaharmahali et al.  investigPopulus deltoides and the fraction of 20 wt % was kept constant for all composites. The degradation temperatures were shifted to higher values for all GNP loadings, but the optimal composition of 0.8 wt % exhibited the highest improvement in the thermal stability and lower water absorption. Although the incorporation of GNP into the PP matrix improves mechanical properties, the SEM micrographs showed that high contents (>3 wt %) of GNP were easily agglomerated, as illustrated in Similar results have been reported by Sheshmani et al. , who evaAnother nanofiller tested in natural fiber-based composites is the carbon nanotube (CNT), which is considered as a graphene sheet rolled up forming 1D structure. However, difficult dispersion of CNT in the polymer matrix and high cost make it not as attractive as other graphene derivatives. Mechanical and fracture properties of ramie fiber reinforced epoxy composites with filler loadings 0.2, 0.4, and 0.6 wt % of CNT were evaluated by Shen et al. . The resLima et al.  reportedIdumah and Hassan  studied A variant of the first strategy in the use of graphene or its derivatives, to produce composites with superior resistance, could be the functionalization of the polymer matrix. This approach is commonly used for producing composites consisting of polymeric matrix modified by GO functionalization. Layek et al.  reportedIn another investigation, Han et al.  exploredDespite not using graphene nor its derivatives, but exfoliated graphite instead, the relevance of that investigation lies in it being the precursor of the functionalization strategy. The use of graphite-based material as coating for natural fiber proved to be a strategy worth of investigation, which regain researcher’s attention in the end of 2020 decade. Such strategy suggests that it could prevent fiber delamination and also delay the crack propagation by redistributing the stress around fibers ,148. DifSarker et al.  discusseStrong adhesion between the GO and alkali treated fiber; andInteraction of GO treated jute fiber with the matrix. The oxygen containing functional groups present in GO could create strong bond with the alkali treated fibers to make them capable of carrying more load from the matrix.The enhanced mechanical properties of GO treated jute/epoxy composites were associated with two main reasons:The authors also observed by SEM fractographic analysis that the untreated fiber failure mechanism is predominantly associated with weak interfacial bonding and fiber pull-out. As for the GO-treated jute fibers, a modification on the failure mode occurred from fiber pull-out or de-bonding to transverse fracture. This indicates an improvement in the interfacial bonding that can lead to higher tensile properties of the composites.Chen et al.  went furWith the increasing of fiber content, the tensile strength, the tensile modulus and the impact strength of the PP composites increased up to 7.5 wt % and then turned down. Higher fiber loading (10 wt %), however, promotes stress concentration and induced more defects, which led to a weak interface adhesion between the fibers and the PP matrix. Meanwhile, the elongation at break significantly decreased with the increase of fiber loading, which was associated with the restriction of molecular chain mobility of PP. In summary, the GO-SF/MAPP-PP composite with 7.5 wt % fiber loading had the highest increase of 36.50, 30.00, 69.73, and 36.27% in tensile strength, tensile modulus, elongation at break and the impact strength comparing to the neat PP.The surface fracture of the tensile specimens of both the treated and untreated composites are presented in It is clear that, to effectively improve the mechanical strength of NLF-GO-PM composites, a previous treatment of the surface is important, which would allow that impurities inherent to natural fibers such as hemicelluloses or waxes be removed. With that, a stronger bonding between the natural fiber reinforcement and the GO-coating would improve the transfer of loads from the matrix to the reinforcement.Garcia Filho et al.  used theDynamic mechanical analysis (DMA) was used to investigate how GO-coated piassava epoxy composites behave at different temperatures for different amount of fiber reinforcement . DMA resThe dynamic modulus, also known as storage modulus, is often associated with the stiffness of the material. It measures the elastic or potential energy stored by the material . It was Sisal fiber (SF)/polypropylene (PP) composites were investigated by TGA and differentials scanning calorimeter (DSC) . The infIt is possible to verify from Damage from heat may be considered one of the major drawbacks for the use of natural fibers reinforced polymeric composites in engineering applications . BecauseTM and TwaronTM, which are aramid-based fibers, or DyneemaTM and SpectraTM, produced with UHMWPE. The ballistic performance obtained in NLFs polymer composites was shown to be a function of different energy absorption mechanisms, Before considering the use of graphene-base materials, the application of NLFs as reinforcement of polymer matrix composites for ballistic protection was investigated ,161,162.Recently, the possible effect of graphene-based material on the ballistic performance of NLF polymer composites was investigated. Target results are illustrated in The improved interface between fiber and matrix might reduce the amount of absorbed ballistic energy by some of the aforementioned mechanisms. Nevertheless, the better physical integrity is a required condition for ballistic protection vests. summarizes the comparison of the GOCF conditions studied by Costa et al.  and seveThe advantage regarding the strategy of GO-incorporation in NLFs polymer composites for ballistic protection application can be seen in The water absorption of sisal fiber (SF) reinforced PP composites, with or without GO, was addressed by Chen et al. . Figure Graphene-coated NLFs can promote new functionalities and can be used as energy storage, filtration, drug release, biosensors, functional clothes, medical implants, and others. For example, in the work by Qu et al. , a novel4, N2H4, C6H8O6, Na2S2O4, and NaOH). Both reducing agent type, concentration, and immersion time had a great influence on the electroconductivity of the cotton fabrics, resulting in an electrical resistivity ranging from 103 to 106 kΩ.cm−1. Among the reducing agents tested, Na2S2O4 achieved the highest level of electrical conductivity, and 30 min reaction was enough for the complete reduction of graphene oxide [Shateri-Khalilabad and Yazdanshenas  also coane oxide . The usene oxide ,50,52 anA different approach was done by Weng et al. , who proSupercapacitors fabricated from graphene/cotton composites as flexible electrodes have also demonstrated good capacitance and low resistance ,169. In It is important to notice that the development of the strategies for incorporation of graphene-based materials into NLFs polymer composites still walks in babies’ steps and many issues, especially in the processing sector, still lack investigation and should be properly addressed. Moreover, it is also clear that the benefits of the incorporation of graphene are much bigger than one could think. Indeed, several properties such as tensile, flexural, impact resistance, ballistic penetration, physical integrity of composites, water resistance, thermal resistance and stability, and many others were shown to be improved by the GO-incorporation.The global market for graphene application is growing rapidly, in association with scalable and more efficient production methods that are continuously being developed. Some applications, such as electronics, semiconductors, and battery are already a reality. Indeed, cell battery based on graphene is already available on the market and can be mass-produced. This battery can charge 3 times faster and has a longer lifetime in terms of charge-cycle than lithium ones. Besides, it generates less heat . As for Electronic textile (e-textile), including natural fabrics, allows the collection, processing, storage, and display of information. These wearable sensors are revolutionizing human-machine interaction in several applications, such as functional clothing, healthcare sensors, interior design, and military applications ,172. E-tSince the 2000s, many publications are dedicated to e-textiles ,173,174.−1 and a measurement range of up 140 kPa, which was considered a relevant performance by the authors [Natural fibers, such as cotton ,184,185, authors . In the  authors , a novel authors .According to Xu et al. , the weaIn the light of the aforementioned investigations, the future wearable electronics must be flexible, resilient, comfortable, and present potential to mass production and low cost. Natural fibers treated with graphene-based materials have been shown as promising candidates ,177,178.2), ammonia (NH3), hydrogen (H2), hydrogen sulfide (H2S), and carbon dioxide (CO2) [Graphene stands out as a material for nano-sensors owing to its single-atom thickness. This makes graphene extremely sensitive to changes in its environment. In addition, the presence of functional groups makes the graphene oxide highly sensitive to water molecules and can also easily react with many other chemicals. Therefore the graphene-based materials can be used for humidity and gas sensors in the detection of various hazardous gases, such as nitrogen dioxide (NOde (CO2) .2. Three types of commercial silk fabric weaving coated with GO thermal reduced were investigated as NO2 sensors by Jung et al. [Using these properties, fiber-based reduced graphene oxide (rGO) gas sensors were fabricated by GO coating in natural fibers textiles and yarns ,189,190.2 sensor was proposed by Kumar et al. [2 with a remarkably low limit of detection at 300 ppt. First, the graphene was grown on Cu foils by a chemical vapor deposition process, then polymethyl methacrylate (PMMA) film was spin-coated forming PMMA-graphene film, which was transferred onto paper and dried at room temperature. After that, the PMMA layer was dissolved in acetone for 5 min, resulting in a graphene/paper composite (g-paper).A simple, low cost, and environmentally friendly NOr et al. . GraphenBased on Scopus metrics, this first overview disclosed that the scientific community might be witnessing the birth of a novel class of natural fiber polymer composites, which is exponentially surging in the present decade. The main characteristic of this class is the incorporation of graphene-based materials to act as reinforcement or to improve the interfacial adhesion between hydrophilic natural fibers and hydrophobic polymer matrix.As a polymer matrix reinforcement, different types of graphene-based: (i) pristine graphene (PG); (ii) nanographene (NG); (iii) graphene flakes (G); (iv) exfoliated graphene/graphite nanoplatelets (GNP) have effectively been incorporated for superior thermal, mechanical, machinability, and viscoelastic properties. Improvement of the interfacial adhesion is attained by coating natural fiber or fabric with graphene oxide (GO) or reduced graphene oxide (rGO). The presence of GO functional groups interacts with hydroxyl groups in the fiber cellulose chains modifying the fiber surface by providing new favorable interfacial bonds.Indeed, among the applications associated with graphene-based materials, the incorporation in natural fiber/fabric polymer composites has very recently attracted special attention. Remarkable properties justify the rapid increase in publications and motivates the attempts to develop engineering products. Indeed, from the aforementioned exponential growth, it is predicted that around 3000 articles on graphene-incorporated natural fiber polymer composites might be published by 2025.Graphene-based materials have been shown to significantly improve stiffness, toughness, electrical conductivity, and as well as the thermal stability of natural fiber polymer composites. Based on the articles discussed in this overview, one should expect a promising future perspective for research works on incorporation and functionalization of natural fibers with GBMs for a new generation of high-performance composites.On the other hand, there are relevant technological challenges to be overcome, especially concerning the graphene, such as its reproducibility to large-scale applications, as well as the homogeneous dispersion in composites and its interfacial interactions. The safety, reliability, and durability of these composites are still subjected to investigations.There is no doubt that graphene incorporated natural fiber composites should promote innovative research works and contribute to industrial developments, but more studies are needed to provide a better understanding of the interaction between components in this surging novel class of composite materials.