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.


TBX5 variation in patients with CHD and AFremains obscure. In this research, by sequencing analysis ofTBX5 in 178 index patients with both CHD and AF, a novelheterozygous variation, NM_000192.3: c.577G>T; p.(Gly193*), was identified inone index patient with CHD and AF as well as bicuspid aortic valve (BAV), withan allele frequency of approximately 0.28%. Genetic analysis of the proband’spedigree showed that the variation co-segregated with the diseases. Thepathogenic variation was not detected in 292 unrelated healthy subjects.Functional analysis by using a dual-luciferase reporter assay system showed thatthe Gly193*-mutant TBX5 protein failed to transcriptionally activate its targetgenes MYH6 and NPPA. Moreover, the mutationnullified the synergistic transactivation between TBX5 and GATA4 as well asNKX2-5. Additionally, whole-exome sequencing analysis showed no other genescontributing to the diseases. This investigation firstly links a pathogenicvariant in the TBX5 gene to familial CHD and AF as well as BAV,suggesting that CHD and AF as well as BAV share a common developmental basis ina subset of patients.TBX5 has been linked to Holt-Oram syndrome, with congenital heart defect (CHD)and atrial fibrillation (AF) being two major cardiac phenotypes. However, theprevalence of a  As the most prevalent type of human birth defect, congenital heart defect (CHD)occurs in about 1% of all live neonates, accounting for nearly a third of all formsof developmental abnormalities , acute kCardiogenesis undergoes a highly complex biological process, and both environmentaland genetic pathogenic factors can perturb this finely regulated process, leading toCHD . HoweverTBX5 variations have recently been involved in atrialfibrillation (AF), the most common sustained cardiac arrhythmia , with AF being the predominant clinical phenotype. TBX5 in multiple patientsaffected with AF. TBX5in a case with AF. TBX5 loss-of-function mutation inan index patient with idiopathic AF. These observational results highlight thepronounced genetic heterogeneity of CHD and AF, which makes it justifiable toinvestigate the prevalence of TBX5 variations in patients with bothCHD and AF, and unveil the molecular mechanism of CHD and AF resulted from novelTBX5 variations.Interestingly, This study subjects comprised 178 unrelated adult patients suffering from bothCHD and AF, who were consecutively recruited between February 2015 and TBX5 were amplified from each study participant’s genomicDNA by polymerase chain reaction (PCR) for a variation scan by PCR-sequencing.The PCR primers were designed as described elsewhere . The PCR program was set asfollows: initial pre-denaturation at 95 °C for 15 min followed by 35 thermalcycles of denaturation at 95 °C for 30 s, annealing at 62 °C for 30 s andextension at 72 °C for 1 min, with final extension at 72 °C for 8 min. Theamplified products were fractionated by electrophoresis on a 1.2% agarose gel,and isolated utilizing the QIAquick Gel Extraction Kit (Qiagen). The purifiedamplicons were subjected to PCR-sequencing under an ABI 3730 XL DNA Analyzer(Applied Biosystems), with the BigDye® Terminator v3.1 CycleSequencing Kit (Applied Biosystems) following the manufacturer’s instructions.The detected sequence variant was validated by bi-directional re-sequencing ofan independent PCR-generated amplicon from the same subject. For an identifiedTBX5 variation, the http://www.1000genomes.org), the https://gnomad.broadinstitute.org), and the Blood samples were collected from each study subject. Genomic DNA of each testsubject was purified from blood cells with the Wizard Genomic DNA PurificationKit . The coding exons and splicing donors/acceptorsof In addition, in order to rule out the potential causative effects of other geneson the diseases, whole-exome sequencing (WES) analysis of the mutation carrier’sfamily members was performed as described previously  and was fully sequenced to confirmthe desired mutation and to exclude any other unwanted sequence variations. Theeukaryotic expression vectors GATA4-pSSRa and NKX2-5-pEFSA, and the natriureticpeptide precursor A-luciferase (NPPA-luc) reporter vector, which expressesFirefly luciferase, were kind gift from Dr. Ichiro Shiojima, at the Departmentof Cardiovascular Science and Medicine, Chiba University, Japan. The α-myosinheavy chain 6-luciferase (MYH6-luc) reporter plasmid, which expresses Fireflyluciferase, was created as described previously  according to theproduct description. To balance transfection efficiency, the internal controlplasmid pGL4.75 (Promega), which expresses the Renilla luciferase, wasco-transfected. Specifically, COS-7 cells were transiently transfected withempty pcDNA3.1 (1.0 μg), or wild-type TBX5-pcDNA3.1 (1.0 μg), or mutantTBX5-pcDNA3.1 (1.0 μg), or wild-type TBX5-pcDNA3.1 (0.5 μg) plus empty pcDNA3.1(0.5 μg), or wild-type TBX5-pcDNA3.1 (0.5 μg) plus mutant TBX5-pcDNA3.1 (0.5μg), together with MYH6-luc (1.5 μg) and pGL4.75 (0.04 μg). To analyze thesynergistic transactivation, the same amount (0.6 μg) of each expression vector was used singly or in combination, in the presence of NPPA-luc (1.0μg) and pGL4.75 (0.04 μg). The transfected cells were cultured for 48 h, andthen were harvested and lysed. The Firefly luciferase and Renilla luciferaseactivities were measured under the GloMax-96 Microplate Luminometer (Promega) byutilizing the Dual-Glo Luciferase Assay System (Promega), following themanufacturer’s manual. The activity of the promoter was presented as foldactivation (ratio) of Firefly luciferase relative to Renilla luciferase. Eachtransfection experiment was conducted in triplicate for three times, and theresults for promoter activity were given as mean ± standard deviation (SD) ofthree experiments in triplicate.COS-7 cells  were grown in DMEM supplemented with 10% fetal bovine serum, as well as penicillin (100 U/mL) andstreptomycin (100 μg/mL), in an atmosphere with 5% COt-test, or one-way ANOVA with Tukey’s post hoc test,when indicated, with a p<0.05 indicating significantdifference.Differences in promoter activities between two groups were compared using theStudent’s In this investigation, a total of 178 unrelated cases suffering from CHD and AF wereclinically analyzed in contrast to a total of 292 unrelated control people . The included cases had bothechocardiograph-documented CHD and electrocardiogram-documented AF, while thecontrols had normal echocardiographs and electrocardiograms, with no evidence ofcardiac diseases. All the 178 patients had positive family histories of CHD andAF; whereas none of the 292 control individuals had a positive family history ofCHD or AF. No study participants had known traditional pathogenic factors forCHD or AF. There was no significant difference between case and control groupsin gender, age or ethnicity. The baseline features of the 178 cases affectedwith CHD and AF are summarized in TBX5 gene, a heterozygous variation, NM_000192.3:c.577G>T; p.(Gly193*), was detected in one out of the 178 patients affectedwith CHD and AF, with an allele frequency of ~0.28% in the patient population.The variation carrier had positive family histories of CHD and AF as well asbicuspid aortic valve (BAV). Genetic studies of the variation carrier’savailable family members revealed that the variation co-segregated with ASD andAF as well as BAV, which were transmitted as autosomal dominant traits. Inaddition, two family members (II-1 and III-1) had also congenital VSD. Thesequence chromatograms illustrating the heterozygous TBX5variation of c.577G>T and its wild-type control sequence are given in TBX5 variation identified in this studywas 1/356 (0.28%) in patients and 0/584 (0%) in controls.By sequencing the whole coding regions and flanking introns of theTBX5 mutation was carried out, and anaverage of 12,973 exonic variants ranging from 11,652 to 14,395 was detected foreach family member. A total of 742 exonic variants were shared by both affectedsubjects, of which 262 were autosomal, heterozygous non-synonymous, nonsense,and splice site variants. After filtered, only the variation c.577G>T inTBX5 was verified by Sanger sequencing and demonstrated toco-segregate with CHD and AF as well as BAV in the family.Additionally, WES analysis of the genomic DNAs from two affected family members(II-4 and III-4) and one unaffected family member (II-3) of the proband whoharbored an identified MYH6 promoter by ~12 fold and ~1 fold, respectively. When half the amount of wild-type andGly193*-mutant TBX5 plasmids (each 0.5 μg) was used, the resultanttranscriptional activity was ~6-fold .As shown in NPPA promoter by ~7 fold and~1 fold, respectively . In combinationwith wild-type NKX2-5, wild-type and Gly193*-mutant TBX5 activated theNPPA promoter by ~30 fold and ~5 fold, respectively; while together with wild-type GATA4,wild-type and Gly193*-mutant TBX5 transcriptionally activated theNPPA promoter by ~22 fold and ~4 fold, respectively.As shown in MYH6 andNPPA promoters. Moreover, the mutation disrupted thesynergistic transcriptional effect between TBX5 and GATA4 as well as NKX2-5.Additionally, WES analysis showed no other genes contributing to the diseases of thefamily. These observational results indicate that the pathogenic variation in theTBX5 gene predisposes to CHD and AF as well as BAV.In the current investigation, a novel heterozygous TBX5 variation, NM_000192.3:c.577G>T; p.(Gly193*), was discovered in a family with CHD and AF as well as BAV.The variation was absent in the 584 reference chromosomes as well as in suchpopulation databases as the TBX5 is located on chromosome 12q24.1, which encodes a518-amino acid protein. The TBX5 protein harbors four functionally importantdomains, including a T-box domain , which functions to bindtarget DNAs and interact with other proteins; a transcriptional activation domain, which is responsible for transactivation of targetgenes; and two nuclear localization signals (NLS) including NLS1 (amino acids 78-90)and NLS2 (amino acids 325-340), which were essential for nuclear localization (NPPA,GJA5, MYH6 and SCN5A,separately or together with GATA4, GATA6, NKX2-5, MEF2C and TBX20 (TBX5 haploinsufficiency is amolecular mechanism of CHD and AF as well as BAV in a subset of patients.In humans, lization . Previoulization . Recent nd TBX20 , and varnd TBX20 . In the TBX5 deficiency contributes to CHD and AF. In mice, TBX5 isabundantly expressed in entire cardiac crescent, heart tube, left ventricle, venacavae, common atrium, and cardiac central conduction system, encompassingatrioventricular bundle and bundle branches (Tbx5 led to murine embryonic death because of failure toundergo cardiac looping as well as left ventricular and sinoatrial hypoplasia; whileheterozygous Tbx5-bull mice showed ASD, VSD, left ventricularhypoplasia, endocardial cushion defect, and conduction system anomalies,encompassing atrioventricular conduction blocks and bundle branch blocks (Tbx5 haploinsufficiency also markedlyreduced the transcription of multiple target genes, including Nppaand Cx40 (Tbx5-mutant mice demonstrated spontaneous AF, and inTbx5-deficient atrial cardiomyocytes, action potentialabnormalities occurred due to a decreased SERCA2-mediated sarcoplasmic reticulumcalcium uptake (TBX5 loss- or gain-of-functionmutations have been causally linked to HOS, including CHD and AF as well as cardiacblock (TBX5 enhances the susceptibility to CHD and AF in humans, andunderscore that TBX5 dosage must be precisely regulated to avoid heartdisorders.It might be ascribed to the aberrant cardiovascular genesis thatacblock .Taken cTBX5 variations tovarious cardiovascular malformations, including ASD, VSD, atrioventricular septaldefect, pulmonary stenosis, hypoplastic left ventricle, mitral valve anomaly (TBX5. Given that loss-of-function mutations in multipletranscriptional partners of TBX5 (Notably, previous studies have causally linked This investigation causally links TBX5 loss-of-function mutation to CHD, AF and BAVfor the first time, which highlights the key role of abnormal cardiovasculardevelopment in the pathogenesis of CHD, AF and BAV, implying potential implicationsfor individualized prophylaxis and management of patients with CHD and AF as well asBAV.