Spinal cord injury healing has been shown to be aided by chondroitinase ABC I (cABCI) treatment. The transport of cABCI to target tissues is complicated by the enzyme's thermal instability; however, cABCI may be immobilized on nanosheets to boost stability and improve delivery efficiency. This investigation's goal was to assess the immobilization of cABC I on graphene oxide (GO). for this purpose, GO was produced from graphene using a modified version of Hummer’s process. the immobilization of cABC I on GO was examined using SEM, XRD, and FTIR. The enzymatic activity of cABC I was evaluated in relation to substrate concentration. The enzyme was then surface-adsorption immobilized on GO, and its thermal stability was examined. As compared to the free enzyme, the results showed that the immobilized enzyme had a greater Km and a lower Vmax value. The stability of the enzyme was greatly improved by immobilization at 20, 4, 25, and 37 °C. For example, at 37 °C, the free enzyme retained 5% of its activity after 100 min, while the immobilized one retained 30% of its initial activity. The results showed, As a suitable surface for immobilizing cABC I, GO nano sheets boost the enzyme's stability, improving its capability to support axonal regeneration after CNC damage and guard against fast degradation. P. vulgaris has yielded two isoforms, cABC I and cABC II, which have been recombinantly produced in Escherichia coli1. Both isoforms may break down members of the glycosaminoglycan family, such as chondroitin sulfate, dermatan sulfate, and chondroitin 4-sulfate, however cABC I has a much higher catalytic efficiency than cABC II due to its 997 amino acids and 112.5 kDa molecular mass2. An injury to the central nervous system (CNS), particularly to the spinal cord, results in permanent tissue damage that activates glial cells at the location of the lesion and creates a glial scar3. Proteoglycans, especially chondroitin sulfate proteoglycans, which serve as a considerable barrier to regenerating axons, make up the majority of the glial scar5. The cleavage of chondroitin sulfate proteoglycan chains by the cABC enzyme opens up a possible therapeutic avenue for the treatment of spinal cord injuries and nerve regeneration. In animal models of CNS damage, therapy with cABC improves motor and sensory function, promotes neuronal connection regeneration, and plasticity, according to experimental findings7. The sustained delivery of bioactive cABC is a serious challenge, requiring highly invasive techniques or the implantation of delivery systems directly into the tissue and is a serious obstacle to the practical use of cABC for spinal cord therapy8. cABC has been shown to be a highly labile enzyme that inactivates after few days in solution9, although weeks after the damage, proteoglycan deposition and the development of the glial scar persist. As the activity of cABC also quickly declines at 37 °C, it is required to increase the thermal stability of cABC2. Otherwise, repeated injections over a period of days to weeks or local infusions may result in infection and a body immune system reaction. To boost the thermal stability and activity of cABC, a variety of techniques have been employed, including the use of stabilizing chemicals, mutagenesis, chemical modification, and immobilization. According to reports, glycerol, sorbitol, and trehalose all help to stabilize cABCI10. Cosolvents also slow down the rate of oxidative and proteolytic inactivation of cABCI11. Moreover, the Proteus vulgaris cABCI's activity, stability, and tertiary structure were examined in glycerol- and betaine-based deep eutectic solvents. The results demonstrated that a glycerol and betaine or choline combination might improve the stability and activity of the enzyme12. In cABCI, mutagenesis is crucial for maintaining the stability of the enzyme. In this manner, the enzyme is deactivated and rendered unstable by the substitution of His475 with Ala and Try476 with His and Ala13. The immobilization technique has become a common way to stabilize the enzyme since the enzyme attachment to a carrier limits the unfolding of the enzyme15. At different temperatures , immobilizing cABCI by trapping it within porous silicon nanoparticles has been shown to considerably boost enzyme stability16. Moreover, cABCI has been immobilized using magnetite nanoparticles, and the findings indicated that the immobilized cABCI's storage stability at low temperatures was greatly improved17. However, due to potential issues with the nanoparticle crossing the structural complexity of the CNS and the blood-spinal cord barrier, careful selection of a specific nanoparticle type is a critical issue. Due to its outstanding qualities, including controllable form and size, cheap cost, and relatively good biocompatibility, graphene derivatives have drawn a lot of attention and research interest in this area19.Monolayer graphene, ultrathin graphite, few-layer graphene, graphene nano sheets, and graphene or graphene oxide (GO) are examples of graphene derivatives. GO is a polar derivative of graphene that is water soluble, and its biocompatibility, large specific surface area, excellent solubility, and low toxicity permits extensive usage in biology and medicine, and it is a suitable host for enzyme immobilization20. Via van der Waals, ionic, and—stacking interactions, the many oxygen-containing groups on GO promote enzyme attachment21. It has been shown that enzyme immobilization on GO sheets is possible without the need for further surface modification or cross-linking agents22. Successful lipase immobilization has also been accomplished on the magnetically separated Fe3O4/GO surface23. Although there have been many advances in enzyme immobilization, designing an immobilization platform for biocatalysis involving nanoparticles that can immobilize the cABCI and enhance its stability and activity is still difficult due to the protein damage caused by the frequently "harsh" reaction conditions required for these reactions25. It has been reported that employing the graphene oxide in scaffold compounds can play a role in regeneration of neuronal tissues26. Also, considering the role of the cABCI enzyme in the neuronal connection regeneration, the immobilization of this enzyme on graphene oxide can be important in the clinical application.The enzyme known as chondroitinase ABC  is responsible for cleaving the chondroitin sulfate’s 1,4-glycosidic bond. As far as we are aware, there has never been a report on the use of GO as a nanocarrier for cABCI. As a result, in the current work, GO was produced utilizing a modified version of Hummer's approach and cABC I enzyme was immobilized on GO through direct (electrostatic) interaction. Finally, the kinetic parameters and thermal stability of the free and immobilized enzyme were investigated.2SO4, NaNO3, NaOH, H2O2 (30%), K3Fe(CN)6, KMnO4, and chondroitin 4-sulfate (USA). All of the chemicals were analytical reagent grades and were used directly out of the package. In this investigation, Ni–NTA agarose from Qiagen in the United States and Isopropyl-β-d-thiogalactopyranoside (IPTG) from Takara in Japan were both utilized. The studies employed substances with an analytical purity grade.From Sigma-Aldrich, we obtained graphite powder, H28. For this purpose, 0.5 g of natural graphite was dissolved in 23 mL of sulfuric acid and 0.5 g of sodium nitrate in an ice bath while being constantly stirred. In order to avoid explosion and overheating, potassium permanganate (3.0 g) was progressively added to the acid mixture while stirring and maintaining the temperature below 20 °C. After 12 h of stirring at 35 °C, the liquid was diluted by adding 500 cc of water while vigorously swirling. The suspension was then treated with a 30% H2O2 solution to verify that the reaction with the KMnO4 was complete (5 ml). After filtering, drying, and washing the resultant mixture with HCl and water, respectively, graphene oxide sheets were produced.GO was created with a modified version of Hummer's approach and was based on earlier researchE. coli host cells, cABC I was expressed using the same technique as described by Daneshjou et al.25. By using SDS-PAGE analysis, the purity and molecular mass were evaluated. The Bradford technique was used to quantify the protein content at 595 nm using bovine serum albumin as the standard29.In BL21 g for 30 min after being incubated on the stirrer at 4 °C for 12 h and being rinsed three times with phosphate buffer. The pellet resuspension and the evaluation of enzyme stability and activity both employed the phosphate buffer.cABC I enzyme solution (concentration of 2.5 mg/mL) was applied to 0.04 mg/ml of GO in order to physically adsorb the enzyme onto the substance. After that, the mixture was put in a sonication bath  to uniformly and thoroughly disperse the nanoparticles throughout the enzyme solution. The resultant solution was centrifuged at 15,000×11. By measuring the product spectrophotometrically at 232 nm in 50 mM phosphate buffer (pH 6.8) at 25 °C, the activity of cABC I was investigated. In a nutshell, 20 μl of pure enzyme were mixed with 290 μl solution containing various concentrations of chondroitin-4-sulfate in 50 mM phosphate buffer. Chondroitin-4-sulfate (C4S) was the substrate. The product's molar extinction coefficient (ε), which is 3800 M−1 cm−1, was utilized to calculate the activity. A unit (U) of cABC I activity is the amount of the enzyme required to convert 1 μmol of chondroitin-4-sulfate into unsaturated disaccharides per minute under the test conditions. Using the Prism version 5.0 software  nonlinear regression function, data were fitted to the Michaelis–Menten equation. The standard deviations were all within 5% after three runs of each experiment.The test for enzymatic activity was carried out in accordance with other investigationscABCI lyase stability was analyzed by incubating the free and immobilized enzyme at − 20, 4, 25 and 37 °C for 3 h, and after various time intervals, the samples were assayed for their residual activity at 25 °C as mentioned above. The experiments were carried out at least in triplicate and the standard deviations has been reported as ± 5%.An image of a GO was obtained by atomic force microscopy (AFM) (Autoprobe CP microscopy). Scanning electron microscopy (SEM), using a Philips XL30 scanning electron microscope, was used to study the morphology of GO and immobilized cABC I. (Netherlands). GO's crystalline makeup and phase purity were examined Using a Panalytical X Pert Pro XRD with Cu Ka 1.5406 A° radiation. The X-ray diffractometer was used to implement the XRD analysis. Raman spectroscopy was performed on GO using an XloRA Plus device . GO's molecular structure was studied using an Fourier Transform Infrared Spectroscopy (FTIR) spectrometer both before and after cABC I immobilization (Varian Inc. 7000e).E. coli host cells. SDS-PAGE analysis determined the molecular mass and purity of cABC I, showing  as the standard.In the current investigation, an N-terminal 6 × His tag was used to express cABC I in BL21 31, while the enzyme with a pI of 8.5 is positively charged16. Therefore, interaction can be visualized as an electrostatic attraction. Eventually, GO was added to the enzyme in phosphate buffer (pH6.8) and swirled for 12 h at 4 °C. To better balance the relationship between enzyme loading and the activity of the immobilized enzyme, a number of enzyme concentrations were examined for their effectiveness in imprisoning within the GO surface. The activity initially increased as the enzyme concentration rose; but, at concentrations greater than 0.2 mg/mL (where about 40 g of enzyme were loaded per mg of GO), the activity remained constant. As seen in Fig. 16.The enzyme was immobilized using a nano substrate made of graphene oxide powder. First, the three-dimensional structure of the enzyme was examined using the YASARA bioinformatics tool Version 20.12.24). Due to the abundance of aspartate and glutamate in the enzyme structure, it was predicted that the ions in graphene oxide are responsible for trapping the carboxylic acid groups from aspartate residues (Asp and glutamate (Glu)) in the side chains of amino acids. The immobilization procedure thus occurred by using the strategy of direct contact of chondroitinase enzyme with GO. It is worth noting that, Enzyme was immobilized on graphene oxide using electrostatic interactions. Zeta analysis shown that graphene oxide has a negatively charge (− 23.9), which was consistent with the studies of Cao et al.0.12.24. 34. Alam et al. found GO lamellar structures in SEM micrographs35. SEM analysis revealed that the graphene nano sheets had thin aggregates that were uniformly distributed and had smaller pores. The GO sheets hung straight and did not budge. SEM images of immobilized cABC I on GO  and scanning electron microscope (SEM) images of graphene oxide (GO) samples are shown in Fig.  GO Fig. B,C demon40. Another diffraction peak observed at 25°,indicates the distance between the graphene layers, which is very consistent with the reported in41 and shows the good order of graphene interlayer spacing40. The diffraction peak at 25° disappears completely when GO is dried42. These phenomenan are similar to our results reported in the research of Yan et al. and Khakpour et al., in which for GO synthesized by the Hummers method, the reemergence of diffraction lines is C(002) at 25°, revealing the typical structure of graphite and multiple layers of graphene43 as well as the polycrystalline nature of graphene that has also been reported in a recent study44. The XRD spectrum of immobilized cABC I on GO is seen in Fig. 45.The XRD spectrum of graphene oxide in Fig. −1 (D band), 1590 cm−1 (G band), which ensure lattice distortions and 2690 cm−1 (2D band) are related to the set of graphene layers46. in the study of Strankowski et al. performed on GO, the most important signals can be seen as strong D and G bands48. The intensity of the D band depends on the number of oxygen atoms on the GO surface49. The results indicate that the synthesis of graphene oxide is consistent with the studies conducted on graphene oxide51. FTIR measurements of graphene oxide (GO) and immobilized cABC I on GO were conducted to confirm the immobilization of cABC I on GO  and immobilized enzyme (no imidazole). At all temperatures studied, the immobilized chondroitinase on GO became more stable in contrast to the free enzyme , it can be concluded that, at 4, 25 and 37 °C, the inactivity rate of immobilized enzyme on GO is slower than immobilized enzyme on porous silicon. In addition, the importance of employing GO in this study is because Graphite Oxide is more biocompatible and more stable than porous silicon. The porous silicon structure is unstable and disintegrates faster than graphene oxide. As a result, graphite oxide is a better option for drug carriers. In general, graphene oxide has a long biodegradation period and may be kept longer with continuous drug release after injection. As a result, drug release can be controlled for a long time, and complications from repeated injections and side effects from high doses can be avoided. It was previously reported that, lipase and dehydrogenase were successfully stabilized by the immobilization on GO54.After enzyme immobilization, some characteristics of the enzyme molecule, such as its catalytic activity or thermal stability, differ from those of its counterpart in solution67. Although Chondroitinase enzyme is also employed for the treatment of spinal lesions, it has low thermal stability and for therefore, it requires repeated injections in the lesion site. However, by immobilizing this enzyme on the GO substrate, the stability of this functional enzyme has increased. Considering the substrate (GO) itself plays a role in the proliferation of neural cells, a promising approach to repair spinal cord injuries can be introduced in the future.In addition to the afromentioned advantages, it has been widely proven that GO plays a very important role in the proliferation and differentiation of neural cellsGO was successfully synthesized in the current work utilizing a modified version of Hummer's technique. XRD, FTIR and SEM were used to characterized graphene oxide and enzyme immobilization. We have shown that immobilized cABC I activity on GO followed Michaelis–Menten kinetics, and had lower Vmax and Higher Km than free enzyme. The findings also indicated that graphene oxide can work as a heat-labile chondroitinase carrier. Because after immobilization on graphene oxide, the immobilized enzyme was considerably more stable . As a result, the GO could be an effective stabilizing medication delivery system. While further experimental testing is required, the findings of this study are highly encouraging and provide an intriguing strategy for future spinal cord injury recovery .Supplementary Information.


The advancement ofoptical technology demands the development ofchiral nanostructures with a strong dissymmetry of optical response.Here, we comprehensively analyze the chiral optical properties ofcircular twisted graphene nanostrips, with a particular emphasis onthe case of a Möbius graphene nanostrip. We use the methodof coordinate transformation to analytically model the electronicstructure and optical spectra of the nanostrips, while employing thecyclic boundary conditions to account for their topology. It is foundthat the dissymmetry factors of twisted graphene nanostrips can reach0.01, exceeding the typical dissymmetry factors of small chiral moleculesby 1–2 orders of magnitude. The results of this work thus demonstratethat twisted graphene nanostrips of Möbius and similar geometriesare highly promising nanostructures for chiral optical applications. Given aright length of the nanoribbon, its opposite ends are rotated by 180°with respect to each other, thus resulting in a Möbius nanostrip.The spectroscopic studies revealed strong optical activity of thefabricated Möbius graphene nanostrips, with the dissymmetryfactors ranging between 10–3 and 10–2. The shape of the Möbius nanostrip is not only chiral butalso topologically nontrivial due to it being a nonorientable surface.33 This feature was shown to give rise to topologicallyprotected edge states and the quantum spin Hall effect in Möbiusstructures.35 Notably, Möbius graphenenanostrips with zigzag edges also feature a spin-polarized groundstate,36 which is ferromagnetic (unlikein plain nanoribbons) because such nanostrips have effectively a singleedge.37 It is natural to wonder how thetopologically nontrivial geometry of Möbius graphene nanostripsaffects their optical activity, which has not been assessed to date.Recently, Segawa et al.synthesized a new chiral graphene nanostructure:graphene nanobelts in the form of a Möbius strip.In this Letter, we comprehensively analyze the optical activityof Möbius graphene nanostrips. In order to account for thenontrivial geometry of the Möbius strip, we introduce a curvilinearspace in which a twisted nanostrip transforms into a plain graphenenanoribbon characterized by modified cyclic boundary conditions. Absorptionof circularly polarized radiation by the nanostrip is described inthe curvilinear coordinates using a topologically transformed light–matterinteraction operator. We establish the selection rules of opticallyactive transitions in twisted graphene nanostrips and confirm thatthe Möbius nanostrip indeed features circular dichroism dueto its chiral shape. We further analyze twisted graphene nanostripsof other dimensions and geometries in search of the structures withthe strongest dissymmetry of optical absorption. It is found thatthe dissymmetry factors of twisted graphene nanostrips can reach values∼0.01, which makes these chiral nanostructures a highly attractivematerial for chiral nanophotonics.L, which is made of atoms arrangedin the armchair configuration,and denote the maximum number of atoms across the nanoribbon’swidth as n. n = 5and the orientation of the Cartesian axes  associated with its surface. A twistedcircular nanostrip is obtained by twisting a plane nanoribbon m times along its x-axis and connectingthe opposite ends. Without loss of generality, we focus on three typesof twisted nanoribbons shown in m = 0, a Möbius nanostrip with m = 1, and a doubly twisted nanostrip with m = 2. The Cartesian coordinates  of atoms in a twisted circularnanostrip are expressed through the Cartesian coordinates  of atoms in theplane nanoribbon asL/(2π)is the radius of the circular nanostrip and χ = ±1distinguishes between the twist directions.Consider a plane graphenenanoribbon of length x, y, z), in which the nanostrip is plainand has simple electronic structure, yet the light–matter interactionis modified by the inverse coordinate transformationY/X).The interaction of a twisted nanostrip with light can be convenientlydescribed using the local coordinates , we find the wave functions ψμk and energies Eμk of the pz electrons. The wave functions are the Bloch waves of the formuμk is the periodic Blochamplitude, μ labels energy bands,and k is the electron wavenumber in the x-direction. This wavenumber determines the local velocity of electronson the surface of the nanostrip as well as the projection of theirangular momentum on the Cartesian Z-axis of the circularnanostrip as ⟨μk|LZ|μk⟩ = ℏρk.By solving the eigensystemproblem for the standard tight-bindingHamiltonian of a graphene nanoribbon,k  is determined by the continuity of the wave functionat the connected ends of the nanoribbon. For even m, which describe the nanostrips of trivial topology, the cross sectionof the nanostrip is oriented the same way at the two ends, as demonstratedin LZ = ℏnx,where nx ∈ Z. For odd m, corresponding to the nontrivialtopology, the cross section of the nanostrip is rotated by mπ upon the twist, as shown in The quantization of the electronwavenumber z because they originate fromthe pz-orbitals of carbonatoms, uμk = −uμk. Therefore, the quantization rule followingfrom y. One can see in uμk is an odd function of y, the wave functionis continuous at the connected ends of the nanostrip, uμk = uμk, and the electron wavenumber k is quantized according to y changes sign upon the rotation by mπ, uμk = −uμk, andthe quantization rule becomesLZ = ℏ(nx + 1/2). We thus obtain that in Möbiusgraphenenanostrips, the Z-projection of angular momentumcan assume semi-integer values of ℏ, contraryto the systems of trivial geometry.Note that the Bloch amplitudesare odd functions of L with respectto the odd states. This prevents vertical transitions between thebands of opposite parities, resulting in the red shift of the absorptionspectrum noticeable for small L.C = 32π3Nc/(3ℏc), Nc is thevolumetric concentration of the nanostrips, the summation is performedover all occupied states |μk⟩ and allunoccupied states Dμk;νk′ = |⟨μk|d|νk′⟩|2 is the transitionprobability, Rμk;νk′ = Im⟨μk|d|νk′⟩·⟨νk′|m|μk⟩ is the rotatory strength of the transition,41d and m are the electric and magneticdipole moment operators, and f(ℏω) is the spectral line shape function.Absorptionand circular dichroism (CD) spectra of randomly orientedcircular graphene nanostrips are given byR =  and P = −iℏ, as42 but not for armchair graphene nanostrips analyzed in this study.On the other hand, armchair-edge Möbius nanostrips made ofmaterials other than graphene may potentially feature topologicallyprotected states due to non-negligible spin–orbit coupling.33 Matrix elements of the dipole moments are evaluatedin the Supporting Information through theconversion of R and P into the curvilinearcoordinates  and subsequent integration of the modified operators over the Blochstates of the original flat nanoribbon. The result is presented in The matrix elementsof the dipole moments are expressed throughthe coordinate and momentum operators in the original Cartesian coordinates, m. Absorption oflight by circular nanostrips with m = 0 occurs viavertical transitions with Δk = k′ – k = 0 (ΔLZ = 0) and diagonal transitions withΔk = ± 2π/L (ΔLZ = ± ℏ). Möbius nanostrips with m = 1 absorb viasix diagonal transitions accompanied by the transfer of momentum Δk = ± π/L, ±2π/L, and ±3π/L, correspondingto ΔLZ = ±ℏ/2, ±ℏ, and ±3ℏ/2. Finally, doubly twisted nanostrips with m = 2 absorb via vertical transitions with Δk = 0 (ΔLZ = 0) and diagonal transitions with Δk = ±2π/L (ΔLZ = ±ℏ) and ±4π/L (ΔLZ = ±2ℏ). While absorption occurs upon both x- and y-polarized transitions, the optical activityis observed only for transitions which are polarized in the y-direction, because chirality of the nanostrip stems fromits rotation in the cross section, i.e., the yz-planein the local coordinates. This means that the CD signal is producedonly by transitions between the bands of opposite parities.The selection rules ofinterband transitions in circular graphenenanostrips are seen to vary significantly depending on the numberof the nanoribbon’s twists g-factor) of optically active transitions43gμk;νk′ = 4Rμk;νk′/Dμk;νk′ and the g-factor spectrum g(ℏω) = 2CD(ℏω)/A(ℏω). The typical g-factors of small chiral molecules and semiconductor nanocrystalsrange between ±(10–3–10–2),45 while the maximum values of g-factor (±2)describe the situation when the system totally transmits one circularpolarization. The data of g-factors of opticallyactive transitions in circular graphene nanostrips scale linearlywith the nanostrip’s radius as gμk;νk′ ∝± qρ, where q = ω/c is the wavenumber of light.The relative strength of the optical activity of circular graphenenanostrips can be conveniently characterized by the dissymmetry factor anddoubly twisted (m = 2) nanostrips with n = 5 and L = 85 nm are shown in x-polarized transitionsoccur over the entire spectrum, whereas the y-polarizedtransitions occur only in the visible range and above. The absenceof the y-polarized transitions in the infrared rangeis due to the fact that the two energy bands near the Fermi levelhave the same parities with respect to the y axis = ψ at the connected ends of the nanostrip composed of an integer numberof unit cells. The density plots in g(ℏω)can reach 10–2, which is close to the experimentalvalue for Möbius graphene nanobelts31 and exceeds the typical values for small chiral molecules and quantumdots by 1–2 orders of magnitude.47 Chiral molecular cylinders, morphologically similarto circular graphene nanostrips, can feature even larger dissymmetryfactors of the order of 10–1, though these valuesare highly sensitive to structural fluctuations.48 In agreement with the above theoretical prediction, g(ℏω) in L. This suggests that smaller graphene nanoribbons are betterfor chiroptical applications. Confirming this, Möbius graphenenanobelts with unusually large dissymmetry factors, fabricated bySegawa et al., were indeed characterized by the radius of only a fewnanometers.31n =3 and n = 5 feature pronounced CD peaks, the spectralpositions of which do not change with the length of the nanostrip.For n = 3 there is a strong signal at 3.4 eV anda weaker signal of the same sign at 3.9 eV, whereas for n = 5 there are two equally pronounced peaks of different signs at1.7 and 2.7 eV. The dissymmetry factors of the doubly twisted nanostripsare seen to be notably larger than those of the Möbius nanostripsof the same dimensions. The difference between m =1 and m = 2 is the least prominent for the widestnanoribbons with n = 7, yet there is more interferencein the optical spectra, with two weak peaks of opposite signs identifiableat 1.5 and 3.2 eV. To investigate the effect of larger widths on chiraloptical properties of graphene nanostrips, in Figure S6 we show dissymmetry factors for n = 9, 11, and 13. This figure reveals similar trends in the opticalspectra of wider graphene nanostrips, including relatively large g-factors of the order of 0.01. Overall, our results demonstratethat short twisted graphene nanostrips with n = 5exhibit the strongest dissymmetry of optical response. This may beattributed to the fact that the armchair graphene nanoribbons with n = 3p + 2  generally have richer electronic properties than other membersof this family of carbon nanostructures.38The two narrower graphene nanostrips with In future studies, the analytical model developed in thisworkcan be extended to include various additional atoms, such as oxygen,to provide more accurate modeling of circular graphene nanostripssynthesized from organic precursors. Our theoretical approach canbe also employed to analyze Möbius graphene nanostrips withzigzag edges, whose nontrivial topological and magnetic propertiescan have significant implications for their chiral optical response.Finally, the coordinate transformation method can potentially describeMöbius nanostrips made of other two-dimensional semiconductors,e.g., transition metal dichalcogenides or hexagonal boron nitride,thus opening up new perspectives for studying the influence of nontrivialtopology on different materials.pz-electrons. Uponthe coordinate transformation, the chirality of the nanostrip is transferredto the curvature of the space, modifying the light–matter interactionoperator. The nontrivial topology of the Möbius nanostrip isaccounted for by the cyclic boundary conditions which distinguishbetween the energy bands of different parities. Further analyticalcalculations revealed that circular dichroism in twisted graphenenanostrips can only be observed upon transitions polarized acrossthe nanostrip, whereas absorption is formed by the transitions polarizedboth across and along the axis of the nanoribbon. We further analyzedthe dependence of the dissymmetry factor on the length and width ofthe Möbius and doubly twisted nanostrips. Among the analyzedstructures, doubly twisted nanostrips of width n =5 and smaller lengths were found to feature the strongest dissymmetryof optical response. Overall, the dissymmetry factors of small twistedgraphene nanostrips can reach values ∼0.01, highlighting Möbiusgraphene nanostrips as highly promising nanostructures for applicationsin chiral nanophotonics.To conclude, we have modeledoptical activity of Möbiusgraphene nanostrips and similar graphene nanostructures with a differentnumber of twists. In order to calculate electronic properties of thesechiral nanostructures, we developed a coordinate transformation thatturns a twisted graphene nanostrip into a plane graphene nanoribbon,whose electronic properties are obtained using the tight-binding Hamiltonianfor


Frontotemporal lobar degeneration (FTLD) belongs to a heterogeneous group of highly complex neurodegenerative diseases and represents the second cause of presenile dementia in individuals under 65. Frontotemporal-TDP is a subgroup of frontotemporal dementia characterized by the aggregation of abnormal protein deposits, predominantly transactive response DNA-binding protein 43 (TDP-43), in the frontal and temporal brain regions. These deposits lead to progressive degeneration of neurons resulting in cognitive and behavioral impairments. Limbic age-related encephalopathy (LATE) pertains to age-related cognitive decline primarily affecting the limbic system, which is crucial for memory, emotions, and learning. However, distinct, emerging research suggests a potential overlap in pathogenic processes, with some cases of limbic encephalopathy displaying TDP-43 pathology. Genetic factors play a pivotal role in both disorders. Mutations in various genes, such as progranulin (GRN) and chromosome 9 open reading frame 72 (C9orf72), have been identified as causative in frontotemporal-TDP. Similarly, specific genetic variants have been associated with an increased risk of developing LATE. Understanding these genetic links provides crucial insights into disease mechanisms and the potential for targeted therapies. The TDP-43 name comes from its discovery when it was identified as a human trans-activation response element (TAR) binding protein . Also, TDescribed initially as a DNA-binding protein, TDP-43 is now known to regulate nucleic acid metabolism through different mechanisms, primarily:In 2001, a study highlighted the participation of TDP-43 in splicing exon 9 of the pre-mRNA encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene ,6. TDP-4Less than 5% of the RNA produced inside cells codes for proteins. It is now known that the remaining 95%, called non-coding RNAs (ncRNAs), play an essential role in regulating gene expression within the CNS, both under physiological and pathological conditions . Micro-Rin vitro neuronal differentiation, TDP-43 was shown to modify Drosha stability, suggesting a potential impact on all; in addition, increased Dicer expression was also observed upon TDP-43 repression in a human neuronal cell line model [The hypothesis that TDP-43 could participate in the regulation of miRNAs was raised by its presence within the Drosha complex and its association at the level of perichromatin fibrils, where pri-miRNAs are born . In agrene model . TogetheAmong the other ncRNAs, long non-coding RNAs (lncRNAs) are involved in numerous cellular mechanisms, particularly at the transcriptional and post-transcriptional levels. Recently, the deregulation of these lncRNAs has been demonstrated, particularly in pathologies related to aging, including neurodegenerative diseases . In thisTransactive response DNA-binding protein 43 was first identified as a regulator of the stability of the mRNA encoding neurofilament lumen (NF-L) and histone deacetylase HDAC6 . StudiesWithin polarized cells such as neurons, transporting mRNAs to axons and dendrites is a fundamental process in maintaining neuronal activity and synaptic plasticity . EvidencFurthermore, the presence of TDP-43 mutations within these iPSCs induces impaired anterograde granule transport, indicating a direct role for TDP-43 in axonal mRNA transport . TDP-43 Drosophila has described a regulation of the translation of the Futsch/22C10 microtubule-associated proteins 1B (MAP1B) and Rac1 proteins by TDP-43 [One of the emerging functions of TDP-43, directly related to its role in mRNA transport to distal regions, is the regulation of local mRNA translation. The first proof of their involvement was highlighted in 2008 when a team observed a TDP-43 translocation at the dendrite level after potassium chloride stimulation within rat hippocampal neurons . Furthery TDP-43 ,29. Thesy TDP-43   or β-amyloid protein experimental model (APPnfv).Type-A FTLD-TDP is distinguished by the presence of numerous DNs and cytoplasmatic aggregates at the level of the second layer of the neocortex. A moderate number of NCI granular inclusions are also present within the dentate gyrus . In addiType-B FTLD-TDP cases show NCI lesions in the outer and deep cortical layers with relatively little DN or NII . Immunohhippocampus. The presence of NII and GCI is sporadic. Type C is the most common pathologic subtype in patients with semantic variant primary progressive aphasia (svPPA).Type-C FTLD-TDP is characterized by long neuritic lesions, mainly in the outer layer of the cortex . Some NCbasal ganglia, thalamus, hippocampus, cerebellum, etc.) can also be heterogeneously affected. This histopathological presentation is rare since it is only found in cases carrying a mutation in the valosin-containing protein (VCP) gene.This pathologic subtype’s hallmark is abundant lentiform NII lesions and short neuritic lesions in the neocortex . VariousThe etiology of most cases of FTLD and ALS is still unclear; yet, these cases are related to each other by the presence of TDP-43 pathology characterized by cytoplasmic delocalization, presence of truncated forms, aggregation, and post-translational modifications of this protein .In brain tissue from patients with FTLD and ALS, TDP-43 hyperphosphorylation and ubiquitination represent one of the main features of the pathology. Indeed, these two modifications preferentially label cleaved, aggregated, and insoluble forms of TDP-43 ,36. ImmuCaenorhabditis elegans.There are 64 potentially phosphorylatable sites on TDP-43, including 41 serine residues, 15 threonine residues, and 8 tyrosine residues. Among these sites, 29 are phosphorylated by CK1 protein kinase (casein kinase 1) . ExperimSimilarly, inhibition of protein kinases involved in TDP-43 phosphorylation, such as cell division cycle-related protein kinase 7 (CDC7) or tau tubulin kinase (TTBK), also reduces the toxicity induced by TDP-43 phosphorylation ,46. TherUbiquitination is another mitochondrial permeability transition (MPT) that affects TDP-43. Indeed, TDP-43 is modified by lysine-linked polyubiquitin chains 48 or 63 . FurtherDrosophila, despite the retention of ubiquitin chains [In contrast, Parkin-mediated ubiquitination induces the translocation of TDP-43 into the cytosolic compartment and may enhance its degradation . Similarn chains .in vitro study indicates that de novo CTF production is insufficient to induce TDP-43 aggregation, and a second event is required for this [TDP-43 CTF between 20 and 25 kDa constitutes one of the biochemical signatures of FTLD-TDP. TDP-43 proteolysis is an event mediated by several mechanisms, including mutations of TDP-43 in its C-terminal region , cellulafor this . It is ein vivo; however, it must be emphasized that these studies do not directly address the nucleocytoplasmic transport of TDP-43, but other mechanisms, such as autophagy. The question arises whether it is the native or malformed form that is responsible for the toxicity induced by TDP-43 in the cytoplasm.Loss of nuclear localization associated with aggregation in the cytoplasm or the neurites is one of the hallmarks of FTLD-TDP. Although most TDP-43 inclusions are extranuclear, we have seen that some are located in the nucleus (NII). TDP-43 typically localizes to euchromatic regions of the nucleoplasm, especially perichromatin fibrils, where transcription and splicing occur . TherefoIntraneuronal aggregates of TDP-43 characterize FTLD-TDP. The presence of protein aggregates is a phenomenon found in many neurodegenerative diseases. However, these inclusions’ protective or harmful role is still highly debated. Indeed, regarding the correlation between aggregation and all the pathological modifications found , it is not easy to designate aggregation as the primary pathophysiological mechanism. TDP-43 is no exception to this observation, and many studies point to differential roles for TDP-43 aggregation in pathology. Therefore, although transgenic animal models expressing the full-length form of TDP-43 show some TDP-43 inclusions, these are relatively rare and do not correlate with the phenomenon of neurodegeneration .Prevention of TDP-43 inclusion formation does not appear to reduce toxicity in a cell model. Nevertheless, it is essential to underline that these studies are based on the presence of visible aggregates of TDP-43. Still, these aggregates may be only the final step involving different TDP-43 species ranging from simple monomers to mature aggregates via oligomers. This hypothesis is supported by studies indicating that the complete form of TDP-43 can form oligomeric structures with amyloid properties. These oligomers are found both in the brain of FTLD-Parkinson disease dementia (PDD) patients and in transgenic mice, and can induce the aggregation of other amyloid peptides such as Aβ .Most of the TDP-43 mutations found in ALS target the C-terminal part.in vivo [CTFs, which essentially contain this prion domain, appear to promote aggregation in vivo .The spread of TDP-43 represents an emerging mechanism in the pathophysiology of FTLD-TDP. This hypothesis is based in particular on the presence of a prion-like domain in the C-terminal part of TDP-43 , but alsin vivo evidence, several studies suggest that TDP-43 can propagate and induce aggregation of native TDP-43 proteins. First, recombinant TDP-43 aggregates, previously formed in vitro, can induce the accumulation of endogenous TDP-43 proteins within HEK293T cells [in vivo , the GRN progranulin gene is located on chromosome 17 at position q21. This gene encodes a 68.5 kDa protein composed of 593 cysteine-rich amino acids. The full-length form of the protein contains 7.5 well-conserved domains, each consisting of a repeat of 12 cysteine motifs  separated by junction regions. Once in the extracellular medium, various proteases can cleave progranulin at the level of these regions to generate fragments ranging from 6 to 25 kDa, called granulins .Progranulin is a secreted protein expressed in many tissues and cell types throughout the body. In the brain, its expression is low during the early stages of development and increases with age. Regarding its distribution in different cell types, progranulin is found mainly in neurons and microglia, although it may be present in small amounts in astrocytes and ependymocytes . Its expThe penetrance of mutations in the GRN gene is incomplete, so knowing the factors that regulate the expression of this gene is essential from a therapeutic point of view. A genome-wide association study (GWAS) of FTLD-TDP cases identified the gene encoding TMEM106B as a modulator of pathology in patients with or without a GRN gene mutation. The genetic variability of TMEM106B notably affects the penetrance of the disease in cases carrying a mutation in the GRN gene, but also the age of onset of the first symptoms . In addiGRN, MAPT, C9ORF72, TARDBP, and VCP, although other genes not yet properly identified for FTLD-TDP but that may be related are HNRNPA2B1, SQSTM1, UBQLN2, and TREM2 [In summary, the genes that are associated with FTLD-TDP are nd TREM2 , as can Progranulin involves many physiological and pathological mechanisms, including cell proliferation, inflammation, and metabolic diseases. On the other hand, its role in the brain remains largely unknown despite intense research since the discovery of its involvement in FTLD in 2006.hippocampus) once again supports the role of progranulin in neurite outgrowth: given the low expression of progranulin during embryonic development, its role in neuritic growth is probably more associated with synaptic plasticity in the adult brain  has been shown to promote neuronal survival and neurite outgrowth in rat primary neuronal cultures ; furtherlt brain .Evidence suggests that progranulin can modulate synaptic biology. Thus, an increase in the number of synaptic vesicles and their probability of secretion has been observed in cultures of rat hippocampal neurons deficient in progranulin. These results were confirmed in the brains of FTLD patients with a mutation in the GRN gene . AlthougDue to the overexpression of progranulin in pathological conditions, several studies have focused on its role in microglia. Thus, a study observed an excessive secretion of cytokines in primary cultures whose microglial progranulin expression is repressed . Furtherin vitro observe proteolysis and aggregation of TDP-43 during progranulin depletion [Transactive response DNA-binding protein pathology is characterized by phosphorylated forms of the complete TDP-43 protein and C-terminal fragments in the brain tissue of FTLD patients. The relationship between progranulin haploinsufficiency and the development of this pathology is unknown. Although several studies epletion ,101,102,epletion . Progranepletion ,105, othepletion .The onset of symptoms is later than for MAPT mutations, with a mean between 59 and 65 years. However, the duration of the evolution is similar, with an average of 9 years. Singularly, there is significant heterogeneity in the clinical symptoms observed in patients, even in individuals with identical mutations or belonging to the same family. Most cases present with clinical signs of bvFTD or svPPA, often accompanied by Parkinsonian syndrome. An association with ALS is found very rarely . The maistriatum [hippocampus [Severe cortical atrophy and parietal lobe involvement are also found in these patients. From a neuropathological point of view, TDP-43 type-A inclusions are mainly found. Inclusions can also appear as NII and are primarily located in the frontal cortex and striatum . Dystroppocampus ,111. FurIn 2011, a GGGGCC hexanucleotide repeat was identified in a non-coding region of the C9ORF72 gene as the major common genetic cause of FTLD and ALS . In factThree hypotheses are proposed to explain hexanucleotide repeats’ pathological mode of action.The presence of many repeats could cause a reduction in the expression of the C9ORF72 gene leading to a loss of its physiological function. It would seem that the expression of the C9ORF72 gene is reduced in patients carrying this type of mutation .It has been shown that brain and spinal cord tissue from FTLD/ALS patients is not distinguished by the presence of nuclear foci composed of GGGGCC RNA but also GGCCCC antisense RNA ,116, simDrosophila demonstrate the toxicity of these DPRs [The transcripts (sense and antisense) produced from the repeated sequences would be the target of an unconventional translation mechanism that does not depend on the presence of an ATG codon. This mechanism, called translation-associated repeat not initiated by ATG (RAN), would be responsible for the production of a series of dipeptides  . These Dese DPRs ,118 ; however, cognitive disorders (bvFTD and svPPA) have been described in two patients with MND with a mutation in the TARDBP gene . A case Mutations in the VCP gene result in a rare syndrome called inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), affecting skeletal muscle, bone, and the nervous system. Valosin containing protein belongs to the triple-A, ATPase family and plays a role in many cellular processes related to protein homeostasis. In particular, VCP promotes the targeting of ubiquitinated proteins to the proteasome, and the degradation of protein aggregates by autophagy. The VCP gene mutation is also at the origin of an alteration in autophagosome maturation . PatientWe have seen that in most cases of FTLD, the tau or the TDP-43 proteins are aggregated; however, in the remaining 5 to 10% of cases, neither of these two proteins is found during neuropathological analysis. In most cases, the FET family proteins  aggregate within neurons. In rare cases, only proteins linked to the ubiquitin proteasome system (FTLD-UPS) could be identified .First, a dysfunction of the Trn1 protein, resulting from a genetic variation of the TNPO1 gene or a post-translational modification, could decrease the efficiency of FET protein transport to the nucleus. However, the absence of other proteins carried by Trn1 within the aggregates, such as heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), makes this hypothesis less plausible .Post-translational modifications, absent under physiological conditions, can also affect the FET proteins. Interestingly, hypomethylation of arginine residues in a region close to the NLS of FET proteins is observed.In 2009, mutations in the FUS gene were discovered in familial cases of ALS (ALS-FUS) ,125. BecThis would promote the affinity of the FET proteins for Trn1 and lead to the formation of an inseparable complex, thus favoring the formation of cytoplasmic inclusions . AlthougThe subtype aFTLD-U corresponds to a group of FTLDs distinguished by the absence of TDP-43 pathology, without clinical symptoms and characteristics of “atypical” pathologies. The onset of bvFTD is generally early in these cases, with a rapid evolution of psychobehavioral disorders without impairment of language and motor pathways.striatum and sclerosis of the hippocampus. Lesions appear NCI-like, particularly small and compact, and are immunoreactive for FET, ubiquitin, and p62 proteins. Regarding their location, they are found mainly in the frontal and temporal neocortex, the hippocampus, and the striatum [gyrus and pyramidal neurons. Compared with other FTLD-FET cases, aFTLD-U shows less pathology in subcortical regions.In addition to frontotemporal atrophy at a macroscopic level, most cases present with degeneration of the anterior part of the striatum ,124. Surhippocampus, lower motor neurons, and various subcortical regions, such as the basal ganglia and brain stem, are affected. So, NII-like vermiform inclusions, similar to those found in aFLTD-U cases, are also regularly seen, but only in the hippocampus, therefore playing a central role in the pathophysiological process in patients with NIFID [NIFID is a rare neurodegenerative disease presenting early FTD symptoms associated with pyramidal and extrapyramidal motor abnormalities . These cth NIFID .basal ganglia and brainstem tegmentum. In addition to BI lesions, NCIs composed of FET proteins are found in many cortical and subcortical regions, such as NIFID [hippocampus and striatum is more variable, and NII is not observed . The morphology of these lesions is variable , and it is possible to detect them using RNA-directed immunohistochemical tools. Although cases with BIBD show atrophy of the frontotemporal cortex, BI lesions are usually more numerous in subcortical regions, such as the as NIFID . Howeverobserved .Among the FET proteins, FUS represents the protein most involved in the familial forms: in fact, many nonsense mutations can affect the C-terminal region of FUS and are responsible for approximately 4% of familial forms of ALS. These mutations favor the appearance of the pathology by altering the nuclear localization signal of FUS. The reduction in the interaction between FUS and Trn1 that results from these mutations leads to the formation of FUS inclusions in the cytoplasm . Thus, Agyrus. These lesions stain positive for ubiquitin and p62 on immunohistochemistry but negative for tau, TDP-43, or FUS. However, it remains to be clarified whether FTLD-UPS results from accumulating an as-yet unidentified protein or a more general lysosomal and endosomal trafficking dysfunction [Despite the discovery of TDP-43 and FET proteins in most FTLD cases without tau pathology, there are still a small number of cases distinguished only by the presence of inclusions immunoreactive for FTLD-UPS. Among these cases, hereditary cases were associated with a mutation in the gene that encodes a protein involved in forming multivesicular bodies, CHMP2B . The firfunction .Several neurodegenerative disorders can be considered FTLD, such as hereditary spheroid leukoencephalopathy or Nasu–Hakola disease ,138. In A study has shed light on a new neurocognitive pathology linked to the TDP-43 protein. Discovered by a college of experts led by Professor Peter Nelson of the University of Kentucky, this new disease has been dubbed “TDP-43 predominantly limbic age-related encephalopathy” . Accordi-amygdala, a region of the brain involved in managing emotions;Stage 1: toxic clumps are formed in the -hippocampus, which plays an essential role in memory processes;Stage 2: protein aggregates spread inside the -gyrus that controls space–time orientation [Stage 3: they reach the median frontal entation ,107,112 This process is usually classified into three stages:Memory loss, especially concerning recent events;Difficulties in carrying out activities and tasks inherent to daily life;Language disorders;Loss of motivation;Spatiotemporal disorientation;Mood swings;Behavior problems ,94.The symptoms are comparable to those of AD. Regarding the latter, the patients present modalities of expression of the pathology that are often very variable and evolutionary ,63. AmonRecently discovered, TDP-43 age-related predominantly limbic encephalopathy is far from having revealed all its secrets. The mechanisms underlying the pathological accumulation of the TDP-43 protein in the different areas of the brain are still unknown. It is known that the appearance of lesions usually occurs in significantly older adults (around 80 years of age). Unlike Alzheimer’s, a disease that occurs at about 65 to 70 years of age, for some unknown reason, the TAR DNA-binding protein TDP-43 begins to malfunction and attaches to nerve cells, causing their death. These mutations would thus be at the origin of irreversible cytoplasmic accumulations. Therefore, for this protein, we seek to understand the mechanisms that underlie these toxic aggregations and develop therapies adapted to the particularities of the clinical signs generated. This toxic aggregate exists in neurodegenerative pathologies such as ALS ,129.The warning signs of age-related TDP-43 limbic-predominant encephalopathy can take on different aspects. If many do not show a pathological character at an advanced age, their recurrence and repetition should lead to physician consultation. Early detection and the fastest possible treatment are two essential conditions to stop the progression of the pathology. Unfortunately, this encephalopathy is not yet the subject of a marked diagnostic pathway. Most cases of LATE, when detected, are similar to AD ,124.As a new diagnostic entity, LATE remains challenging to differentiate from AD, which shares many commonalities. That is why researchers have estimated that about one third of 85-year-olds diagnosed with the best-known degenerative condition may have TDP-43 age-related predominantly limbic encephalopathy. Physicians currently have very few clinical data to support their diagnostic process, and no known ante-mortem biological biomarkers can reveal the disease with certainty. Therefore, the diagnosis is neuro-pathophysiological and can only be formally confirmed by performing a pathological study of the brain tissue after the patient’s death (a post-mortem diagnosis). But work continues to establish objective diagnostic criteria based on formal medical examinations ,63.amygdala and hippocampus [Medical neuroimaging seems to have a promising future since recent studies have shown that people affected by this particular encephalopathy will present atrophy in the medial temporal lobes and frontal cortex, as well as a particularly damaged pocampus .Thus, regarding the differences between FTLD and LATE, the first corresponds to several anatomopathological entities that have as a common consequence frontal and anterior temporal degeneration. Behavioral FTLD typically has dysexecutive disorders at the forefront with relative preservation of memory and visuospatial domains, while the second is characterized by an isolated amnestic syndrome, which progresses slowly in the absence of other comorbidities, and more rapidly in the presence of Alzheimer’s disease (AD) or other dementia pathology, specially FTLD. In the absence of specific markers, the combination of imaging data  and plasma markers may contribute to the differential diagnosis .The precise pathophysiological mechanisms of frontotemporal-TDP and limbic age-related encephalopathy are not fully understood. Nevertheless, age-related changes in the brain, including protein misfolding and neuroinflammation, are considered contributing factors. These disorders have a complex interplay of genetic and environmental factors in their development and progression. Familial cases of frontotemporal-TDP indicate the involvement of specific gene mutations, while sporadic cases point to potential genetic predisposition and other unidentified triggers. Understanding the pathophysiological and genetic basis for both disorders is crucial for early diagnosis, targeted therapies, and the development of disease-modifying treatments in the future. Further research into these neurocognitive disorders holds promise for enhancing the quality of life for affected individuals and their families.


U), which provides integral information about the system of defects and quality of the material, makes it possible to identify chips with low graphene quality and with inhomogeneously distributed areas of compressive stresses by the type of frequency dependence SU(f).The quality of graphene intended for use in biosensors was assessed on manufactured chips using a set of methods including atomic force microscopy (AFM), Raman spectroscopy, and low-frequency noise investigation. It is shown that local areas of residues on the graphene surface, formed as a result of the interaction of graphene with a photoresist at the initial stage of chip development, led to a spread of chip resistance (R) in the range of 1–10 kOhm and to an increase in the root mean square (RMS) roughness up to 10 times, which can significantly worsen the reproducibility of the parameters of graphene chips for biosensor applications. It was observed that the control of the photoresist residues after photolithography (PLG) using AFM and subsequent additional cleaning reduced the spread of R values in chips to 1–1.6 kOhm and obtained an RMS roughness similar to the roughness in the graphene film before PLG. Monitoring of the spectral density of low-frequency voltage fluctuation (S Unique properties of graphene such as large surface area, high adsorption capacity and mobility, low thermal and 1/f noise characteristics, high elasticity, good charge-transfer ability, and ferromagnetic properties are well known [Graphene is a single or few atoms thick sheet of spll known ,9,10,11.The graphene industry has been developing rapidly in recent years. However, a number of issues remain which need to be addressed to improve technological progress of the graphene-based biosensors . FirstlyIt should be noted that the largest number of stages of graphene processing is typical of biosensor fabrication, including viral biosensors ,7,8,9,10−5 pM [Briefly, the main stages of creating biosensors include the formation of a graphene film, the formation of chips with contact pads (graphene resistors or transistor) by photolithography (PLG), controlled treatment  of the graphene surface in chips, immobilization (attachment) of biomolecules (antibodies), and the implementation of an antibody–antigen reaction ,6,7,8,9.−5 pM . However−5 pM . It is n−5 pM .As was shown earlier, the non-reproducibility of graphene properties in chips and films can occur at any stage of chip manufacturing ,12. The It is shown that the photoresist interacts with graphene creating local regions with resist residues (LRRs) not completely removed from the graphene surface ,19,20,21The presence of LRRs impairs graphene morphology, enhances the heterogeneous deformation distribution and non-reproducibility of biosensor parameters even before graphene functionalization, antibody immobilization, and antigen (virus) detection ,17,18,19One of the methods to resolve the problem is to use a protective  layer between the graphene film and a photoresist to limit the interaction between them ,20,21. IIn this work, we report on the quality assessment of processed graphene chips, obtained by traditional PLG and PLG with additional LRRs cleaning, and on the reproducibility of their parameters for biosensor applications. Additional LRR cleaning is based on the use of piranha solution, which is widely used in microelectronics and in silicon technology to remove organic contaminants, but was not used in graphene PLG, according to our data. We assess the quality of graphene in chips using traditional AFM ,22 and RIt is very important that the LFN method  integralFluctuations in population levels form a tail of the density of states near the boundaries of the conduction and the valence band. These fluctuations can be caused by various imperfections of the crystal lattice, including clusters, extended defects, local stresses, inhomogeneities of composition, and non-ohmic contacts.2·V−1·s−1) showed that in most of the studied devices the dominant contribution to 1/f noise was made by mobility fluctuations arising from fluctuations in the scattering cross-section σ [An independent investigation of 1/f noise in a wide range of graphene devices  as well as that of a low-frequency voltage fluctuations (SU) on frequency have the form SU (or SI) ~ 1/fγ in the frequency range 1–100 Hz. The quality of graphene and the reproducibility of its properties in the chips in general can be judged by comparing the magnitude of low-frequency noise at a frequency of 1.22 Hz between the chips with the same geometry and from the same film. We discuss the impact of the identified reasons of non-reproducibility of graphene parameters in chips on the properties of chips for biosensor applications.Low-frequency 1/f noise caused by mobility fluctuations can occur as a result of the superposition of elementary events in which the scattering cross-section of the scattering centers σ fluctuates from σThe graphene films were formed on semi-insulating 4H-SiC substrates by thermal decomposition of the (0001) Si surface in a graphite crucible with induction heating . The met2O2 (30%) + H2SO4 (concentrated) 1:2,  at 25–27 °C was used. Details of the graphene films and the processing and mounting of chips on holders can be found elsewhere [2.The ordinary photolithography process was applied to form a topological pattern of as-grown graphene/SiC films utilizing AZ1318 photoresist . The photoresist was removed in acetone, and the graphene surface was examined by AFM followed by additional cleaning of the LRRs. Additional cleaning based on a piranha solution Hlsewhere ,30. ChipThe presence of graphene films on the SiC surface was confirmed, and their structure was characterized by Raman spectroscopy. Measurements were performed at room temperature in the backscattering geometry using a Horiba LabRAM HREvo UV-VIS-NIR-Open spectrometer  equipped with a confocal microscope. A YAG:Nd laser  with a wavelength of 532 nm was used as an excitation source. The laser beam was focused on the area with ~1 µm diameter using an Olympus MPLN100x  objective lens (NA = 0.9). The laser power nm was limited to 4.0 mW to prevent damaging and modification of graphene films.www.tipsnano.com, accessed on 11 August 2022) in a resonant mode of operation. The AFM probe knocks on the surface scanning frequency 0.6 Hz. The stiffness coefficient of such a cantilever is 3.5 N/m, the radius of curvature is less than 10 nm, and the scanning field size is 256 × 256 points.The AFM measurements of the graphene surface morphology were carried out on the Ntegra AURA setup . The AFM studies were carried out using the HA_FM cantilever (U) was measured for the graphene chips mounted on a holder in a frequency range from 1 to 50 kHz. The studied chips were connected in series with a low-noise load resistor RL whose resistance varied from 100 Ohm to 13 kOhm depending on the current passing through the chip. The voltage fluctuations SU at the resistors RL were amplified by a low-noise preamplifier SR560  and subsequently measured by an SR 770 FET NETWORK Analyzer . The background noise of the preamplifier did not exceed 4 nV/√Hz at 1 kHz, which is approximately equivalent to the Johnson–Nyquist noise of a 1000-Ohm resistance. The I–U characteristics of the chip were measured using the KEITHLEY 6487  power source.The power spectral density of voltage fluctuations  + H2SO4 (concentrated) 1:2,  was used. After the cleaning, 50 chips fabricated from the as-grown graphene/SiC film (EG417 series) were evaluated based on the AFM characterization, the results of resistance measurement, the level of spectral density of a low-frequency noise (SU) at a frequency of 1.22 Hz, as well as analyzing the shape of SU frequency dependencies. No RMS values larger than 2 nm were observed in graphene in the vast majority of chips. The typical RMS values are about 0.60 nm . We chose this technique because SU values provide integral information about the state of the defect system of the whole graphene film in the chip, rather than some local areas. The histogram of the distribution of SU values is shown in U values are as low as SU = (1 − 4) × 10−13 V2/Hz, which indicates a good quality graphene. Selective check-up of the Raman spectra for these chips shows that they are identical to those shown in To find out the reasons for the resistance spread in the range of 1–1.6 kOhm, measurements of the spectral density of voltage fluctuations SU values was in the range of 6 × 10−13 to 8 × 10−11 V2/Hz  were investigated. It is known that the shape of noise spectra SU ~1/fγ (γ > 1) reveals the information about inhomogeneous deformations in the material [U ~1/fγ having different values of γ up to 1.4 with a maximum SU value at 1.22 Hz for the EG417-C5 chip as shown in U ~1/fγ (γ > 1) in graphene films and the chips is consistent with the Raman spectra data that demonstrate an inhomogeneous distribution of compressive stresses in graphene [For the remaining 18% of chips, the spread of S11 V2/Hz a. Assumimaterial ,27. Indegraphene . StudyinAs was shown previously , the hetU values as low as SU = (1 − 4) × 10−13 V2/Hz, the dependence SU ~1/fγ (γ > 1) is not observed. A typical dependence for these chips is illustrated by the EG417-D6 chip in U ~1/f. A weaker dependence SU ~1/f0.8 is observed for higher frequencies f >100 Hz. We believe the SU dependence at a higher frequency may be due to a superposition of 1/f noise and generation-recombination (GR) noise. These features mean that, in addition to the system of defects typical to low-dimensional materials, there are single Shockley–Reed–Hall defects. It should be noted that this SU behavior is observed in graphene in chips with the RMS values close to those on the as-grown graphene film before PLG (see For 82% of the chips from the same film (EG417) with S PLG see a. AFM anThe results of comparing the quality assessment of processed graphene in chips after conventional PLG and PLG with additional cleaning, as well as the reproducibility of the parameters of these chips, which are the basis for viral biosensors, are presented. Conventional PLG with the removal of the photoresist in acetone is accompanied by the formation of local regions with resist residues (LRRs). Significant non-reproducibility of graphene parameters in chips after conventional PLG is observed. The presence of LRRs on the surface of processed graphene result in a spread in RMS values from 0.5 to 10 nm at an AFM scan of 10 µm × 10 µm and also in the resistance from 1 to 10 kOhm of processed chips from the same as-grown graphene/SiC film.The introduction of additional cleaning of the graphene surface in the chips after PLG resulted in RMS values close to the values on as-grown graphene/SiC film for the vast majority of the chips (up to 80%). As a result, the spread of chip resistance values decreased to 1–1.6 kOhm.U > 10−12 V2/Hz at 1.22 Hz is typical for processed graphene with rather large local LRRs, clusters, or cracks or a high level of deformation. Moreover, the noise spectra SU(f) show the ranges of SU ~1/fγ (γ > 1), indicating the non-uniform deformations in graphene. At the same time, the value of γ allows us to compare the level of deformation in different chips and at different stages of graphene-based biosensor production. The revealed features of processed graphene with SU > 10−12 V2/Hz lead to a deterioration in the adsorption properties of graphene, and the inhomogeneous attachment of biomolecules led to the deterioration of the detecting properties of the biosensor. In I vast majority of the chips after additional cleaning (R < 1.6 kOhm and SU < 5 × 10−13 V2/Hz), regions with this type of dependence were not detected. Influenza viruses were successfully detected on chips with these parameters [It has been shown that measurements of low-frequency noise are an effective tool for monitoring the non-reproducibility of graphene properties in a chip and finding out the causes of non-reproducibility. The spectral density of voltage fluctuations Srameters . Thus, t


High capacitance devices (Supercapacitors) fabricated using two-dimensional materials such as Graphene and its composites are attracting great attention of the research community, recently. Synthesis of 2D materials and their composites with high quality is desirable for the fabrication of 2D materials-based supercapacitors. Ultrasonic Assisted Liquid Phase Exfoliation (UALPE) is one of the widely used techniques for the synthesis of graphene. In this article, we report the effect of variation in sonication time on the exfoliation of graphite powder to extract a sample with optimal properties well suited for supercapacitors applications. Three different graphite powders  were sonicated for duration of 24 h, 48 h and 72 h at 60 °C. The exfoliation of graphite powder into graphene, GO and GrO was studied using XRD and RAMAN. AFM and SEM were further used to examine the layered structure of the synthesized nanocomposite. UV–visible spectroscopy and cyclic voltammetery were used to measure the band gaps, and capacitive behavior of the samples. Sample B exhibited a remarkable specific capacitance of 534.53 F/g with charge specific capacity of 530.1 C/g at 1 A/g and energy density of 66 kW/kg. Power density varied 0.75 kWh/kg to 7.5 kWh/kg for a variation in current density from 1 to 10 A/g. Sample B showed capacitive retention of 94%, the lowest impedance and highest degree of exfoliation and conductivity as compared to the other two samples. These devices exhibit high energy density or high power density well suitable for current and future portable electronics. However, now a days, achievement of high energy density and power density with same material is desirable2. Super-capacitors exhibit high specific capacitance and high energy density making them a suitable candidate for energy storage applications.Energy storage devices have always been a blooming research area due to continuous rise in energy demand. Different type of energy storage devices has been realized which store energy chemically, electrochemically, mechanically, kinetically, magnetically and thermally etc. For example, fuel cells, batteries, fly wheels, hydro pumps and super magnets etc. Due to constant rise in fossil fuel prices (fear of depletion), and rise in eco-friendly energy has increased demand of energy storage devices. Electrochemical energy storage devices use chemical energy in their active material to generate electrical energy through oxidation reduction reactions, efficiently and cost effectively3 hybridized. These carbon atoms are arranged in hexagonal honeycomb layers with weak Van der Waal's forces between the layers. Graphene can be exfoliated from graphite due to this weak interlayer bonding. Graphene shows extraordinary properties like flexibility, unbeatable thermal conductivity, and electronic behavior. Graphene monolayer is a zero-bandgap material with a sheet of sp2 hybridized carbon atoms. Due to its zero bandgap, its use in semiconducting devices is limited.Two Dimensional materials  have thickness in nanoscale and they exhibit superior electronic and mechanical properties such as electron mobility, conductivity and mechanical strength. Numerous 2D materials are candidates for energy storage applications such as Graphene, Transition metal dichalcogenides (TMDCs), Transition metal carbide or nitrides (MXenes) and hexagonal boron nitride (h-BN) etc. Mostly 2D materials are exfoliated from their 3D precursor, for example, graphene is mechanically exfoliated from Graphite. In graphite each carbon atom is covalently bonded with three other carbon atoms and each carbon atom is sp3. Andre Geim and Kostya Novoselov used micromechanical cleavage technique to extract graphene from graphite4. Micromechanical cleavage or scotch tape method is a top down approach in which bulk material is used and exfoliated to nanoscale. It gives monolayer of graphene but it is not a scalable process and is very tedious with impurities attached with graphene sheet. Different bottom up techniques are used for graphene fabrication such as chemical vapor deposition (CVD), Plasma enhanced chemical vapor deposition (PECVD), Liquid phase exfoliation (LPE) and Ultrasonic Assisted Liquid Phase Exfoliation (UALPE). Chemical vapor deposition(CVD)used for deposition of graphene on metallic substrates and its transfer on different substrates like SiC are introduced and optimized6. The maximum size of single crystal grown on dielectric substrate by CVD is in range of a micron7. Plasma enhanced chemical vapor deposition(PECVD)overcomes disadvantages of CVD by growing graphene at lower temperature and less time while compromising the quality of the domain size9. 3D graphene films are grown on nanoporous anodic alumina by using PECVD10. Liquid phase exfoliation (LPE) is a simple, cheap and scalable method for exfoliation of graphene. In this method there is no temperature constraint and graphene is obtained in form of suspension and can be used for fabrication of electronic devices. However, some solvents may require large sonication time for exfoliation and graphene quantity in dispersion is very low. Recently, graphene nanoplatelets are exfoliated from graphite in a relatively less sonication time of 3 h using graphite oxide as dispersing agent11. Graphene like material is obtained by intermittent shearing of commercial graphite powder in DI water using Ultra Plus Konzentrat surfactant12. Use of simple dispersing agents is very crucial for commercializing graphene synthesis. In Ultrasonic Assisted Liquid Phase Exfoliation (UALPE), powder is dispersed in a solvent and ultrasonic sound waves are passed through the dispersion to produce graphene dispersions.Monolayer graphene was first theoretically explored by Wallace in 194713. Due to these functional groups, hydrophilicity is introduced in GrO. When GrO is exfoliated it results in formation of graphene oxide (GO). GrO shows promising prospects for mass scale production of graphene14. Oxygen intercalation during exfoliation of graphite powder results in high inter layer spacing and also helps in easy exfoliation of GrO15. GO shows better capacitive behavior and properties than graphite due to its lower bandgap, increased surface area which results in more accessible sites for redox reactions16. Oxygen provides inter layer distance creating a facile charge transfer network which increases interlayer distance and enhances surface area which facilitates remarkable charge transfer and additional pseudocapacitance17. The oxygen functional groups are responsible for the change of carbon atoms hybridization from sp2 to sp318. To increase capacitive behavior of graphene, pseudocapacitance property is compulsory which is induced by addition of oxygen functional group in graphene. GO has been synthesized using electrochemical exfoliation process in which two graphite rods were immersed in SDS solution and exfoliated for 24 h by applying different voltages20.In 1958 the famous Hummer’s modified method was introduced. Strong chemicals used in this method change graphite to graphite oxide (GrO) and introduce functional groups to the lateral plane like hydroxyl (OH) and epoxide groups221. Graphene is a promising candidate when used as electrode in supercapacitors due to high specific capacitance and high energy density in different electrolytes. Chenguang et al. reported specific capacitance of 154.1 F/g and specific energy density of 85.6Wh/kg using high surface area graphene22. To improve the specific capacitance of graphene, surfactant stabilization was used which gave specific capacitance upto 194 F/g23. Santhakumar et al. reported a graphene based supercapacitor with specific capacitance of 195 F/g and energy density of 83.4 Wh/kg24. Ideally, specific capacitance for a monolayer of sp2 bonded carbon atoms (monolayer graphene) can reach upto ~ 550 F/g which sets an upper limit on graphene’s specific capacitance25. Laser scribed graphene showed a specific capacitance of 276 F/g26. Although graphene based supercapacitor electrodes shows good performance, they are limited by specific capacitance of around 300 F/g. To improve specific capacitance its composites and hybrids are being used.Mitra et al. reported graphite as an electrode for supercapacitors with capacitance values ranging from 0.74 to 0.98 mF/cm27. Hsieh et al. used GO in Na2SO4 and Li2SO4 and achieved specific capacitance of 98.8 F/g and 238 F/g respectively28. Lobato et al. showed that reduced GO showed a specific capacitance of 84 F/g in an H2SO4 electrolyte29. A well-defined composite of Graphene/GO/GrO can achieve both high specific capacitance and energy density. Graphene composites with other materials like oxides, carbon-based materials CNTs etc. have higher capacitive properties. To the best of our knowledge few studies have investigated the effect of GO and GrO in supercapacitors and whereas no study has been reported yet on Graphene/GO/GrO nanocomposite.Yanwu Zhu et al. reported a specific capacitance of 166 F/g by using GOIn this article, we have studied the effect of sonication time on exfoliation of graphite powder for cost effective and mass scale production of graphene. Three different samples were collected at sonication time of 24 h,48 h and 72 h and characterization techniques such as XRD and RAMAN were used to confirm the exfoliation of graphite powder into graphene, GO and GrO. AFM, SEM and EDS were further employed to observe layered structure of the nanocomposites. UV–Visible Spectroscopy was performed to analyze the effect of sonication on the bandgap of nanocomposite material. I-V, CV, GCD and EIS analysis were also performed to test the electrical and capacitive behavior of the nanocomposite. This article provides a scalable, green, and low-cost method to prepare graphene and its composites for supercapacitor applications.Graphite powder (1000 mesh flakes), Polyvinylpyrrolidone , Iso-propyl alchohal (Molecular Weight 60.10) and Nickel foam were purchased from Sigma- Aldrich Chemical Co.Ultrasonic Assisted Liquid Phase Exfoliation of Graphite (UALPE) was used in order to produce graphene of high quality which is economical and scalable. Graphite powder was dispersed in Isopropyl alcohol (IPA) and Polyvinylpyrrolidone (PVP) was added as a stabilizer and this suspension was sonicated in a bath sonicator at different time stamps at 60 °C and atmospheric pressure. After sonication, the graphite layers are isolated by overcoming the van der Waal’s forces which are quite weak and graphene flakes are dispersed in the suspension. For 20 mg of Graphite powder , 1.5 mg of PVP  were dispersed in 20 ml of IPA. This suspension was then centrifuged at 4030 rpm for 1 h. After centrifugation, the dispersion is settled into two distinct layers, hereafter referred as top and bottom portion (residue). The top layer forms 70% of the dispersion with graphene flakes suspended in IPA and the bottom layer (30%) is residue material. The top 70% suspension is separated. The bottom 30% part is diluted in IPA 5% by volume. More details are given in supplementary information Fig. The electrode fabrication process involved the dissolution of 80% active material, 10% Polyvinylidene fluoride (PVDF) as a binder agent, and 10% carbon black in mass in NMP (n-methyl-2-pyrrolidone) to form a slurry. The slurry was coated on Ni foam and dried in a vacuum oven overnight. The resulting electrode was then pressed at 5 MPa to ensure proper functioning. Supplementary Fig. XRD diffractometer  was used for the structural analysis of composite having Cu Kα as an X-ray source. Raman shifts were measured using Xplora ONE, Horiba Scientific Raman diffractometer with an exciting laser source of 532 nm. UV–Visible spectrophotometer  was used for measuring absorbance spectrum of samples over the range of 100 nm to 350 nm. Scanning Electron Microscopy (SEM) SEM, 6490A, JEOL Japan) and energy dispersive spectroscopy (EDS) were used to study morphology and elemental distribution. SEM was performed at a resolution of 5 µm, 2 µm, 1 µm and 500 nm. Two probes method electrical measurements were performed with sourcemeter . Gamry cyclic voltammeter  was used for electrochemical analysis. Three electrode setup was used for measuring specific capacitance using platinum wire as counter electrode and Ag/AgCl as reference electrode. Bath sonicator  was used for sonication.30. According to literature, single graphene layer has ~ 0.335 nm thickness and bilayer graphene has 0.81 nm thickness with an interlayer distance of 0.14 nm between two single layer graphene nanosheets32. Using these values, we calculated that 14.1 nm thick graphene contains 17 layers of carbon atoms i-e 17 monolayer graphene nanosheets. Layers stacked on top of one another with thickness ≥ 100 nm are graphite. In nutshell multi-layered graphene was synthesized as represented in Fig. Sample A was sonicated for 24 h and its SEM images were taken at 2 μm, 1 μm, and 500 nm. SEM images show coagulated particles as illustrated in Fig. AFM probe with a tip of 10–20 nm in diameter was used for AFM scanning. AFM images Fig. a–c can b34. This indicates the presence of graphene in the suspensions. These peaks appear due to π–π* transitions. The shift shown in red colored line is observed in all samples which represent change of electronic configuration due to exfoliation of graphite to graphene. The number of graphene layers can be represented by variation in the intensity of peaks35. Sample A shows very small absorbance peaks, indicating a low concentration of graphene flakes in the solution. This suggests that there is insignificant exfoliation of graphene from graphite powder in this sample. On the other hand, samples B and C show higher intensity peaks, indicating a higher concentration of exfoliated graphene flakes in the suspension. This suggests a relatively higher degree of exfoliation of graphene from graphite in these samples. Overall, UV–visible spectroscopy results suggest the formation of graphene in the suspensions, which is consistent with the results obtained from SEM and AFM.Based on the UV–visible spectroscopy results Fig. , it is oBand gap calculation was done on absorption spectrum of all samples by using Tauc Plot method using:This trend was consistent when calculations were performed for the bottom 30% solution. It's worth noting that the top 70% solution was too dilute to perform certain characterizations like XRD and IV due to sensitivity issues with the equipment. As a result, the bottom 30% solution was further analyzed and which is discussed.36. The calculated value of d-spacing for sample A is 0.3342 nm corresponding to The XRD pattern of the samples is shown in Fig. 38. When new atoms are incorporated or substituted for some of the host atoms, the structure expands or contracts, depending on the newly incorporated atoms. From the X-ray diffraction results, it is observed that the samples contain GO. The (001) peak of GO at 39. These functional groups make bonds with the π electrons of the carbon atoms resulting in sp2 to sp3 hybridization of carbon atoms. The degree of oxidation, or the amount of functional groups attached to the basal plane of GO, can be monitored through the interplanar distance, which increases with higher oxidation levels and vice versa. Moreover, the (001) peak in the X-ray diffraction pattern of GO is sensitive to changes in interplanar distance, and its intensity can be used to infer the degree of oxidation of GO. Sample B has the most intense (001) peak, indicating a higher degree of oxidation compared to samples A and C. The decrease in intensity of the (001) peak in sample C suggests a lower degree of oxidation compared to sample B. These partially oxidized flakes have un-oxidized sp2 hybridized graphitic domain in them surrounded by sp3 hybridized GO40. This results in the presence of both GO and graphite in the diffraction spectra of the samples. The crystallinity and arrangement of crystallographic planes in the formed nanocomposite material vary in sample A, B and C. Specifically, the XRD peaks for graphene oxide (19°), and graphite flakes increase in intensity from sample A to sample B, whereas for sample C, the GO and GrO peaks vanish completely while the graphite oxide peak becomes very weak. Additionally, the GO (100) peak also disappears in sample C. The amount of graphene in this nanocomposite is small, which may be why it was not detected in the XRD results. Full scale image of XRD is given in Supplementary Fig. The increase in the value of d-spacing in graphite is attributed to the incorporation of oxygen atoms on the basal planes of graphiteScanning Electron Microscope (SEM) was used to examine the structure of three samples taken from the bottom 30%  at a resolution of 1 µm. Sample A did not show any distinct layered structure and exhibited coagulation which is clear in Fig. Energy Dispersive X-ray Spectroscopy (EDX) was used to map and analyze the elemental composition of samples A, B, and C. Table −1 corresponds to the D-band, which may be assigned to the A1g mode of vibrations. The formation of the D-band is possibly due to defect-generated phonon mode vibrations or disorders in the honeycomb graphitic structures such as vacancies, bond-angle disorder, edge defects, and bond-length disorder  and the level of oxidation in the samples. Figure 41. As shown in Fig. 42. The blue shift of the G band observed from sample A to B suggests an increase in the level of oxidation, which leads to the disruption of graphitic stacked ordering and increased disorder in the material. The increased disorder is reflected in the shift of the G band to a higher frequency, as the phonon frequency of the G band is affected by the degree of sp2 hybridization and the degree of disorder in the material. Therefore, the blue shift observed in the G band from A to B indicates the presence of GO or GrO, which have a higher level of disorder and more oxygen-containing functional groups compared to pristine graphene. The increase in the intensity of the G band from B to C is observed because the samples were subjected to coagulation and annealing at an increased temperature and pressure for 72 h. Coagulation can result in the stacking of graphene oxide layers, leading to an increase in the number of graphitic domains, which could increase the intensity of the G band. Additionally, the increased temperature and pressure may have caused further reduction of the graphene oxide, resulting in more ordered graphitic domains and a higher G band intensity.The presence of functional groups like hydroxyl and epoxide on the basal plane of GO nanosheets can also contribute to the occurrence of the D-bandD/IG ratio is the ratio of the intensities of the D band and the G band in Raman spectra. This ratio is commonly used to estimate the degree of disorder in graphene-based materials and size of sp2 domains in the GO based systems43. Different relations like the Knight and White formula44 and Tuinstra and Koeng’s relation45 can be used to calculate the average sp2 domain size in GO based systems, but in our study, we used a general relation by Cancodo et al. to measure the average crystallite size of the sp2 domain46. These sp2 domains gives us information about extent of oxidation of graphite into GO and GrO. This relation gave us a value ofa is the average crystallite size of the sp2 domains, λl is the input laser energy, ID is the intensity of the D-band, and IG is the intensity of the G-band. The ID/IG ratio increases from A (0.42) to B (0.50) indicating an increase in disorder, number of defects, exfoliation, and oxygen content, which confirms the presence of GO and GrO. This increase in ID/IG ratio also reflects a decrease in the size of the in-plane sp2 domains and the partially disordered crystal structure of the nanosheets47. The decrease in the ID/IG ratio from B (0.5) to C (0.13) is due to the highly disordered structure and loss of some crystallinity. The crystallite size is observed to be maximum in sample B, while it decreases in sample A and sample C. This reflects the shrinking of graphitic domains  and formation of more grain boundaries or lateral defects, which may occur due to expulsion of graphene layers from domains. Therefore, the ID/IG ratio can be used to determine the size of sp2 domains in GO-based systems.The ID/IG ratio is an indicator of the degree of disorder and the size of sp2 domains in GO-based systems. ID/IG ratio increases in low defect density regime and decreases in high defect density regime48. A higher ID/IG ratio corresponds to a greater degree of disorder and a smaller size of sp2 domains. Therefore, sample A, with an ID/IG ratio of 0.42, has larger sp2 domains and is more ordered than sample B, which has an ID/IG ratio of 0.50. Similarly, sample C, with an ID/IG ratio of 0.13 lies in high defect density regime and has the smallest sp2 domains and is the most disordered of the three samples. Overall, the Raman spectroscopic analysis provides further evidence for the presence of GO and GrO as well as the level of oxidation in the samples. Therefore, the Raman spectroscopy results support the EDX findings and provide additional information about the level of disorder and defects in the samples.The I49.UV–Visible spectroscopy in the range of 100 nm to 350 nm was conducted to examine the impact of exfoliation on graphite powder. The results indicate that all three samples, A, B, and C, have their maximum absorbance peak at 220.09 nm, 218.90 nm, and 218.90 nm, respectively as shown in Fig. 50. In sample B, with the increase in oxygen content the donor states increase which increases the conduction states as compared to sample A. Other factors like formation of graphite flakes, GrO and GO might also be contributing factors in reduction of band gap. Sample A has the highest bandgap value of 2.06 eV, while sample B has band gap of 1.82 eV and C have 1.76 eV band gap as shown in Fig. The increase in oxidation leads to the formation of the more donor states which in turn can increase the number of conduction states in the material resulting in decreased band gap value. Higher oxygen coverage on surface of graphite causes lattice relaxations which results in the change of band gapThe samples of 5 μm thickness were prepared and subsequently I–V measurements were carried out to observe electrical properties. For I–V measurements, the voltages in the range of − 2 V to 2 V were applied. The I–V curve for each sample was found to be a straight line, indicating Ohmic behavior as shown in Fig. All samples A, B, and C (bottom 30%) were tested using CV analysis and all of them showed pseudocapacitive behavior, this suggests that these materials have the potential to be used as electrode materials in electrochemical capacitors, such as supercapacitors. Gamry cyclic voltameter with a three-electrode setup was used to measure charge–discharge process, where a platinum wire was used as the counter electrode, Ag/AgCl as the reference electrode, and 1 M KOH as the electrolyte.Each sample was analyzed at different scan rates ranging between 0.5 and 10 mV/s as shown in Fig. The presence of oxygen in the form of GO and GrO is the main reason for the pseudocapacitive behavior observed in the nanocomposite material. Sample B shows better results than sample A because it has a higher level of oxidation, which makes it more conductive and hence able to participate in more redox reactions. On the other hand, sample C shows weaker pseudocapacitive behavior, as indicated by the relatively weak redox peaks observed in the CV analysis. Specific capacitance decreases with the increase in scan rate due to increased number of inaccessible sites on the surface of the electrode. Conversely, a lower scan rate enables access to more sites on the surface of the electrode, resulting in a larger specific capacitance value. For sample C, the highest specific capacitance value of 195.12F/g was observed at a scan rate of 0.5 mV/s, as demonstrated in Fig. 3 hybridization and more oxygen contents, which result in better charge transfer and enhanced Faradaic reactions at the electrode site. When GO is synthesized from graphite, the carbon to carbon double bonds break to incorporate the incoming oxygen and other oxygen-containing functional groups. This process leads to the disruption of graphitic stacked ordering and the formation of more grain boundaries or lateral defects, as observed through Raman analysis.Graphite is an insulator in its pure form. Graphene shows excellent electronic properties but its use in energy storage devices is limited because of its weak capacitive behavior due to its limited surface accessibility. Graphene oxide (GO) can enhance its capacitive behavior due to increased sp2 to sp3. Degree of oxidation or the amount of functional groups attached to the basal plane of GO can be detected by monitoring the interplanar distance. Higher oxidation levels leads toward the increased d-spacing and vice versa. The addition of functional groups to graphene oxide results in more donor sites, which facilitates charge transfer and can lead to improved pseudo capacitive behavior. The increased interlayer spacing in GO compared to graphite and abundant sp3 domains are another factors that can contribute to improved pseudo capacitive behavior. This increased spacing provides a better charge transfer network throughout the basal plane, which can enhance conductivity and capacitive behavior. GrO increases the surface area causing more active sites for charge transfer during charging and discharging process. The presence of oxygen in the form of functional groups on GO and GrO is a key factor contributing to the pseudocapacitive behavior in the formed nanocomposite. Furthermore, the lower band gap resulting from oxygen addition can increase the electrical conductivity of the nanocomposite.These defects may occur due to the expulsion of graphene layers from domains, leading to a smaller average size of the graphitic domains. These functional groups form bonds with the π electrons of the carbon atoms thus resulting in hybridization of carbon atoms from spOverall, the experiment provides valuable information about the electrochemical properties of composite residual material electrodes made on Nickel foam substrate. The pseudocapacitive behavior exhibited by all samples indicates that these electrodes have promising potential for energy storage applications. The energy density of sample C was found to be lower than that of samples A and B, likely due to its decreased surface area and poorer conductive behavior.−1, 5 Ag−1, 2 Ag−1 and 1 Ag−1 and represented in Fig. Galvanstatic charge discharge (GCD) technique was used to further analyze charge storage mechanism of prepared samples. Nonlinear capacitive GCD curves for sample A, B and C are measured at different current densities of 10 AgElectrochemical impedance spectroscopy (EIS) was employed to measure electrochemical conductivity and analyze the stability and impedance behavior of samples A, B, and C. Figure A cost-effective method for production of Graphene, GO and GrO nanocomposites was developed by tuning sonication time durations for exfoliation. Three graphite powders were sonicated for duration of 24 h, 48 h and 72 h at 60 °C and the resulted nanocomposites were analyzed using AFM, SEM, XRD, Raman, UV–visible spectroscopy and cyclic Voltammetery. The sample B (sonicated for 48 h at 60 °C) showed low resistance and the best pseudocapacitive behavior among the three samples with a specific capacitance of 534.53 F/g specific charge capacity of 530.1 C/g and energy density of 66.2 Wh/kg. Cyclic stability test was performed for over 5000 cycles giving 94% capacitance retention. Morphological and structural characterization suggest that sample B has highly exfoliated graphite and a reasonable amount of graphene, GO and GrO and the conductivity of the Sample B also increased because of presence of exfoliated graphite, GO and GrO. The nanocomposite can have wide spectrum of application in energy storage applications.Supplementary Information.