Graphene, an allotrope of carbon, consists of a single layer of carbon atoms with uniquely tuneable properties. As such, graphene-based materials (GBMs) have gained interest for tissue engineering applications. GBMs are often discussed in the context of how different physicochemical properties affect cell physiology, without explicitly considering the impact of adsorbed proteins. Establishing a relationship between graphene properties, adsorbed proteins, and cell response is necessary as these proteins provide the surface upon which cells attach and grow. This review highlights the molecular adsorption of proteins on different GBMs, protein structural changes, and the connection to cellular function. Graphene-based materials are widely studied in biomedical applications, but a full picture of their interactions with proteins and cells remains elusive. Here the molecular basis for the effect of graphene-based materials on cell fate and in tissue engineering is reviewed. In our previous review article, we highlighted the influence of GBM physical , chemical , and mechanical properties on cell response, without explicitly discussing how GBMs influence protein or biomolecular interaction3. The effect of graphene physicochemistry on biomolecular interactions of proteins has been reviewed separately6. Since proteins on biomaterial surfaces are a key mediator of subsequent cell behavior for nanomaterials9, the unique physicochemical properties of graphene offer exciting opportunities to control protein adsorption, orientation, conformation, and ultimately cell fate.Over the past decade, graphene and graphene-based materials (GBMs), such as graphene oxide (GO), reduced graphene oxide (RGO), and their chemical derivatives, have gained substantial interest for the development of biomaterials for tissue engineering applications. The unique physicochemical properties of graphene and GBMs have been shown to significantly influence cell response, as previously reviewed10. Studies have demonstrated that a material’s ability to adsorb proteins , fibronectin (Fn), collagen, and laminin) from the serum of cell culture media plays a vital role in cellular attachment and function11. Moreover, adsorption of the same protein but with different conformations, which exposes different domains to cells, has been shown to mediate integrin-based cell adhesion and trigger downstream signaling, directing cellular function and differentiation16. Thus, one can surmise that adsorbed proteins and other bioactive molecules from culture media influence cellular response in such a way that is tightly controlled by the underlying physicochemical properties of the biomaterial surface.The adsorbed protein distribution and conformation on biomaterial surfaces provide the cell-interfacing topographical, physical, and biochemical cues for cells to interact with and respond to a material17, there is a clear need to link the physicochemical interaction of the graphene surface with protein adsorption, protein structure, and cellular response. Developing such a multiscale understanding will involve integrating experimental, theoretical, and simulation studies.The initial protein-substrate interactions are critically important when designing biomaterial substrates, since they set the stage for how proteins will attach to the substrate surface. Surprisingly for GBMs—given their strong interest from the research community—a complete picture detailing (i) molecular conformation of proteins on different GBMs, (ii) how this influences cell–substrate interaction, and (iii) the connection to cellular response remains elusive. As GBMs are expected to become promising next-generation biomaterials for tissue engineering applicationsIn this review, we attempt to thread a line through the above-mentioned areas (i–iii) and present how graphene physicochemical properties can be tuned to control cell fate via the proteins coating the graphene surface. We hope this review will encourage more researchers to work in this burgeoning subfield, and provide quantitative experimental results with direct connection between the different areas (GBM–protein and protein–cell) that link cell function and GBM physicochemical properties.19. Common methods for graphene synthesis include chemical vapor deposition (CVD) and mechanical exfoliation18. Other techniques for preparation of GBMs, such as GO , RGO , and their derivatives, include chemical exfoliation, chemical and thermal reduction, and chemical functionalization20.Since the discovery of graphene, numerous techniques and methods have been reported for production of graphene and GBMs21. Measuring the interaction of graphene and GBMs with numerous different cell lines and animal models to understand the mechanism of cytotoxicity is only now becoming routine22. Hydrophobic suspended graphene nanoparticles in aqueous media showed more toxicity than hydrophilic GO particles, due to rapid agglomeration of hydrophobic graphene covering the cell surface, purportedly limiting nutrient supply and inducing oxidative stress, causing cell death. Upon interaction, graphene sheets result in physical damage to the cell membrane23. Also, the uptake of nanoscale, non-functionalized graphene shows more cytotoxicity compared with large and functionalized graphene23. The biological response of graphene and GBMs—as reviewed below—must be taken in the context of potential complications with cytocompatibility, which depends on particle size, concentration, chemistry, processing methods, and purity of graphene substrates.Application of graphene and graphene-derived materials in biomedical research has been limited due to latent cytotoxicity concerns25. Bussy et al. demonstrated the effect of a GO suspension on different cell lines in serum-free HEPES-buffered salt solution (BSS), Dulbecco’s phosphate-buffered saline (PBS), and Dulbecco’s modified Eagle’s medium with serum (DMEM-S). Cell membrane ruffling and shedding damage upon interaction with GO occurred in both BSS and PBS, but not in DMEM—presumably due to adsorption of proteins26. These results show that different parameters affect graphene-substrate cytotoxicity in both the absence and in the presence of proteins.While cytotoxicity studies with GBMs and particles are growing, relatively few studies have shown how the interaction of graphene-based particles with proteins in the culture media can influence cell survival. In one example, graphene particles in suspension were shown to exhibit cytotoxicity due to direct interactions with the cell membrane, and the cytotoxicity effects of graphene and GO in suspension can be mitigated when pre-incubated with FBS. Pre-incubation in FBS was shown to form a thin protein coating on GBMs in suspension, limiting their direct interaction with cells, thereby minimizing cytotoxicityWe begin by focusing on the protein–graphene interaction, and review example studies highlighting what is now known about this interaction. An obvious starting point is the interaction of serum proteins with different graphene-based surfaces, as serum is present in nearly all cellular experiments.28. GO, a graphene derivative with rich, oxygen-containing functional groups on graphene, has been found to interact and adsorb many proteins found in serum-based culture media. GO, having negatively charged oxygenated functional groups at physiological pH, as well as the hexagonal aromatic graphene structure, promoted hydrogen bonding, electrostatic, hydrophobic van der Waals, and π–π interactions allowing it to interact with various proteins in serum. As a result, GO showed very high amounts of serum protein capture on its surface27. Chong et al.29 demonstrated interaction of different serum proteins: bovine fibrinogen (BFG), immunoglobulin (Ig), transferrin (Tf), and bovine serum albumin (BSA) with GO and RGO29. Both GO and RGO showed serum protein adsorption in the following order: BFG > Ig > Tf > BSA (most to least) with GO showing higher adsorption compared with RGO. The difference in protein adsorption was attributed to differences in surface chemistry of GO and RGO, with GO offering a greater variety of different interactions compared with RGO; RGO would offer less hydrogen bonding and electrostatic interactions.Carbon-based nanomaterials, especially graphene and its derivatives, have been shown to interact strongly with different serum proteins30. BFG and Ig showed structural heterogeneity on the GO surface in comparison with RGO, which was attributed to the presence of multiple oxygenated moieties on GO and differences among native protein structures29. Interestingly, BSA and Tf showed structural rearrangement from α-helical to enhanced β-sheet conformation after adsorption on GO surface, as depicted by circular dichroism (CD) spectra in Fig. 31. Chong et al.29 found that proteins with exposed aromatic residues content like Trp, Tyr, and Phe residues first align with the graphene surface via π–π stacking as illustrated in Fig. 29. The authors highlighted that solvent-exposed aromatic residues are involved in the initial protein adsorption, followed by later interaction of buried residues once the protein partially unfolds on the graphene surface29. Their results are suggestive of a positive correlation between protein adsorption and the amount of aromatic hydrophobic residues in a protein29. In another experimental study, Shi et al. showed how the reduction state of GO affected serum protein adsorption differently for different proteins32. In that study, neat GO, partially reduced GO (pRGO) , and completely reduced GO (RGO)  were prepared by thermal reduction, and adsorption of different proteins such as fibronectin and BSA was compared. Their results showed that pRGO and GO exhibited better serum protein adsorption in comparison with RGO surfaces, consistent with Chong et al.29. Interestingly, pRGO showed the most adsorption of serum proteins, purportedly due to the presence of partial oxygen groups and partial hexagonal carbon structures. Oxygenated functional groups on pRGO surface introduced charged and electronegative regions for protein interaction through electrostatic and hydrogen bonding, while aromatic hexagonal carbon background of pRGO accommodated hydrophobic interactions with proteins as schematically shown in Fig. 29 strongly suggest that strongest adsorption of serum proteins on pRGO was attributed to the mixture of electrostatic forces, hydrogen bonding, and hydrophobic interactions in pRGO with proteins32.Consistent with the two graphene substrates having different available protein interaction mechanisms, adsorbed proteins were found to interact differently on GO and RGO surfaces. The presence of polar groups such as hydroxyls, carboxyls, and epoxides on GO promoted adsorption mainly through electrostatic interactions, whereas RGO protein adsorption was mediated primarily by van der Waals interactions2 carbon hybridization and distort the hexagonal benzene ring structure of graphene. This in turn affects protein binding due to steric hindrance and potential wrinkling of the graphene layer. Ebrahimi et al. used molecular dynamics (MD) simulations to study the role of graphene wrinkles and roughness on collagen interaction and adsorption. Their simulation results revealed that rough graphene surfaces show more adhesion of collagen fibers in comparison with smooth surfaces. As might be expected, the rough and wrinkled graphene surface provided more contact points between the graphene and collagen molecules for strong interaction33. Consistent with this finding, another theoretical study showed that the RGD cell adhesion peptide exhibited stronger interaction on vacancy defects in graphene (missing carbon atom in the lattice) in comparison with neat graphene34. The defects in graphene served as highly reactive sites for interaction between COO– of RGD peptide and vacancy defects on graphene as depicted in Fig. 35.Chemical defects, which are often created during functionalization and modification of graphene, are known to be “hot spots” for protein interaction. Structural defects on graphene typically alter the sp37. A molecular simulation study demonstrated how insulin hormones present in serum can interact with graphene ribbon-like surfaces of different sizes36. The width of graphene ribbons showed direct correlation with insulin adsorption, and strong π–π interaction between phenyl rings in insulin and graphene surfaces was shown to promote stepwise conformational  changes of insulin. On the other hand, Atabay et al. found that during adsorption of insulin on GO, insulin underwent configurational  rearrangement mediated by hydrophilic Ser, His, and Thr residues in chain B37. Interestingly, the anchored residues ultimately showed weak electrostatic interaction with the hydrophilic GO surface, which allowed the protein to rearrange after adsorption and restore its native structure. These studies again demonstrate the multiplicity (complexity) of ways in which even a single protein can interact with graphene, depending on the graphene surface chemistry.In addition to serum proteins, graphene substrates have also shown strong propensity to interact with hormones present in serum38. The exceptionally high adsorption ability of graphene for dexamethasone can be again attributed to π–π stacking between the aromatic rings in the dexamethasone and the graphene basal plane. Also we previously showed that polyethyleneimine (PEI)-functionalized GO (GO/PEI) showed high affinity to adsorb the osteogenic growth factor β-glycerolphosphate39. This was due to the presence of cationic PEI molecules on GO that provided electrostatic attraction to attract anionic phosphates like β-glycerolphosphate. In another study, GO’s ability to adsorb a high amount of transforming growth factor‐β3 (TGF‐β3) was highlighted. GO having both graphene aromatic ring domains and oxygenated functional groups supported interaction with TGF‐β340.Another important aspect of cell culture is use of specific growth factors or differentiation supplements in culture media. Interestingly, graphene-based substrates have also shown adsorption and interaction with growth factors and supplements present in culture media. Lee et al. reported that graphene showed more adsorption of osteogenic growth factor dexamethasone in comparison with GOπ–π interaction to orient hydrophobic residues toward the graphene surface. Defects and surface nano wrinkles on graphene further influence protein interaction and morphology. Thus, it is very important to know and understand the specific surface properties of graphene substrate in order to appreciate the influence it has on protein interaction and conformation.In summary, different serum proteins (hormones and growth factors) have complex interactions with graphene-based substrates. Depending on the physicochemical properties of graphene substrates, biomolecules interact and orient differently as highlighted collectively in Fig. 42. However, there is no consensus at this time about how different physicochemical properties of graphene substrates influence stem cell fate. The general paradigm for biomaterial–cell interaction is that, in the presence of media and serum, protein  adsorb and orient at the surface prior to cellular interaction8. It is virtually impossible to neglect the molecule –graphene interaction from the cell–graphene interaction. Ultimately, protein adsorption, conformation, and activity, in addition to other graphene physicochemical properties , control subsequent cell interaction44. Our goal in this section is to offer a perspective on cell response to graphene with a rationale starting from graphene–protein interaction on different graphene substrates and how the graphene-coating proteins affect cell behavior.Having reviewed how graphene physicochemical properties affect protein, hormone and growth factor adsorption, and structure, we move to the next level for evaluating graphene as a biomaterial, which is the graphene–cell interaction. Over the years, graphene-based particles have received substantial attention as future materials for tissue engineering applications, and authors have highlighted numerous physicochemical properties  of graphene substrates that influence, e.g., stem cell differentiation45. Proteins show conformational (globular vs. elongated) change upon adsorption to nanoroughened surfaces where surface roughness is smaller, or larger, than dimension of protein molecule46. In addition, it was observed that the adsorbed protein monolayer surface topography/morphology was directly related to the surface topographical profile of the underlying material47. Thus, the adsorbed protein has been suggested to provide the ultimate topographical cues for cells.Surface nanoroughness has been shown to play a critical role in protein adsorption and conformation48. Surface roughness at the nanoscale translates into increased surface area for protein adsorption and orientation, providing increased number of cell-binding contact points50. On a similar note, Subbiah et al. showed that coating pure titanium (Ti) with GO provided nanoscale roughness for strong, homogeneous FN protein adsorption on the GO-coated Ti51. Subsequent biological studies showed more pre-osteoblasts attached, and more osteogenic differentiation features, on FN adsorbed to GO-coated Ti in comparison with pristine Ti or non-FN, GO-coated titanium (Tigra). The authors suggested that the availability of RGD motif of FN on GO surface caused better cell attachment and organization of the cytoskeleton. However, no direct measurements of FN molecular structure and conformation on the GO surface, nor information about cell-binding domain exposure, were presented in that work.Wrinkled graphene, which is essentially roughened graphene, was shown by Nayak et al. to have significant influence on stem cell response52. GO showed high affinity for albumin and fibrinogen proteins on its surface in comparison with glass, as depicted in Fig. 52. This shows how graphene roughness can modify protein structure and downstream cell function.In another study by Hank et al., platelet aggregation in the presence of albumin and fibrinogen on hydrophilic GO and glass surfaces, with different roughness, was found to be distinct Fig. 52. GO sh55. Lee et al. showed that graphene and GO substrates, with different surface chemistry and wettability, had different effects on differentiation of stem cells due to different interactions with adsorbed proteins and other biomolecules38. Their study demonstrated that GO promoted adipogenic differentiation of stem cells compared with graphene as shown in Fig. 56. High affinity and adsorption of insulin on GO surfaces was attributed to electrostatic interaction and hydrogen bonding with polar oxygenated functional groups on GO. After adsorption on a GO surface, insulin was shown to retain its three-dimensional conformation , protein morphology, and biological response on (hydrophilic) GO-modified chitosanion Fig. , and minion Fig. ) of oste58. They cultured myoblasts on GO and RGO, and both showed elongated morphology in comparison with polygonal shape on glass. The authors suggested that elongated and cytoplasmic extension was caused by surface nanotopography provided by wrinkled structure of GO and RGO. However, when comparing RGO and GO surfaces, one sees that myoblasts on GO showed more myogenic differentiation by forming more multinucleated myotubes even though both had near-identical surface wrinkle roughness  showed strong binding of FN to cellular integrins and exhibited substantial differentiation into myotubes59. However, the FN conformation was not probed in these studies, so the connection to differentiation is still somewhat unclear62. The common undercurrent in all examples presented in this section is that the exact mechanism underlying all the examples shown here is ambiguous due to the complexity of the system.In a final example from this section, we highlight work by Ku et al. where they showed that graphene-based substrates were capable of influencing myogenic differentiationess Fig. . Hence, ess Fig. . The aut63—both cells where membrane  polarization is fundamental for function. Several reports have described the interplay of electrical and chemical cues imposed by graphene to influence cell differentiation66. In one example, Li et al. showed that graphene supported neuron growth by promoting better attachment and neurite sprouting of mouse hippocampal neurons when compared with standard polystyrene. The authors attributed this result to the complex interaction of graphene and chemicals in the culture medium in combination with electrical phenomena of graphene67. Feng et al., who investigated the effect of GO and RGO on neurogenic differentiation of the adipose-derived stem cells (ADSCs)68, showed more effective differentiation of ADSCs into neuron-like cells on GO. This was despite GO having lower conductivity in comparison with RGO, encouraging the authors to suggest that chemistry “won” the competition between conductivity and chemical properties. The authors posited that the presence of oxygenated functional groups on GO surface influence protein adsorption, which mediated better interaction with cells to influence neuronal differentiation69. Thus, GO, with a rich amount of oxygenated functional groups and promoting protein adsorption in addition to the presence of more edge defects on GO, might have provided synergistic, localized electric fields for neuronal differentiation of ADSCs as illustrated in Fig. In addition to surface chemistry and topography of graphene, tissue engineering researchers have utilized graphene’s surface conductivity/charge-carrying ability. Graphene’s unique electrical and charge transfer properties have been reported to influence musculoskeletal and neuronal cell response70. Coating graphene on the germanium surface was suggested to promote increased blood plasma protein (Fibrinogen) adsorption. Fibrinogen adsorption was further suggested to facilitate electron transfer from fibrinogen to graphene to germanium. Electron loss from fibrinogen has been shown to unfold and lead to formation of the fibrinopeptide and fibrin monomer, competent for polymerization71, and fibrin is known to have a strong interaction with platelets through the αIIIβIIb integrin72. Thus, graphene acting as an electron acceptor and transporter, resulting in electron extraction from fibrinogen, could lead to enhanced platelet activation as illustrated in Fig. 73 and stimuli to support different types of cell growth, harnessing the combined effects of electrical and chemical properties of graphene substrates is very attractive76.In addition to the intrinsic conductivity of graphene, its charge, doping, and metal coating of graphene substrates can also be exploited to bias cell fate. Geng et al. revealed that coating germanium surfaces with graphene induced platelet adhesion and activation39. Lee et al. reported that graphene showed more osteogenic differentiation in comparison with GO due to high adsorption of osteogenic growth factors such as dexamethasone and β-glycerolphosphate38. Similarly Jung et al. exploited the ability of RGO-coated Ti alloy to interact with osteogenic dexamethasone (Dex) via π−π stacking on the graphitic domains of RGO to enhance osteogenic differentiation  was exploited to promote chondrogenic differentiation of adult stem cells. TGF‐β3 retained its native structure on a GO surface for interaction with TGF‐β receptors on a stem cell surface, and significantly influenced stem cell adhesion and chondrogenic differentiation as illustrated in Fig. 7b40.To conclude this section, we would also like to highlight how different graphene-substrate–growth factor interactions can act to pre-concentrate various growth factors and differentiation chemicals in order to influence stem cell response. One key aspect of differentiating stem cells to specific lineages is using specific growth factors, or differentiation supplements, in culture media. Adsorbed growth factors/differentiation supplements on graphene-based surfaces are reported to further influence the biological outcome of stem cellsion Fig. 77. In an78. The high degree of oxidation of GO promoted more VEFG binding, and the highly basic surface charge of the heparin-binding domain of VEFG played a significant role in binding to GO surfaces through electrostatic and hydrogen interactions. Upon binding, VEFG showed structural changes with a decrease in secondary β-structure and increase in random coil structure. The authors went on to show that strong binding of VEFG by GO from plasma hindered the interaction with VEGF cellular receptors, inhibiting the proliferation, migration, and tube formation of human umbilical vein endothelial cells as illustrated in Fig. Growth factor adsorption has also been shown to promote undesired biological outcomes. For instance, Lai et al. showed that GO can efficiently bind a vascular endothelial growth factor (VEGF), depending on the oxidation stateBiomaterials influence cell function primarily through cell-material surface interactions where the identity and structure of adsorbed molecules, namely proteins, hormones, and growth factors, strongly determine the bioactivity of the material in context. The underlying physicochemical properties of the biomaterial ultimately regulate the entire cascade of interactions from molecular adsorption and structure to what cells ultimately “feel”. Graphene, as an emerging material with unique physicochemical properties, has been shown to stimulate different cell response and protein interaction due to the multiplicity of its interaction pathways. The potential of graphene for biomaterials is enormous because the biomaterial community can exploit the unique topographical, chemical, and electrical properties of the material for specific purposes. In this review, we try to highlight the diversity of graphene’s properties and biomolecular interactions to emphasize the opportunities, and complexity, of using graphene substrates as biomaterials. Specifically, because of graphene’s unique nature, harnessing its power requires understanding what is adsorbed, how adsorbed molecules look , and the (specific) cell response.Going forward, the synergetic effort of experimental, theoretical, and simulation studies will be needed to develop methodologies and rational models, which allow connecting the substrate–protein interaction to cell function on different graphene substrates. Use of advanced analytical instruments like nano IR-AFM may provide protein topography and conformation in combination with IR spectroscopic information of graphene substrates at nanoscale. This information, along with the ultimate cell response, may provide the functional coupling of topography and molecular structure of the adsorbed proteins with cell function on graphene substrates having different physicochemical properties. Finally, additional research is needed to evaluate cellular response on different graphene surfaces with well-defined biochemical environments and serum-free media formulations to reduce interference from these proteins, so that a clear conceptual picture can be drawn. In writing this review, we often found that unequivocal statements were hard to find due to the multiple ways graphene can affect protein structure, and how protein structure affects cell fate. Overall there is much to be done in the graphene–biomolecular interaction–cell response context that can help provide design criteria for graphene biomaterials for specific needs.Peer review file


Graphene oxides with different degrees of oxidation are prepared by controlling UV irradiation on graphene, and the charge transport and the evolution of the transport gap are investigated according to the extent of oxidation. With increasing oxygenous defect density  Graphene is a two-dimensional semimetal with high conductivity and mobility ,2, and ioribbons , a bandgoribbons ,5,6 suchC = 50%) ,8,9,10.The most common method to produce GO is based on wet chemistry, consisting of oxidation of graphite in strong acids, followed by a liquid exfoliation ,11. The Transport studies of GO and observation of metal–insulator transition via oxidation or reduction reactions have been reported ,13,14,18T < 400 K. The graphene device with Hall-bar geometry was investigated after UV oxidation by using a quantum design PPMS for lower T down to 2 K and magnetic fields up to 7 tesla.Our experiments were conducted on seven graphene field-effect-transistors (FETs) fabricated using exfoliated graphene on SiOD I(D)D peak phonon, and Here, periment ,27 with A,D=3 nm . As showG peak intensities, G peak intensities can be fitted with the following Equation , consistent with our observation.Regarding or model . Here, Vlows σ∝n a,b, whillat area c–f. Thisgraphene ,29 and σgraphene . The estσh2e2·1πn,32 givese)–hole(h) puddle [The change in the transport mechanism observed in GO FETs happens because the scattering with oxygenous defects becomes more frequent as the extent of the oxidation increases. =1e·dσdn ), decreas method ,33. On t) puddle . The traFπΔnflat ,38,39, o nD=0.29×12 cm−2 aT dependence of T < 400 K. Pristine graphene exhibits metallic behavior in which T. For GO samples with T, and the insulating behavior develops further with increasing l is the mean free path with T-dependence of E) is the density of states of graphene [T < 300 K. T < 145 K, T < 300 K as shown in the inset of T < 15 K, deviating from the linear dependence.In (2D VRH) ,41, follncreases ,41. On tT < 300 K, the T-dependence of the conductivity can be explained by considering 2D VRH and thermal activation conduction (TA) together. The data are fitted well with T-dependence of electrical resistivity In the temperature range of 145 < e regime .(3)ρ=ρcHere, lue of TK. In FiguB increased up to 7 teslas. The negative MR is analyzed with the following equation [equation  includinHere, In summary, we have prepared graphene-oxide FETs with different degrees of oxidation by controlling the UV irradiation time on graphene, and investigated the charge transport and the evolution of the transport gap according to the extent of oxidation. With increasing oxygenous defect density


A major hurdle for single particle cryo-EM in structural determination lies in the specimen preparation impaired by the air-water interface (AWI) and preferential particle-orientation problems. In this work, we develop functionalized graphene grids with various charges via a dediazoniation reaction for cryo-EM specimen preparation. The graphene grids are paraffin-assistant fabricated, which appear with less contaminations compared with those produced by polymer transfer method. By applying onto three different types of macromolecules, we demonstrate that the high-yield charged graphene grids bring macromolecules away from the AWI and enable adjustable particle-orientation distribution for more robust single particle cryo-EM structural determination. Air-water interface and preferential orientation problems are crucial challenges in cryo-EM specimen preparation. Here, the authors utilize graphene-coated EM grids functionalized by salts with various electrostatic properties, successfully overcoming preferred orientation. However, high-quality cryo-specimen preparation still faces many challenges, exemplified by the preferential orientation and air-water interface (AWI) problems2, reducing the success rate and efficiency of high-resolution structure determination by cryo-EM. To alleviate the preferential orientation problem, tilting the cryo-specimen during data collection has been applied3, which, however, may result in large beam-induced motion and inaccurate defocus estimation during data processing, thereby impairing the high-resolution reconstruction. Many efforts have been also made to solve the preferential orientation as well as AWI problem by introducing supporting films9, among which graphene membrane is the most promising one.Cryo-electron microscopy (cryo-EM) technique has been successfully applied to reveal the molecular basis of many essential macromolecules at atomic resolution10, an atomically thin film comprised of sp2-bonded carbon atoms, has a superior electrical and thermal conductivity, mechanical strength and negligible background noise. Graphene membrane of sole nature, however, might induce preferential orientation problem of particles adsorbed to its surface11. The fabrication of clean graphene grids also remains a practical challenge for cryo-EM specimen preparation. Although the graphene functionalization has been used to specifically capture and anchor target particles to avoid the AWI14, the current protocols of the bioactive functionalization process of graphene, based on the electrophilic reaction or conjugation interaction with the π-π bonds, are sub-optimal due to contamination built-up on the graphene surface during the transfer process or storage.GrapheneIn this work, to overcome the graphene functionalization and transfer challenges and solve the preferential orientation problem, we develop a method to produce robust graphene grids functionalized with different electrostatic charges, which provide various electrostatic interaction interface for target macromolecules to bind in and enrich orientations. Applied onto 20S proteasome and ribosome complex, the modified graphene with different charge properties enables particle adsorption on the surface with favorite orientation distribution, thus solving the AWI and preferred orientation problems in cryo-EM specimen. Finally, we successfully determine the cryo-EM structure of Ll.LtrB RNP at 3.2-Å resolution, the highest resolution of the endogenously purified group II intron RNP, whose structural reconstruction is hindered by severe preferential orientation problem on conventional EM grids.15, theoretically making it easier to be cleaned from the graphene surface.As the clean transfer of graphene remains one of the major practical barriers for the application of graphene-based supporting films in cryo-EM, we aimed to develop a robust procedure to transfer graphene on EM grids without contamination. We found that paraffin is an ideal transfer mediator. Paraffin is a chemically inert alkane in solid state with relatively lower melting temperature (50–60 °C) compared to other commonly used transfer mediator such as PMMA, which is normally dissolved and removed by organic solvents. Moreover, the graphene-adsorption energy of paraffin is reported to be much smaller than PMMA16 and then used paraffin to support the graphene during the fabrication procedure  was found on the Raman spectra of the functionalized graphene, suggesting the presence of the modification-induced defects  and SO3--functionalized graphene (SFG) grids have decreased water contact angles of ~67° and ~66°, respectively  to characterize the elemental composition of the functionalized graphene and found the presence of N 1 s Fig. , upper a. p Fig. , down siely Fig. , allowinWe used the charged graphene grids to make cryo-EM specimens of 20S proteasome  The electrostatic interaction is much stronger, compared with other interaction modes such as the van der Waals force, allowing us to effectively manipulate the orientational distribution of target macromolecules. 2) We adopted a dediazoniation reaction to functionalize graphene in moderate conditions with high efficiency. 3) Graphene was modified prior to its transfer onto EM grids to reduce the chance of contamination and then transferred to EM grids with a paraffin-assistant graphene grid fabrication method.The charged graphene grids provide different surfaces for protein molecules or macromolecule complexes to interact with, based on the target molecule’s surface charge properties, therefore allowing multiple orientations on various charged graphene grids and avoiding impairment by the AWI.4, and the mixture was stirred at 0 °C for 5 min. 1 ml 2.2 M sodium nitrite solution was added into the mixture at 0 °C and the solution was stirred vigorously for 1 hr followed by filtration to obtain a crude product. We used acetone to dissolve the solid, used ether to precipitate the product, and finally filtered the product again to get pure diazonium salt. The benzene-sulfonic acid group diazonium salt was dissolved in deionized water and benzene-anilinic group diazonium salt was dissolved in DMSO, both at a final concentration of 20 mM. For better reaction efficiency, 20 µl 1% (by wt) anionic surfactant SDS aqueous solution was added into 100 µl 20 mM diazonium salt solution to form the reactant solution for later graphene functionalization procedures.0.22-gram p-phenylenediamine and 0.35-gram p-aminobenzene sulfonic acid were dissolved in 5 ml deionized water, and then added by 1 ml 50% HBF1H NMR spectra were recorded at room temperature on a Bruker AVANCEIII 400 MHz spectrometer in D2O (for 4-diazoniobenzenesulfonate tetrafluoroborate) or DMSO-d6 (for 4-aminobenzenediazonium tetrafluoroborate), using TMS or solvent peak as a standard. Low-resolution mass spectral analyses were performed with Waters AQUITY UPLCTM/MS.The Graphene was grown on copper foil by the chemical vapor deposition (CVD) method. We firstly cut a piece of graphene-coated copper foil and dropped the diazonium salt solution onto graphene surface, followed by a 30-min incubation at 40 °C. Afterwards, graphene membrane was washed by 1 ml deionized water for 5 times and immersed into deionized water for an additional 0.5–1 h to ensure the complete removal of residual diazonium reagent from graphene surface.2 (11.5 sccm) and Ar (35 sccm) atmosphere for 30 s. We then heated the copper foil to 65 °C, and added a small piece of paraffin (Sigma-Aldrich 18634) onto its functionalized graphene side, which immediately melted and spread to cover the whole foil. After the paraffin spread as a thin film on top of the graphene, the copper foil was cooled down to room temperature and the paraffin solidified. Afterwards, the paraffin-graphene-copper was gently put on the surface of 0.5 M ammonium persulfate (APS) solution with the paraffin side facing up, and incubated for 5 h. After the copper foil was thoroughly etched off, the paraffin-graphene was rinsed with deionized water several times, then transferred into a 40 °C incubator and incubated for at least 1 h, to stretch and diminish wrinkles in the graphene membrane. After that, we used Quantifoil grids  to scoop out the paraffin-coated graphene from the 40 °C deionized water and air-dried the graphene grids in a clean chamber at 40 °C overnight. The paraffin-graphene-grids supported by oil-absorbing paper  were then incubated at 65 °C for 5–10 min to remove most paraffin coating. Afterwards, the grid was soaked into petroleum ether (preheated to 65 °C) and stirred at 60 rpm for 3 h to completely remove the paraffin. Finally, the grids were washed by acetone, isopropyl alcohol (IPA) and water consecutively to clean off residual petroleum ether and air-dried.We firstly removed the unfunctionalized graphene grown on the backside of the copper foil by placing the foil in a Gatan plasma machine (Model 950) with its backside up, and performed the plasma treatment at a O15 as control, to compare and demonstrate the cleanliness of graphene grid fabricated in this work. To better describe the cleanliness of graphene in the holes on EM grid, here we define a hole with more than 5 contamination spots with the area of >400 nm2 as highly contaminated hole, and we found almost all of the holes on the graphene grids fabricated by PMMA-assisted transfer method were highly contaminated. For graphene grid fabricated by paraffin-assisted method, we imaged 141 holes and found 123 holes (~87%) with no or no more than five contamination spots, and 98 holes (~70%) with no or no more than three contamination spots.We used graphene membrane transferred by PMMA-assisted methodX-ray photoelectron spectra was collected on a ESCALAB Xi + (ThermoFisher Scientific), with the monochromatic Al Kα X-ray beam spot of 500 μm. Raman spectra was measured on a LabRAM HR-800 (Horiba) with the 633-nm laser wavelength. For water contact angle measurement, we applied 0.7 μl deionized water onto the grid surface and used OCA15Pro (Dataphysics) to collect the water contact angles. The roughness of the suspended graphene surface was characterized using AFM .Thermoplasma acidophilum 20S proteasome was expressed and purified from Escherichia coli BL21(DE3) strain by following previously reported protocols19. Briefly, we transformed the plasmids into Escherichia coli competent cells, which contained α and β subunits of 20S proteasome, with the N terminal of β subunit tagged by six histidines. After induced expression, Escherichia coli cells were sonicated and the cell lysates were then applied onto affinity nickel column to get purified 20S proteasome sample.Lactococcus lactis strain IL1403 according to the published methods17. We firstly constructed a plasmid containing Ll.LtrB intron with the ORF region in its DIV depleted, its encoded protein LtrA and a chitin binding domain (CBD), which was then transformed into Lactococcus lactis strain IL1403 and allowed for induction. The cell lysates were loaded onto affinity chitin column, followed by dithiothreitol elution and ultracentrifugation to get purified Ll.LtrB RNP sample.Ll.LtrB RNP were purified from E. Coli ribosome was purchased from NEB company (P0763S).For sample vitrification, we firstly pipetted 7 μl sample buffer  onto the functionalized graphene grids, and incubated the grids for 2 min at a chamber of high humidity. Afterwards, the grids were edge-blotted by filter papers to remove the extra buffer, immediately followed by adding 4-μl sample solution, incubated for 2 min and then transferred into Vitrobot Mark IV (ThermoFisher Scientific). The humidity of Vitrobot chamber was set as 100%, and the temperature as 8 °C. The grids were blotted by filter papers (Ted Pella) for 1 s with a force of −2, and then plunge-frozen into liquid ethane. The vitrified specimens were stored in liquid nitrogen. To prepare conventional graphene-supported cryo-specimen, we pipetted 4 μl sample solution onto freshly glow-discharged graphene grid, and blotted the grid for 1~2 s with a force of −2, using Vitrobot Mark IV (ThermoFisher Scientific). After blotting, the grid was frozen into liquid ethane and stored in liquid nitrogen.-/Å2. The movies were motion-corrected by MotionCor220. The CTF values were estimated by CTFFIND421 and the resulted micrographs were imported to Relion3.1 for particle picking and 2D classification22. After discarding junk particles grouped in bad 2D classes, the remaining particles were imported for 3D classification and refinement, to obtain the final 3D reconstructions. The final particle number used for the reconstruction of Ll.LtrB was 399,660, and the resolution was 3.2 Å, estimated by the Fourier Shell Correction (FSC) = 0.143 cutoff criteria. Structural visualization and analysis were performed in UCSF Chimera23.The cryo-EM datasets were collected on a Titan Krios (300 kV) microscope equipped with a Gatan K3 Summit direct electron detector. The pixel size was 0.97 Å and every movie contained 32 frames with a summed dose of 50 e24. We collected 8 frames at each tilt angle, with a summed dose of 3 e-/Å2, which were further motion-corrected by MotionCor220. We used IMOD software25 to align and reconstruct the tomograms.Tilted series were acquired on a Titan Krios (300 kV) microscope equipped with a Gatan K3 Summit direct electron detector, from −60° to +60° with a step of 3° using SerialEMFurther information on research design is available in the Supplementary InformationReporting Summary


Idiopathic pulmonary fibrosis (IPF) is frequently accompanied by comorbidities, with the management of these comorbidities crucial for clinical outcomes. This study investigated the prevalence, incidence, changes over time, and clinical impact of comorbidities in IPF patients, based on nationwide claims data in South Korea.This retrospective cohort study utilised nationwide health claim data in South Korea between 2011 and 2019. Patients with IPF were defined as those with ICD-10 code J84.1 and Rare Intractable Disease code V236 who made at least one claim per year. Patients were classified by sex, age, pirfenidone use and burden of comorbidities, and differences among groups were determined.The yearly prevalence rate of IPF increased from 7.50 to 23.20 per 100,000 people, and the yearly incidence rate increased from 3.56 to 7.91 per 100,000 person-years over time. The most common respiratory comorbidity was chronic obstructive pulmonary disease (37.34%), followed by lung cancer (3.34%), whereas the most common non-respiratory comorbidities were gastro-oesophageal reflux disease (70.83%), dyslipidaemia (62.93%) and hypertension (59.04%). The proportion of some comorbidities differed by sex, age and use of pirfenidone. The proportion of lung cancer was higher in patients treated with pirfenidone, whereas the proportion of anxiety and depression were lower in patients not treated with pirfenidone. Charlson comorbidity index ≥ 4 was associated with increases in hospitalisations and total medical costs.The yearly prevalence and incidence of IPF and comorbidities in Korea increased over time. These comorbidities affected the use of pirfenidone and medical resources.The online version contains supplementary material available at 10.1186/s12890-023-02340-8. Idiopathic pulmonary fibrosis (IPF), defined as a chronic, progressive, fibrotic lung disease of unknown causes, is the most common type of interstitial lung disease . AlthougTreatment options for patients with IPF are often limited, despite the use of antifibrotic agents such as pirfenidone and nintedaninb , 6. BecaThis was a retrospective cohort study based on nationwide health claims data in South Korea. De-identified nationwide health claims data between 2011 and 2019 were collected from the database of the Korean Health Insurance Review and Assessment (HIRA), which includes all health insurance claims from the Korean National Health Insurance System (NHIS) and therefore covers most of the population. The HIRA database presents all information on healthcare utilisation by outpatients and inpatients, including their demographic characteristics, diagnoses, and prescribed medications. Diagnostic codes were determined using the 10th revision of the International Classification of Disease and Related Health Problems (ICD-10). The study was approved by the Institutional Review Board of Asan Medical Center (No. 2020-1000), which waived the requirement for informed consent due to the retrospective nature of the study. The study was performed in accordance with the principles of the Declaration of Helsinki.Patients in the HIRA database were defined as having IPF if they had made at least one claim per year under the ICD-10 code J84.1 of the medical care system. Because patients in South Korea with confirmed IPF were enrolled as the Rare Intractable Diseases (RID), we additionally used RID code V236 to define patients with IPF patients. In South Korea, patients assigned RID code V236 can benefit from reduced medical costs from the NHIS. Hospitals charge the NHIS for the remainder, but if their diagnosis does not meet RID diagnostic criteria, the NHIS refuses to pay these hospital costs. This study therefore assumed that patients were accurately diagnosed at the time of RID registration. Therefore, IPF in the present study was defined as a composite of ICD-10 code J84.1 and RID code V236. The prevalence was estimated by including patients who had made at least one claim per year for IPF. The earliest claim date for IPF was defined as the index date, with incidence calculated based on the year of earliest claim.The demographic characteristics, comorbidities, prescribed pirfenidone, and medical costs of patients with IPF were analysed. Comorbidities were classified as respiratory or non-respiratory. Respiratory comorbidities included COPD, lung cancer, pulmonary embolism, pulmonary hypertension, and obstructive sleep apnoea, whereas non-respiratory comorbidities included GERD, dyslipidaemia, hypertension, diabetes mellitus, ischaemic heart disease, anxiety, depression, and congestive heart failure. The prevalence of comorbidities was assessed at (i) the time of diagnosis, defined as the period from 1 year before to initial diagnosis of IPF; (ii) 1 year after diagnosis, defined as the period from initial diagnosis of IPF to 1 year later; and (iii) 3 years after diagnosis, defined as the period from 1 to 3 years after the initial dignosis of IPF. The occurrence of comorbidities was defined by at least one claim for disease-specific ICD-10 codes , sex, CCI (0–3 vs. ≥ 4) and pirfenidone use, with the latter classified into two groups, those treated with pirfenidone more than 3 months (pirfenidone user group) and those treated for less than 3 months or not treated with pirfenidone (pirfenidone non-user group). In pirfenidone user group, we classified into two groups, those treated with 600 mg oral pirfenidone three times daily for more than 3 months (standard group) and those treated with < 600 mg oral pirfenidone three times daily for more than 3 months (low dose group). Because the cost of pirfenidone has been covered by the NHIS in Korea since October 2015, this classification applied to patients after 2016.p values < 0.05 regarded as statistically significant.Categorical variables are presented as number and percentages, and continuous variables as means and standard deviations. Annual prevalence was calculated as the number of the prevalent cases per 100,000 people. Annual incidence was calculated as the number of newly diagnosed IPF patients per 100,000 person-years. The denominator of prevalence and incidence was based on the annual population census obtained from the Korean Statistical Information Service . PatientThe demographic characteristics of patients with IPF are shown in Table The prevalence of comorbidities was evaluated in the 21,111 patients with IPF by age and sex Tables . The mosClassification by sex showed that, of the respiratory comorbidities, COPD (40.58% vs. 29.32%), lung cancer (4.11% vs. 1.45%), and obstructive sleep apnoea (0.37% vs. 0.16%) were more common in men than in women, whereas pulmonary hypertension (2.43% vs. 1.11%) was more prevalent in women than in men. Evaluation of the non-respiratory comorbidities showed that the rates of diabetes mellitus and ischaemic heart disease were higher in men, whereas GERD, anxiety, depression and congestive heart failure were more frequent in the women group. A higher proportion of patients aged ≥ 70 years had COPD (39.39% vs. 34.20%) and pulmonary embolism (1.92% vs. 1.11%)  and low (CCI 0–3) burdens of comorbidities within 90 and 365 days showed that, within 90 days, patients with high CCI had utilised more resources associated with admission  was the most common respiratory comorbidity, followed by lung cancer (3.34%) and pulmonary embolism (1.60%), whereas the most common non-respiratory comorbidities were GERD 70.83%), dyslipidaemia (62.93%), and hypertension (59.04%). The prevalence rates of these comorbidities were comparable to those in previous studies, although the reported ranges of comorbidity rates were very wide 3%, dysli. When co% was theOf the 12,943 IPF patients since 2016 in this study, 5449 42.10%) had been treated with pirfenidone. This relatively low rate of pirfenidone use may be due to the lack of insurance coverage for this medication prior to October 2015 and/or to our definition of pirfenidone use as treatment with this agent for more than 3 months. A recent real-world cohort study of a Korean population found that only 656 48.77%) of 1345 patients with IPF had been treated with antifibrotic agents, including pirfenidone and nintedanib . This lo.77% of 1.10% had Several studies have evaluated the relationships between comorbidities and clinical outcomes in patients with IPF , 9, 20. The present study had several limitations. First, IPF was defined based on the ICD-10 code at the time of diagnosis. Although actual diagnosis was associated with the operational definition based on ICD-10 code, our use of the latter may overestimate or underestimate the prevalence and incidence rates of IPF and comorbidities . To imprIn conclusion, this study found that the prevalence and incidence rates of IPF in the Korean population increased annually. Many patients with IPF had respiratory and non-respiratory comorbidities, although the prevalence was dependent on sex, age and use of pirfenidone. The prevalence of these comorbidities was higher 3 years after than at the time of IPF diagnosis and affected the use of pirfenidone and medical resources. Additional studies are needed to clarify causal relationships.Additional file 1. International Classification of Diseases, 10th revision codes (ICD-10 codes) for the diseases evaluated in this study.Additional file 2. Prevalence of comorbidities in patients with idiopathic pulmonary fibrosis during the 3 years after initial diagnosis.Additional file 3. Clinical characteristics of patients with idiopathic pulmonary fibrosis based on the pirfenidone dose since 2016.


The burden of cataracts was substantial in the current aging world. However, few epidemiological studies have examined the associations between climate and weather conditions and cataract in older populations. We aimed to investigate the associations of air relative humidity and temperature with cataracts in older adults in China.We used the cohort data from 2002, 2005, 2008, 2011, 2014, and 2018 waves of the Chinese Longitudinal Healthy Longevity Survey (CLHLS). A total of 62,595 Chinese older adults aged between 65 and 105 years were included in the analyses. City-level annual average air humidity and temperature during 2001 and 2017 (before the survey year) was used to measure population exposure. A cataract was self-reports based on the medical record or the doctor's diagnosis and 8,071 older adults had cataract. Covariates included socio-demographic, health status, lifestyles, and chronic conditions. We adopted the Generalized estimation equation (GEE) model to analyze the associations of relative humidity and temperature with cataracts.We found that the average relative humidity  in the past year was inversely associated with cataract likelihoods in older adults and a positive association between temperature  in the past year and cataract likelihoods in older adults. The associations were robust in stratified analyses by sex, urban/rural residence, and education level. Furthermore, we found a nonlinear J-shaped relationship between temperature and cataract prevalence.Our findings provide the evidence that higher temperature and low relative humidity may be associated with cataracts in older adults. Cataract, defined as any opacity of the crystalline lens in the eye that affects clear vision, is a common condition in later life , 2. CataEnvironmental factors-induced adverse health outcomes were well-documented . A largeIn light of the increasing occurrence of extreme climates and extreme temperatures, it is essential to examine their impacts on health, especially that of aging individuals, which will be directly related to future healthy aging and the creation of age-friendly environments. However, epidemiological studies on cataracts in older adults are rare. This study examined the associations of humidity and temperature with cataracts among a nationally representative sample of older adults in China.Data used in this study were derived from the Chinese Longitudinal Healthy Longevity Survey (CLHLS) waves of 2002, 2005, 2008, 2011, 2014, and 2018. The CLHLS applied a multistage, stratified cluster sampling design in 23 out of 31 provinces in China. The goal of CLHLS was to understand better the determinants of healthy longevity in Chinese older adults. Between 1998 and 2018, the CLHLS was conducted in half of the counties and cities (randomly selected) in 23 out of 31 provinces in China. Details of the CLHLS have been described . Hourly data was annually or seasonally averaged to measure residential temperature and relative humidity exposure for each participant in CHLHS, matching by the administrative codes of the cities where the CLHLS samples are resident (non-publicly available information). The temperature and humidity exposures adopted in this study are (1) Average relative humidity over the past year and (2) Average temperature in the past year. To reduce the seasonal bias, we also used (3) Average temperature in the cold months and (4) Average temperature in the warm months to measure the participants' temperature exposure. Similar with the previous publications  and temperature (°C) are derived from the China Meteorological Data Network  for continuous variables and as frequencies and percentages for categorical variables. Associations of relative humidity and temperature with cataracts were analyzed by generalized estimation equation (GEE). We chose GEE because using the observed correlational structure of the data, we can obtain efficient and unbiased regression parameters . We usedA total of 62,595 observations were included in this study. The mean age of the participants was 85.78 ± 11.27 years; the gender composition was generally balanced (44.4% men). The prevalence of cataracts was 12.89%. Nearly half (47.6%) of the older adults lived in urban areas, and nearly half (48.6%) lived in east China. More than half (55.6%) of the participants were illiterate, and about 36.2% of them were married. About 25.4% of the participants have disability in activities of daily living, and 20.9% of them had cognitive impairment .The average relative humidity and temperature are also summarized in The average relative humidity over the past year was negatively correlated with the prevalence of cataracts. In the past year, each 1% rise in average relative humidity was associated with a 1.4% decrease in cataracts . In contrast, the average temperature in the past year was positively related to the risk of cataracts. There was a 4% increase in cataracts with each 1°C increase in average temperature over the past year . Based on the subgroup analyses, we found that the associations were robust across subgroups of cataract risk factors, including sex, urban/rural residence, and level of educational attainment. However, a non-significant effect of average temperature was found for urban population .via following analyses: using the average temperature in the warm months (Model I), the average temperature in the cold months (Model II), the average daily maximum temperature (Model III), and the average daily minimum temperature (Model IV). The results were consistent with our main findings  and 1.04  for annual average relative humidity and temperature, respectively. Furthermore, we found a nonlinear J-shaped relationship between temperature and cataracts prevalence. The associations indicated that extreme heat and low humidity may be linked to a higher incidence of cataracts in older individuals.We found that lower humidity and higher temperatures were associated with an increased risk of cataracts. Although limited epidemiological studies have explored the health effects of humidity, similar findings have been reported in previous studies on other eye diseases. Based on a study of 100,636 participants, relative humidity was negatively related to allergic conjunctivitis , there iRegarding temperature, Miranda et al. reported that senile cataract develops earlier and is more prevalent in warm regions, and that cataract prevalence increases with increasing temperature . AccordiLow humidity may affect cataract prevalence by drying out the airways, resulting in hyperosmolarity, which stimulates nerves to produce reflex responses and may release inflammatory biomarkers . It has 2.5 and NO2) on cataracts. Furthermore, further research from the perspective of molecular biology is needed to investigate the causal relationship between relative humidity and temperature and cataract development.This study contains several limitations. First, relative humidity and temperature are only measured from ambient data at the city level (not from indoors), since personal exposure varies with the ventilation in a house and the movement of people inside the house. Second, except for humidity and temperature, we were unable to assess the possible confounding effects or interactive effects of other environmental exposures  and Natural Science Foundation of Hunan China (2020JJ4087), and the State Laboratory of Sub-tropical Architecture of China (2020ZB10).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The physical fundamentals for polariton hybridizations depend on the evanescent fields coupling originating from the constituent materials as well as the phase match condition, which can be severely affected by the α-MoO3 thickness and actively tuned by the gate voltages. Hybrid polaritons propagate with in-plane anisotropy that exhibit momentum dispersion characterized by elliptical, hyperboloidal and even flattened iso-frequency contours (IFCs) in the THz range. Our results provide a tunable and flexible anisotropic polariton platform for THz sensing, imaging, and modulation.Electromagnetic field confinement is significant in enhancing light-matter interactions as well as in reducing footprints of photonic devices especially in Terahertz (THz). Polaritons offer a promising platform for the manipulation of light at the deep sub-wavelength scale. However, traditional THz polariton materials lack active tuning and anisotropic propagation simultaneously. In this paper, we design a graphene/α-MoO In THz, the optical response of α-MoOequation : (1)εj=εeference . As showGraphene’s surface conductivity is modeled using the Kubo formula ,22: (2)σe is the electron charge, FE is the Fermi energy, ℏ is the reduced Planck constants, and τ is the momentum relaxation time. The relaxation time of charge carriers in graphene is set to 0.5 ps, which is typical for encapsulated graphene used in optical experiments at ambient conditions. The permittivity of graphene can be written as ε0 is the vacuum permittivity. The real parts of the permittivity tensors of graphene with different Fermi levels are negative in the frequency range from 7 THz to 12 THz  of the real part of the z component of the near-field amplitude Ez. Due to the opposite in-plane permittivity signs of α-MoO3 along [001] and [100] crystal directions, polariton propagation along the [100] direction is forbidden at the low frequency band, which resulted in hyperbolic Ez distribution centered along the crystallographic directions of [001]  and the dispersion of PhPs (without PPs). If there is a crossing point between the dispersions for a given configuration, there will be coupling. Therefore, the graphene Fermi level, α-MoO3 thickness, spectra range, and polariton momenta must be optimized. The permittivity results suggest the potential formation of hybrid plasmon-phonon polariton modes in two excitation bands. We thereby construct a platform for hybridizing isotropic graphene PPs with anisotropic α-MoOlaritons . However3, 3 are not considered in this calculation. The dispersion relation of electromagnetic modes in biaxial α-MoO3 slabs can be described by the following analytical approximations  direction, explaining the canalization of hybrid polaritons along this direction e. When t3 heterostructure can be strongly enhanced, and more importantly, the hybrid polaritons can be actively tuned from elliptic to flattened wavefronts by electrical gating. Moreover, the precise control of light localization is very important in nanophotonics, and the electromagnetic field concentration can be further enhanced by fabricating nanostructures that act as Fabry-Perot resonators. For this purpose, we precisely shaped graphene/α-MoO3 heterostructures into square nanostructures for tailoring electromagnetic field localizations. We adopted a phenomenological cavity model to simulate the near-field amplitude and phase patterns within graphene/α-MoO3 nanostructures [3 nanostructures, the excited polariton wave propagates outward and is reflected by the nanostructure boundaries. The dipole-launched polariton waves interfere with the reflected waves perpendicular to the boundaries. The total field detected can be described as: E0 and p,jE are the dipole-launched polariton wave and that reflected from four boundaries, respectively. Reflected waves are given by R0 and Δφ are the reflectivity and phase shift of the polariton wave, respectively. pγ, jr and pλ represent the polariton damping rate, distance between the nanostructure edge and dipole tip, and the polariton wavelength, respectively. In simulations the amplitude of the dipole-launched polariton wave E0 was set to 1 and the damping rate pγ to 0.4 eV, whereas the polariton wavelength pλ is set based on π was used for the phase shift of the polariton wave [The near-field simulations review found that the hybrid polaritons in graphene/α-MoOructures ,26. A po3 micro-flakes at the cross points in pλ. x and y axes at different wavelengths. Except for the flattened-band dispersion at 10.1 THz, polariton propagation along y axis is forbidden, which results in interference fringes only appearing in the horizonal direction. As the frequency decreases, the number of bright spots decreases and the field localizations are strongest at the regions close to the corner of the nanostructures. The realization of electromagnetic field localization in two dimensions using graphene/α-MoO3 nanostructures can offer several advantages in terms of efficient focusing and manipulating of light at the nanoscale, which can further be integrated with photonic or optoelectronic devices to enhance light-matter interactions. In addition, due to the elliptical-type dispersion, hybrid polaritons propagate along 3 heterostructures. It was found that the polariton hybridization between isotropic graphene PPs and anisotropic α-MoO3 PhPs strongly depends on the α-MoO3 thickness and can be actively tuned by the gate voltages. In addition, the hybrid polaritons propagate with in-plane anisotropy that exhibits momentum dispersion characterized by elliptical, hyperboloidal and even flattened IFCs in the THz range. The near-field results suggest that the localized electromagnetic fields can be enhanced in nanostructures, which provide a flexible anisotropic polariton platform and can further be integrated with photonic crystals or quantum well structures to ulteriorly enhance THz-matter interactions. Our findings demonstrate that active tuning of THz polaritons with certain propagations can be used to steer THz light along specific directions at the nanoscale. More significantly, these results will provide guidelines for THz device fabrications and experiments with dynamically controllable properties.In conclusion, we designed and investigated polariton hybridizations in graphene/α-MoO


In order to maintain the flexibility of PCM without volume shrinkage under the external force, cross-linked graphene aerogel was prepared by the cysteamine vapor method. The cross-linked graphene aerogel had a high stress–strain durability and chemical stability for infiltrating PCM to produce a form-stable PCM composite. The latent heat of PCM is one of the elements to estimate the capacity of PCM thermal energy storage (TES) during the phase transition process. The cross-linked graphene aerogel-supported PCM composite showed a great TES to be utilized in thermal-to-electrical energy harvesting. The cross-linked graphene aerogel also had an excellent mechanical property of preventing damage at a high temperature. The porous materials exhibit internal structures that can infiltrate plenty of solid–liquid working materials into the internal space ,2. That However, one of the recent problems in the graphene aerogel is the volume shrinkage while infiltrating a polymer into the internal space ,16. The It is necessary to utilize graphene aerogel for fabricating form-stable PCM composite due to the leakage problem under the solid–liquid melting process ,30. The In this study, the importance of modified graphene aerogel shows not only the reduction of volume shrinkage but also the prevention of the leakage problem under external force. There are conventional graphene aerogel, PDMS embedded graphene aerogel, and cross-linked graphene aerogel with some typical morphologies, and cross-linked graphene aerogel supported PCM composite shows excellent form-stability under the external force at a high temperature. For the microencapsulation method of fabricating form-stable PCM composite, the replacement of supporting shell material causes a decrease in pure PCM weight fraction, which affects the TES capacity during the phase transition process. The graphene aerogel-supported PCM composite has a high PCM weight fraction and keeps a high TES capacity for utilizing stored energy sufficiently. It indicates that cross-linked graphene aerogel is an optimized supporting material and can be employed to fabricate tremendous form-stable PCM composites.4 oxidized GO had high values in the range of 4–10 pH values, as shown in 0 high-pressure region  that the PEG can be melted at 80 °C. Graphene aerogels need high mechanical properties to sustain the melted PEG in the internal pores. The pure PEG weight fractions of each PEG composite were presented in The fabricated graphene aerogels exhibited high porosities and appropriate mechanical properties. The form-stable test indicates that the graphene aerogels can act as excellent supporting materials to prevent the leakage problem a. In ordIt was demonstrated that the graphene aerogel-supported PEG composites had excellent form stabilities during the melting process. In order to observe the durability of external force at a high temperature, all of the samples were placed on the hot plate under 80 °C. An external force of 2 N was applied, and the resulting deformation of the PEG composites was presented in In this work, we fabricated the graphene aerogel, PDMS embedded graphene aerogel, and cross-linked graphene aerogel (GCA) for 3D porous supporting materials. The modified graphene aerogel, especially the cross-linked GCA sheet, showed a high value of contact angle to infiltrate pure PCM effectively. All the graphene aerogel exhibited nearly 370 of their specific surface area under the BET measurement. High dimensional stability and durability of graphene aerogels are required to infiltrate a large amount of pure PCM, which is over 98%, into the internal space without any volume shrinkage. The modified graphene aerogels exhibited great flexibility to reduce the volume shrinkage effectively. Since the form-stability of PCM composites at high temperatures is essential for applications of PCM. The mechanical properties of the graphene aerogels supported PCM composites were analyzed under the 2N external force during the phase transition process. The cross-linked graphene aerogel could retain the PCM composite without any leakage, even at 80 °C. The cross-linked porous aerogel showed a large potential as a light-weight container for aerospace applications.3), sulfuric acid (H2SO4), potassium permanganate (KMnO4), perchloric acid (HClO4), and citric acid were purchased from Sigma-Aldrich .Polyethylene glycol  was selected as a phase change material (PCM). Graphene nano-platelet , Polydimethylsiloxane (PDMS), nitric acid (HNO3) and potassium permanganate (KMnO4)/perchloric acid (HClO4) solution to oxidize initial GO powders. After removing the excess KMnO4 by adding citric acid, the oxidized GO was purified by centrifugation and freeze-drying process. The oxidized GO aerogel was obtained by mixing with GNP, evaporating solvent, and utilizing the cysteamine vapor method to cause a chemical reaction with GO functional groups [The first step of fabricating porous graphene aerogel was synthesizing graphene oxide (GO). The preparation of GO was followed by the modified Hummers’ method after grinding the graphite ,42. The l groups ,46. As al groups .The graphene oxide (GO) and Graphene nano-platelet (GNP) dispersion abilities were measured by a Zetasizer . The fabricated graphene sheets were put in the contact angle goniometer  to obtain hydrophilic properties. The maximum stress–strain durability of graphene aerogels was observed by a Universal Testing Machine . Field-emission scanning electron microscopy  was used to measure the internal surface structures of graphene aerogels and PCM composites under a 5 kV accelerating voltage. A Brunauer–Emmett–Teller (BET) analyzer  was provided to measure the graphene aerogel internal structures. The intrinsic peaks were confirmed using Fourier-transform infrared spectroscopy  and X-ray diffraction  from 10° to 70° with 3°/min rate (2θ). The phase transition field and latent heat (∆H) of PCM composites were obtained using differential scanning calorimetry  under the range of 15 °C to 90 °C with 10 °C/min. The thermal stability was confirmed by using a thermal gravimetric analyzer .


Spider silk is one of the hottest biomaterials researched currently, due to its excellent mechanical properties. This work reports a novel humidity sensing platform based on a spider silk-modified quartz tuning fork (SSM-QTF). Since spider silk is a kind of natural moisture-sensitive material, it does not demand additional sensitization. Quartz-enhanced conductance spectroscopy (QECS) was combined with the SSM-QTF to access humidity sensing sensitively. The results indicate that the resonance frequency of the SSM-QTF decreased monotonously with the ambient humidity. The detection sensitivity of the proposed SSM-QTF sensor was 12.7 ppm at 1 min. The SSM-QTF sensor showed good linearity of ~0.99. Using this sensor, we successfully measured the humidity of disposable medical masks for different periods of wearing time. The results showed that even a 20 min wearing time can lead to a >70% humidity in the mask enclosed space. It is suggested that a disposable medical mask should be changed <2 h. Spider silk is well known for its outstanding mechanical properties. One of them is that spider silk is particularly sensitive to water . When weQuartz tuning forks (QTFs) have been traditionally used as a timing reference in wristwatches. The QTF is a bimorph cantilever based on the piezoelectric properties of the quartz . The senIn this work, a sensitive and cost-effective humidity sensor was developed by using quartz-enhanced conductance spectroscopy (QECS) . CompareQTFs were purchased from the Shenzhen XinChuangYue Electronic components Factory, with a resonance frequency of ~32.7 kHz and a Q factor of >60,000 in a sealed vacuum metallic package and ~10,000 in the air after removal of the metallic housing. The geometrical parameters were measured by an optical stereomicroscope. The prong length, prong width, and prong spacing of a QTF were 3.8 mm, 0.6 mm, and 0.3 mm, respectively. The effective spring constant of the QTF is ~20 kPa . The effTo evaluate the humidity sensing performance of the SSM-QTF, we employed the experimental setup shown in f of the QTF can be expressed as [QTFk, QTFm, and QTFE are the spring constant, effective mass, and Young’s modulus, respectively. The t, l, and w are the QTF prong thickness, QTF prong length, and QTF prong width, respectively. We considered that the mass of the spider silk was negligible, i.e., QTFdm≪QTFm. The resonance frequency shift, after modification by the spider silk, can be expressed as:The resonance frequency essed as ,51:(1)fQQTFdk value, which results from the spider silk spring constant ksilk. Due to the fact that the QTF and the spider silk were connected in parallel, the effective spring constant effk of the SSM-QTF can be expressed as:The resonance frequency shift can be attributed to the The electrical parameters of the SSM-QTF were measured by the abovementioned system in f = 241.3 Hz. This can be attributed to the mass increase caused by the spider silk and the glue.A bare QTF without modification was also evaluated, under the same experimental conditions. The frequencies of the sine signals were scanned from 32,500 Hz to 32,560 Hz and from 32,725 Hz to 32,805 Hz with a step of 0.3 Hz for SSM-QTF and standard QTF, respectively. The resonance curves of the bare QTF and SSM-QTF are shown in f = 25.95 Hz. This can be attributed to the modulus of spider silk being inversely proportional to the humidity; the effective modulus of the SSM-QTF decreased with the increase in the humidity [f was caused by the spider silk, not the QTF itself.humidity . The curQ factors of the SSM-QTF with different humidities. As the humidity increased, the spider silk absorbs water molecules, increasing the additional mass attached to the QTF. In addition, as the humidity increased, the damping effect by the water absorption into spider silk increased; thus, the quality factor of the SSM-QTF decreased [Q factor decreased from 5253 to 2790, by >2400. For comparison, the Q factor change of a bare QTF without spider silk modification was <200, shown in the inset of ecreased . As a reThe relative humidity (RH) was converted into absolute humidity by the Vaisala humidity calculator. With an absolute humidity change from 8042 ppm to 24,056 ppm, the detection sensitivity of the SSM-QTF was calculated to be ~12.7 ppm. This humidity dynamic range was limited by our gas stream system, which can be improved by a better humidifier. The comparison of the SSM-QTF with current technology such as optical fibers, cantilever, and string resonators are summarized in −3 Hz at the optimum time of ∼30 min. With the optimal integration time, the detection sensitivity can be improved to ~0.8 ppm.The resonance frequency of the SSM-QTF was measured with a fixed relative humidity. The humidity in the gas chamber was well controlled by using a mass flow controller  with a relatively low flow rate but higher resolution. With a relative humidity of 20 ± 0.1% RH, a resonance frequency of 32,542.88 Hz was obtained. Since the relative humidity is sensitive to temperature, the ambient temperature condition has to be properly maintained throughout the experiment. For the tested humidity range, the temperature in the humidity chamber has been monitored to be ~24 °C. It can be seen from The COVID-19 pandemic has forced the global population to adopt new ways of living, including the possible use of masks. Research has been conducted to evaluate the potential consequences of wearing masks for long periods ,67. AmonIn the measurement, the humidity inside a disposable medical mask made of polypropylene was evaluated. The disposable medical masks were worn by the author Leqing Lin for different periods; then, they were taken off quickly to cover the SSM-QTF sensor in a sealed space. The humidity of the covered air inside the medical mask was then measured. For each measurement, the medical mask was replaced by a new one. The time clock was restarted. Except for the wearing time, all the other conditions were the same in the repeated experiments. The enclosed spaces were set up to prevent humidity interference from the outside atmosphere. Resonance frequencies of the different wearing time of the masks were recorded by a LabVIEW program. The obtained results were shown in This work featured a novel humidity sensing platform based on a quartz tuning fork (QTF) with a section of natural spider silk. The experimental results showed that the higher the environmental humidity, the lower the resonance frequency of the spider silk-modified QTF. The variation of the resonance frequency of the QTF modified by spider silk was closely correlated with the change in the environmental humidity. The evaluation was conducted in relative humidity from ~25% RH to ~80% RH. The resonance curves were recorded to obtain the SSM-QTF frequency variations by Lorentz fitting. With a 1 min working time, the detection sensitivity of ~12.7 ppm was obtained.As proof of concept, the humidities of disposable medical masks, worn for different periods, were evaluated. The results suggested that the masks should be changed every 2 h due to excessive humidity, which may cause potential facial skin discomfort. Currently, the acquisition time for the resonance frequency and Q factor requires ~1 min. This time can be shortened by narrowing the frequency scanning time and range. Allan deviation was measured over a long time with a fixed humidity. With 30 points of integration, the detection sensitivity can be improved to ~0.8 ppm. Future work will include the development of a wearable respiration sensor to measure the humidity inside a medical mask. Compared to the current electrochemistry sensor ,76, the


An acceptable recovery has been revealed in the real sample analysis. The electrochemical sensing behaviour of the composite indicates that it may be a promising candidate for a glucose sensor and it significantly extends the range of applications in the electrochemical field.In this work, a novel electrochemical sensor was developed by electron-withdrawing substituent modification of 1-phenyl-3-methyl-4-(4-fluorobenzoyl)-5-pyrazolone on a graphene-modified glassy carbon electrode (HPMpFP-graphene/GCE) for glucose detection. The results of characterizations using a scanning electron microscope, Fourier transform infrared spectroscopy, Raman spectroscopy, and nuclear magnetic resonance spectroscopy showed the successful fabrication of HPMpFP-graphene nanocomposite, which served as an electroactive probe for glucose detection. The electron transfer ability of HPMpFBP-graphene/GCE has been successfully revealed using cyclic voltammetry and electrochemical impedance spectroscopy results. The good electrochemical performance was shown by well-defined peak currents of square wave voltammetry under various parameters, including pH, HPMpFP and graphene composition, and scan rate effect. A high electrochemically evaluated surface area using chronoamperometry suggested that the present glucose detection response was intensified. The chronoamperometry results at a work potential of 0.4 V presented a wide linear range of 1 × 10 Clark and Lyons proposed the use of glucose oxidase entrapped inside a semipermeable dialysis membrane built on an oxygen electrode almost 50 years ago . After tFood composition information, including glucose content, is vital for the modern food industry as low-quality foods are associated with increased morbidity, mortality, human suffering, and economic burden . Hence, Numerous articles described the use of the enzymatic electrochemical sensing technique for glucose detection in food and beverage samples –9. Many 2 carbon atoms organized in a honeycomb-like form. Graphene has 260 times the surface area of graphite and twice that of a carbon nanotube, making it the most densely packed material known ). As shown in Ep=Epc − Epa) of 0.367 V. The redox peak potentials were raised by 0.158 V and 0.139 V with the addition of graphene and HPMpFBP-graphene composite. For bare GCE, the current ratio of the oxidation peaks to the corresponding reduction peaks (ipa/ipc) was 1.04. For graphene/GCE, the current ratio was 0.93, and for HPMpFP-graphene/GCE, the current ratio was 0.97. As the ratios were close to one, these results were due to a reversible voltammogram (ipa/ipc = 1)  with 1.0 M KCl. The direct part of the semicircle depicts the diffusion process, while the whole semicircle represents an active control barrier that acts to reduce the flow of electrons at the electrode contact, which is also known as electron transfer resistance (Rct) [3 [Fe(CN)6] at the bare GCE showed a small semicircle in the high-frequency area and is a straight line in the low-frequency region, as shown in Rct. The Randles equal circuit viable with the impedance spectrum is shown in the inset of Rs, Cdl, and Zw.The EIS was carried out to take into consideration the electrochemical characteristics of the modified electrodes via the use of a redox probe of 4 mM Kce (Rct) . The EISRct for HPMpFBP-graphene/GCE, which was found to be lower (20.86) than graphene/GCE (43.14) and bare GCE (80.97). The electrode with the lowest Rct value has the best conductivity. It tends to be summed up that the arrangement of Rct on the electrodes expanded in the request for HPMpFP-graphene/GCE < graphene/GCE < unmodified GCE. The astounding conductivity of HPMpFP-graphene/GCE demonstrates the extraordinary electron move capacity. In the development of electrochemical sensors, this is an important factor to consider. The electron move rate steady (kapp) values obtained from Equation (3 [Fe(CN)6] undergoes a redox reaction, the amount of moles of electrons transported (n) is equal to one.Using the Randles equivalent electrical circuit, it was possible to calculate the value of e/GCE 43.4 and barkapp value achieved for HPMpFP-graphene/GCE was greater than the corresponding values found for graphene/GCE and bare GCE. Consequently, it can be concluded that the HPMpFP-graphene/GCE, which benefited from the synergistic impact of the HPMpFP complex and the graphene composite, has produced excellent electrochemical sensor characteristics as a result of its use.From The electrochemical effectiveness of various prepared electrodes was determined using CV with the presence and absence of 0.1 mM glucose, which was tested in 0.1 M PBS (pH 7) electrolyte solution. The experiments shown in π-π strength and hydrogen bonding to form the composite, but it is also used to construct a conductive interconnivance network that can prevent graphene coagulation and increase surface area. Third, introducing electron-withdrawing substituents such as fluorine into the 1,3-β-diketone position of the HPMpFP may improve the ligand's electrical characteristics, thereby enhancing glucose detection at the modified electrode surface.This modified electrode's electrocatalytic reaction to glucose is beneficial for the following reasons. First, the outstanding electrical conductivity of graphene helps increase the glucose-attracting ability of the resulting electrode, thereby increasing its large surface area. Second, graphene is not only integrated with HPMpFP aromaticity that is adsorbed on graphene surfaces through Ep) and pH levels is shown. It is possible to describe this connection mathematically using the linear regression equation, which may be expressed as follows:Using SWV, the impact of pH (6.8 to 7.6) on the peak glucose potential at the HPMpFP-graphene/GCE in 0.1 M PBS was investigated . The resThe slope was neared to the theoretical value of 58.6 mV/pH. This indicates that during the glucose redox process the total number of electrons and protons taking part was identical . FollowiµL resulted in a substantial rise in peak current, while increasing the dispersion volume further to 2.0 µL resulted in a considerable decrease in peak current. This is likely to become more difficult due to the excessive thickness of sample dispersion and electrode surface diffusion. Therefore, 1.0 μL of HPMpFP-graphene composite dispersion was selected for electrode preparation in this study.A droplet of HPMpFP-graphene composite was cast on the GCE surface based on the electrode preparation stage, and a thin layer of the sample was left behind on the GCE due to solvent evaporation. Therefore, by changing the amount of dispersion cast on the GCE surface, the thickness of the HPMpFP-graphene composite layer could be altered. The SWVs of prepared electrodes with different quantities of composite HPMpFP-graphene cast on GCE are shown in Epa) and cathodic peak (Epc) potentials shifted positive and negative, respectively. The values of peak separation (ΔEp = Epc–Epa) were calculated to be between 0.215 and 0.455 V at different scan rates, which illustrates the sluggish electron transport kinetics. The difference between the anodic and cathodic peaks in these redox reactions is related to ion transport resistance in these processes  is 7.6 × 10−6 cm2/s, and the electron number is one [The surface area of the working electrode is indicated by r is one .Q and t1/2 was used to derive the following equation for unmodified GCE, graphene/GCE, and HPMpFP-graphene/GCE, which was found to follow with the findings presented in Figure 8(a) for all three electrodes.The slope of the linear connection between −3 mC/s1/2 (bare GCE), 1.126 × 10−3 mC/s1/2 (graphene/GCE), and 2.365 × 10−3 mC/s1/2 (HPMpFP-graphene/GCE), A was calculated to be 1.12 × 10−4 cm2, 1.01 × 10−3 cm2, and 6.13 × 10−3 cm2 for unmodified GCE, graphene/GCE, and HPMpFP-graphene/GCE, respectively. In addition to increasing the number of electrode reaction sites, increasing the adsorption capacity, and intensifying the current response of glucose, the increased electrode surface may also improve the sensitivity and detection limit of this sensor.According to the slopes of 0.124 × 10Q was plotted against time t1/2 ), it revealed a linear connection with the following equation, where the slope was 1.471 × 10−3 mC/s1/2 and the intercept (Qads) was 0.098 × 10−3 mC.In addition, HPMpFP-graphene/GCE was also subjected to chronocoulometry analysis in the absence and presence of 0.1 mM glucose for 30 minutes. When Q and t1/2, the intercept of the linear relationship corresponds to Qads. As n = 2, A = 5.31 × 10−4 cm2, and C = 0.1 mM, the D was estimated to be 1.6194 × 10−4 cm2/s. The adsorption capacity (Γs) of glucose at the HPMpFP-graphene/GCE was calculated to be 9.58 × 10−7 mol cm−2, following with equation:When the background is subtracted from the linear connection between The dependence of the potential applied to the amperometric signal of HPMpFBP-graphene/GCE is shown in µM. As shown in µM, then 15 µM, and finally 25 µM, before being stopped. Later, the increments were raised by 45 µM at a time, until the concentration reached 90 µM. Finally, 100 µM increments were made until the final 1000 µM step-in addition.Rather than using SWV or CV, the chronoamperometry approach was utilized in this research for quantitative glucose analysis since it has better sensitivity and resolution than other methods. After the first administration, a 50-second interval was given among the following doses, and the glucose concentration was varied from 5 to 1000 μM. There were two linear regions in this range (μA) = 0.07 + 1.46 C (R2 = 0.996) with a sensitivity of 11.42 μA/mM/cm2. Meanwhile, the current continued to rise in the second area, starting at 88 µM, when the linear regression equation was found to be I (μA) = 0.032 + 6.074 C (R2 = 0.998) and the sensitivity was measured as 5.22 μA/mM/cm2. It was estimated that the detection limit was 0.74 μM (S/N = 3). Compared with other carbon-based materials reported in the literature . The reproducibility was tested using six identically produced electrodes, and the relative standard deviation (RSD) was determined to be 2.52 ± 0.51% . Ten conThe stability of the as-prepared sensor was determined by monitoring the current response to glucose detection after two weeks of operation . The curt-test analysis to determine whether any of the findings obtained in the presence of a known amount of glucose added to the PBS solution were legitimate. As indicated in p < 0.05, there was no statistically significant difference, while for p > 0.05, there was a statistically significant difference. These findings suggest that nonenzymatic HPMpFP-graphene composite sensors may be employed in glucose determination in beverages.To show the viability of nonenzymatic HPMpFP-graphene/GCE measurements in real-world glucose, this precise sensor was used to detect glucose concentrations in orange juice and milk samples. In summary, the fabrication of HPMpFP-graphene/GCE sensors for selective glucose detection was described in this study. Under optimal conditions, the peak current for glucose detection on HPMpFP-graphene/GCE was significantly higher than the peak currents on graphene/GCE and unmodified GCE. As a result, the integration of the greater surface area and unique graphene conductivity of the composite with the HPMpFP complex significantly improved the performance of the sensor. Diffusion-controlled redox processes of glucose were shown to be more common in the presence of HPMpFP-graphene/GCE. Low detection limits for glucose and a wide concentration range for glucose were shown by the HPMpFP-graphene/GCE, demonstrating good selectivity and sensitivity towards glucose. The HPMpFP-graphene/GCE was effectively utilized to detect glucose in certain orange juice and milk samples, with a recovery rate ranging from 90 to 103%.