Background: Idiopathic pulmonary fibrosis (IPF), a chronic, progressive lung disease characterized by interstitial remodeling and tissue destruction, affects people worldwide and places a great burden on society. Cellular senescence is thought to be involved in the mechanisms and development of IPF. The aim of this study was to predictively investigate subtypes of IPF according to cellular senescence-related genes and their correlation with the outcome of patients with IPF, providing possible treatment and management options for disease control.Methods: Gene expression profiles and follow-up data were obtained from the GEO database. Senescence-related genes were obtained from the CSGene database and analyzed their correlation with the outcome of IPF. A consensus cluster was constructed to classify the samples based on correlated genes. The GSVA and WGCNA packages in R were used to calculate the immune-related enriched fractions and construct gene expression modules, respectively. Metascape and the clusterProfiler package in R were used to enrich gene functions. The ConnectivityMap was used to probe suitable drugs for potential treatment.Results: A total of 99 cellular senescence-related genes were associated with IPF prognosis. Patients with IPF were divided into two subtypes with significant prognostic differences. Subtype S2 was characterized by enhanced fibrotic progression and infection, leading to acute exacerbation of IPF and poor prognosis. Finally, five cellular senescence-related genes, TYMS, HJURP, UBE2C, BIRC5, and KIF2C, were identified as potential biomarkers in poor prognostic patients with IPF.Conclusion: The study findings indicate that cellular senescence-related genes can be used to distinguish the prognosis of patients with IPF. Among them, five genes can be used as candidate biomarkers to predict patients with a poor prognostic subtype for which anti-fibrosis and anti-infection treatments could be suitable. Idiopathic pulmonary fibrosis (IPF) is a chronic, fibrotic interstitial pneumonia characterized by progressive hypoxemia and deterioration of lung function with overall poor prognosis . IPF is Cellular senescence, particularly alveolar epithelial cell senescence, is involved in the pathogenesis of IPF . AlveolaTo investigate the relationship and effect of cellular senescence-related genes on IPF, in this predictive study, we used cellular senescence-related genes to identify subtypes of IPF distinguished by prognosis, to determine specific biomarkers for clinical prediction, and screened probable drugs for targeted treatment based on IPF subtype. The results provided a new perspective to improve the treatment and management of IPF.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE70867). All patients were diagnosed as IPF based on American Thoracic Society and European Respiratory Society criteria in a retrospective study , and clinical data with survival-related information  of 176 pve study , regardlhttp://csgene.bioinfo-minzhao.org/)  pathway enrichment analysis was performed on significant genes using the clusterProfiler package in R  enrichment analysis was carried out using Metascape (https://metascape.org/gp/index.html#/main/step1)  . After rao.org/) . The surage in R , with p-n/step1)  to identp-values <0.05 considered to be significant. Age and sex information was performed by the Wilcoxon and Chi-squared tests respectively, with p-values <0.05 as significant.Using the ConsensusClusterPlus package in R , a consivia single-sample gene set enrichment analysis (ssGSEA) for the two subtypes using the GSVA package in R and published literature for immune cell types and pathways . Gene Ontology (GO) and KEGG analyses were conducted on the DEGs using Metascape. GO analysis included three aspects: biological processes, molecular functions, and cellular components. p-values <0.01 were considered to be significant pathways from the above enrichment analysis. By searching for similar expression signature caused by drug treatment, drug predictions for both subtypes were performed using the Connectivity Map (https://clue.io/) based on the DEGs  , 2012. TThe study workflow is shown in . Prognosp = 0.455 > 0.05) and sex (p = 0.584 > 0.05). This indicated that the subtyping constructed by cellular senescence-related genes can effectively distinguish prognosis of patients with IPF, that is, subtype S2 has a worse prognosis than subtype S1.To better manage the treatment of patients with IPF, samples were clustered into two prognostic subtypes designated as S1 and S2 based on the expression of the aforementioned genes . Next, tGiven that pro-inflammatory cytokines are an important component of SASP  and immuThe DEGs  were comr = 0.19; p = 0.02; 95% CI: 0.03-0.34)  , suggest03-0.34) . Hence, Cellular senescence is implicated in the pathogenesis of IPF, and many studies have shown that it plays a pivotal role in the development of the disease. Most current research on senescence and IPF is based on experimental animal models. Senescence in epithelial cells, basal cells, and fibroblasts may be mechanically associated with the development of pulmonary fibrosis . HoweverCellular senescence is thought to have both protective and harmful effects on biological processes. This corresponds with the study findings in which prognostic senescence-related genes had a bilateral effect on patient outcomes. Gene regulation networks involve complex crosstalk, with each gene playing a particular role in the same biological process. For example, KL expression has been shown to exert a protective effect against overall mortality in non-diabetic, pre-dialysis chronic kidney disease , whereasIn the classification system applied in the present study, subtype S2 showed a prominent feature of IPF progression in which cellular senescence leads to increased fibrosis, resulting in poor prognosis. The deposition of ECM is an important process in fibrosis . TGF-β iStreptococcus and Staphylococcus aureus can be detected in the BAL of patients with AE-IPF in contrast to those in stable condition. This microbiome change is associated with disease progression (The study findings also support another hypothesis, in that cellular senescence may lead to defects in epithelial cell function, causing impaired barrier function and susceptibility to infection that can lead to progression or even death in patients with IPF. Patients with a high proportion of neutrophils in BAL reportedly had a higher risk of early mortality . In addigression  and bactgression . Unlike gression . Interesgression . These fFurthermore, the present study provides insight into drug screening for both subtypes. Leflunomide is a common drug used as a background therapy for rheumatoid arthritis, and another study reported that it can attenuate fibrosis in systemic sclerosis . AlthougFive genes were identified to clinically predict subtype S2, including TYMS, HJURP, UBE2C, BIRC5, and KIF2C. Among them, TYMS, KIF2C, UBE2C, and HJURP are associated with unfavorable prognosis in hepatocellular carcinoma, possibly due to the malignant growth of cells . TYMS, win vivo or elucidate the underlying molecular mechanisms. Further, poor prognosis due to infection-induced progression of fibrosis does not exclude other causes of death induced by the infection itself, such as acute respiratory distress syndrome. Moreover, the specific effects of the identified drugs remain to be experimentally confirmed. Therefore, in vitro and in vivo experiments will be performed in the future to verify and evaluate the results of the present study in terms of immune cell infiltration, immune pathways, and candidate drugs. Clinical trials are also needed to determine the importance of infection in the adverse outcomes of patients with IPF. Similarly, the diagnostic genes for subtype S2 should be evaluated in large clinical samples.This study had some limitations. The lack of survival information data for patients with IPF prevented finding a validation cohort to confirm the reliability of the two subtypes. In addition, the study findings do not confirm whether cellular senescence produces considerable pro-fibrotic effects In summary, this predictive study provides new insights into the etiology, treatment, and management of patients with IPF. For such patients with potential rapid progression and poor prognosis based on the classification system described herein, clinicians should intervene early, paying greater attention to general conditions, clinical symptoms and signs, and objective examination indicators, offering assistance in standardized management and drug therapy as appropriate during clinical follow-up. For patients with a long-lasting dry cough and a suspicious, new ground-glass shadow on CT scans, clinicians should be vigilant about the progression of IPF and intervene with treatment at an early stage.This predictive study demonstrates a cellular senescence-based IPF subtyping approach that distinguished patients with poor prognosis and identifies anti-fibrosis and anti-infection agents as possible options for targeted treatment and management. TYMS, HJURP, UBE2C, BIRC5, and KIF2C were identified as potential biomarkers for poor prognostic patients with IPF.


Densityfunctional theory (DFT) corroborates the experimental results andreveals the intercalation mechanism, whereby P2O5 dissociates into small fragments catalyzed by defects in the graphenethat then permeates through lattice defects and reacts at the heterointerfaceto form P2O5. This process can also be usedto form new confined metal phosphates .While the focus of this study is on P2O5 intercalation,the possibility of intercalation from predissociated molecules catalyzedby defects in graphene may exist for other types of molecules as well.This in-depth study advances our understanding of intercalation routesof large molecules via the basal plane of graphene as well as heterointerfacechemical reactions leading to the formation of distinctive confinedcomplex oxide compounds.Intercalation is the process of inserting chemical speciesintothe heterointerfaces of two-dimensional (2D) layered materials. Whilemuch research has focused on the intercalation of metals and smallgas molecules into graphene, the intercalation of larger moleculesthrough the basal plane of graphene remains challenging. In this work,we present a new mechanism for intercalating large molecules throughmonolayer graphene to form confined oxide materials at the graphene-substrateheterointerface. We investigate the intercalation of phosphorus pentoxide(P To date,much of the research surrounding the intercalation of monolayer graphenehas focused on the intercalation of metals  and smallgas molecules . However, the intercalation of moleculesthrough the basal plane of monolayer graphene that are significantlylarger than the lattice parameters of graphene remain challenging.This is because the size and energy barrier for intercalation inhibitsthe permeation of such large molecules.26 The abilityto intercalate large molecules could further expand the toolsets forsubsurface and heterointerface engineering of 2D layered materialsand therefore enrich the material choices available for the fabricationof heterostructures and intercalation compounds used in energy storage,optoelectronics, thermoelectric, catalysis, etc.29Intercalation is a topotactic insertionprocess of organic or inorganicchemical species  between theinterfaces and heterointerfaces of two-dimensional (2D) layered materials.This process is known to form a variety of intercalation compounds,notably in graphite, encompassing a diverse range of intercalantswithin the host of the 2D layered bulk crystal.x1–Gex,30 Fex1–Cox,31 etc.) and compounds  have been formed at graphene-substrate heterointerfacesvia chemical reactions. In all of these cases, however, chemical speciesare individually introduced in a sequential manner when intercalatinggraphene. This is done to prevent prereactions in the vapor phasebetween the individual elements prior to intercalation. Such prereactionsusually lead to the formation of clusters at defect sites on the graphenesurface rather than intercalating through the lattice itself and forminga compound at the heterointerface.Chemicalreactions enabled by confinement to the heterointerfacesof 2D layered materials provide a pathway to circumvent the limitationon intercalating large molecules directly through the basal plane.That is, individual chemical species or multiple small fragments ofthe initial state of the molecule would first intercalate throughtypically available pathways in the graphene lattice, and then recombineat the heterointerface via chemical reactions. So far, a variety ofalloys (Sn2O5) molecules directly from the vapor phase to form P2O5 at a graphene-substrate heterointerface. The formation ofconfined P2O5 was confirmed using a varietyof chemical analysis and spectroscopic techniques as well as microscopicsurface and cross-sectional imaging. Density functional theory (DFT)was used to corroborate experimental results and to deduce the mechanismfor the intercalation of P2O5 molecules throughthe basal plane of monolayer graphene. First, P2O5 dissociates into small fragments  when interacting withthe graphene surface. These small fragments consistently showed lowerenergy barriers for intercalation through defects commonly observedin the graphene lattice. Once the small fragments intercalate, theyreact at the heterointerface to form P2O5. Weshow that the intercalation of P2O5 at the graphene-substrateheterointerface can tune the electronic structure of the grapheneoverlayer through strain and charge transfer. In addition, we showthat this process can also be used in the formation of new confinedmetal phosphates by intercalating P2O5 intoa graphene-substrate heterointerface initially containing a confined2D indium metal to form InPO4. Although this study focuseson the intercalation of P2O5, the concept ofintercalation from predissociated molecules catalyzed by defects ingraphene could be possible in other classes of molecules. Ultimately,this study provides an important steppingstone for advancing the understandingof intercalation pathways of large molecules though the basal planeof graphene as well as heterointerface chemical reactions to formunique confined complex oxide compounds.In this work, we report amechanism of vapor phase intercalationof large molecules through the heterointerface of monolayer grapheneto form confined oxide materials underneath graphene with a chemicalcomposition that resembles the initial state of the large intercalantmolecule itself. Unlike prior work, where chemical species and/orelements were sequentially introduced into the heterointerface, singlemolecules were introduced directly into the vapor phase for intercalationthrough the basal plane. This mechanism was revealed by investigatingthe intercalation of phosphorus pentoxide (P2O5) can be deduced from our DFTcalculated ground state structure whenit is adsorbed onto the surface of pristine monolayer graphene . The formation of such large poresin graphene is, however, energetically unfavorable.40 In fact, other theoretical studies have predicted largeenergy barriers for permeation of many atoms and molecules throughpristine graphene.26 In our case, we experimentallyshow that it is possible to intercalate P2O5 through monolayer graphene transferred onto germanium substrateswithout the need for large pores to serve as intercalation pathways.In our studies, germanium was used as a substrate due to the limitedsolubility41 and low diffusivity42 of phosphorus into germanium, allowing to fullycapture and study the formation of P2O5 at graphene-germaniumheterointerfaces. To intercalate P2O5, we preparedmonolayer graphene on germanium substrates which were then annealeddownstream of SiP2O7 at 950°C . From scanning tunneling microscopy(STM) imaging of the bubble surface (46 Upon further inspection of the surface using energy-dispersive spectroscopy(EDS), strong phosphorus and oxygen signals confined to the graphenebubbles were observed . During the ion sputteringprocess, the graphene surface was first etched. This resulted in arapid decrease in the C 1s core-level peak intensity . The peak intensities for the Ge,O, and P core levels increased upon sputtering of graphene , which serves as additionalevidence on the confinement of phosphorus oxide at the graphene–germaniumsubstrate heterointerface after samples were exposed to a flux ofP2O5.As shown in  surface 1c, insetobserved 1d. This observed 1f,g also–1, is highlighted in 2) heat map, which is related to the localabsorption spectra of the material, and therefore, useful for determiningthe chemical environment of the intercalant.482O5 using high-resolutionsynchrotron-based nano-FTIR measurements (see –1), the P–O bridgingmode (1060–1125 cm–1), and the P=Oterminal oxygen stretching modes (1135–1235 cm–1).49 The intensity of the P=O bondin the heat map . A near-field white light IR image of a regionof the sample containing graphene bubbles, spectrally averaged over∼610–1400 cments see . Evidentheat map 2b reache2O5, while the XPS corelevels in 2O5  intercalates through the basal plane of graphene,we first examined the adsorptive properties of these molecules onfive different graphene systems, consisting of pristine graphene andfour graphene lattice defect configurations (Supporting Information). This survey was performed to factor in differentinteraction parameters of distinct chemical environments on the intercalationprocess. The configurational space of the adsorptive states (chemisorptionvs physisorption) was thoroughly explored by including a multitudeof initial interacting geometries for both P2O5 and P4O10 on each of the different graphenelattice defect configurations  forces. In comparison, interactionsbetween the more stable P4O10 molecule and allgraphene systems were vdW in nature. In the case of covalent bondingbetween P2O5 adsorbents and graphene, P atomsconsistently demonstrated a higher affinity toward graphene. Thisis shown by the greater adsorption energy of P2O5 to graphene through the P atom (denoted by P2O5(P) in 2O5 approachinggraphene with its O atom (designated by P2O5(O)) either relaxed to drastically different interaction geometriesto allow bond formation between carbon and phosphorus or had a muchlower total adsorption energy when compared to P2O5(P). The only exception was P2O5(O)chemisorbed to S defects, in which both the atoms of the O and P bondedcovalently to the graphene defect site. Such trends can be rationalizedby the fulfillment of covalency and increased charge transfer stabilizationof carbon atoms at defects when bonding to less electronegative Patoms in P2O5.To gain a deeper understanding into the mechanism by which phosphoruspentoxide Purations 3b. As su46 and the presence of defects is thus theprecondition for P2O5 intercalation. A quantitativedescription of intercalation through defects is complicated by thecompetition between diffusion and bonding with carbon atoms at defectsites. This gives rise to three different energy profiles correspondingto distinct pathways.51 To address thesecomplexities, we employed a case-by-case strategy by calculating thepenetration barriers for fragments of P2O5 ofvaried size and chemistry based on calculated and experimental evidencefrom the literature.45 Based on experimentalevidence of P2O5 confinement at the graphene-germaniumheterointerface, the intercalation of molecular P2O5 in its entirety was first investigated. Point defects ofvarious sizes were examined, with the largest created by removing9 adjacent carbon atoms in an isolated graphene sheet . In all scenarios, P2O5 decomposed into fragments solvated by carbon atoms at thedefect sites. Notably, the energetic profile of P2O5 permeating through large defects showed a negative reactioncoordinate energy curve with small intermediate energy barriers asa result of the bonding of the fragmented P2O5 molecule to the defect site. Despite this negative reaction coordinateenergy curve, a barrier of at least 7.7 eV was still present for fragmentsto desorb from the defect and diffuse into opposite sides of the monolayergraphene . From this, we ascertain that intercalationis only possible from significantly smaller molecular fragments ofP2O5. Developed from these findings, we proposea mechanism involving three consecutive steps of realistic energybarriers to intercalate P2O5 through the basalplane of monolayer graphene. First, chemisorbed P2O5 decomposes into atoms or small molecular fragments. The resultingspecies then intercalate separately through point defects and thenreact underneath the graphene monolayer to form P2O5 at the confined heterointerface.Pristine graphene has been predicted to be impermeableto mostgaseous species under nonextreme conditions,2O5 intoPO2 and PO3 by cleaving the single bond betweenP and bridging O. Energy barriers for both reactions are shown in 2O5. These findings were consistent with the results ofadsorption state calculations, as covalent interactions and chargetransfer between the defects and adsorbed molecules weaken the intramolecularbonds. Having substantiated the possibility of the catalytic dissociationof adsorbed P2O5 at defect sites, we then calculatedthe energetics of smaller fragments intercalating through point defects.Phosphorus monoxide (PO), the simplest oxide of phosphorus, was chosenas the subject of this study. Given the positive correlation betweenan intercalant size and the energy barrier required for its permeationthrough graphene point defects, we postulate that the intercalationof PO provides a baseline for the minimum energy barrier applicableto the diffusion of any potential phosphorus oxide molecules throughmonolayer graphene. In the focused reaction coordinate diagram  defects without the introduction of defects of larger sizes.When penetrating through S- and D-defects, the molecule decomposedinto individual atoms with the rate limiting step having an energybarrier as high as 8 eV. On the other hand, PO wassurprisingly permeable at Q-defects. It diffused through the Q-defectwithout further fragmentation, with a barrier of approximately 2 eV, comparable to the energy required to break down chemisorbedP2O5. Finally, to compare atomic phosphorusand oxygen diffusing through the four different types of defects ingraphene , further corroboratesthe formation of the P2O5 nonplanar cage-likestructure at the graphene-substrate heterointerface after intercalationof phosphorus oxide fragments as suggested from our DFT calculations.Furthermore, it is possible to deconvolute the effects of charge transferand strain on the doping level of graphene by performing a correlationanalysis of the G and 2D peak positions between the nonintercalatedand intercalated samples  dI/dV measurements of the intercalated graphene surface  of the sampleat the tunneling site. Two local minima were observed in all dI/dV curves, that is, one at V = 0 which was the phonon scattering gap (green dashed line), andthe another at the Dirac point where the LDOS of graphene reachesits minimum (yellow dashed line).63 From V = 0) after intercalation, which indicatedthat P2O5 led to p-type doping of graphene.Graphene inherits highly delocalized π-electrons. Any modificationto the spatial extent of the charge density of these π-electronsor tilt of the π-orbitals leads to significant changes to thephysical properties of graphene.O–Pbond S3, furthO–Pbond S3, furthO–Pbond S3, furth66 the impact of possible deformation on the electronicstructure of monolayer graphene was studied. Our density of states(DOS) calculations based on (4 × 8) supercells of graphene suggestedthat the electronic structure near the Fermi level did not undergosignificant change, despite an increase in the overall system energywith respect to the increased strain. While compressive and tensilestress led to horizontal stretches and compression of the total DOS,respectively, the overall electronic structure near the Fermi leveldid not vary . We observed thatboth pristine and deformed graphenes behaved as semimetals. As a result,strain alone did not significantly contribute to the experimentallyobserved p-type conductive properties in the Raman and STS measurements.While nonuniform strain and formation of bubbles on the graphene monolayercould cause variations in the distance between graphene and underlyinggermanium substrate, the impact of the germanium surface on the electronicstructure of graphene becomes negligible when the separation reaches∼2 nm . Notably, evenin regions where the graphene monolayer was only a few angstroms fromthe top layer of the germanium surface atoms, the presence of germaniumstill does not induce semiconducting behavior in graphene. Similarly,while the exposure to P2O5 caused changes inDOS of graphene far above and below the Fermi level, the overall electronicproperties of pristine graphene with adsorbed molecules remained semimetallic,with characteristic converging overlap between the bottom of the conductionband and the peak of valence band  chemisorbed to S-defect being themost notable case  to bond with germanium 69 with excellent dielectric properties that initially gaininterest as a gate material for InP formed via surface oxidation.73 However, the formation of dimensionally confined indium phosphatesremains largely unexplored, thus impending a more comprehensive understandingof their physical properties and potential applications. In our case,we demonstrated the successful formation of confined indium phosphatesby intercalating P2O5 into a graphene-SiC heterointerfaceinitially containing confined mono- to bilayer indium also formedby intercalation.74 We identified the compositionof the confined indium phosphate at the heterointerface by investigatingthe In and P core-levels using high-resolution XPS .In addition to the typical peaks for indium metal (black dashed line),the XPS spectra exhibited new peaks around 445.8 and 453.3 eV in theintercalated sample (blue dashed line). These two new peaks were up-shiftedby 1.1 eV from the peak position of the In2O3 reference (red dashed line).75 Furthermore,a strong phosphorus signal was detected in samples intercalated withP2O5 3 and other metastable oxide phases could alsoform at such confined spaces68 and therefore are important to investigate in future studies.Although phosphorus has limited solubilityermanium 2i. To futudy see . Indium ithP2O5 6b. There2O5, can permeate the basalplane of graphene andintercalate into the heterointerface. This was evident from the strongP and O signals that were detected underneath the graphene surfacefrom spectroscopic measurements and microscopic cross-sectional imaging and analysis in TEM. Althoughthe physical size of the molecule was larger than that of the graphenelattice, fragments of the molecule could intercalate through typicalpathways in the graphene lattice. This was revealed by calculatingthe penetration barriers for fragments of P2O5 of varied size and chemistry through pristine and defected grapheneof different configurations. The complexity of the interaction processof large molecules through the basal plane of graphene cannot be generalizedinto a single step process. However, a two-step mechanism, based onrealistic permeation energy barriers, was proposed for the intercalationof P2O5: (i) P2O5 firstdissociated into small fragments  catalyzed by pointdefects in the graphene lattice; (ii) these fragments once intercalatedcan chemically react at the confined graphene–substrate heterointerfaceforming a condensed phase that chemically resembles the initial stateof the molecule. It has been shown that the intercalation of P2O5 at the graphene-substrate heterointerface couldeffectively tune the doping level of graphene via charge transfer.Moreover, the intercalated P2O5 (and/or itsfragments) can also act as reactants and further contribute to otherinterfacial reactions, for example, the conversion of 2D indium into2D indium phosphate at the heterointerface between graphene and SiC.Although our findings only focus on indium phosphates, our resultsdemonstrate the potential of this approach for the conversion of othermetals  and alloys. Furthermore, spatial controlover the distribution of defects, such as localized irradiation usingplasma processing,76 can promote the selectivearea intercalation of metals and/or alloys under specific regionsof graphene. While the focus of this study is on P2O5 intercalation, the possibility of intercalation from predissociatedmolecules catalyzed by defects in graphene may exist for other typesof molecules as well. This in-depth study advances the comprehensionof intercalation routes of large molecules via the basal plane ofgraphene as well as heterointerface chemical reactions leading tothe formation of distinctive confined complex oxide compounds.In summary, we demonstrated that largemolecules, such as P17 SiP2O7 (Saint-GobainInc.) was utilized to produce an upstream flux of P2O5 to the samples. First, the center of the furnace was setto 950 °C at a ramping rate of 55 °C/min (stage I) and thendwelled at 950 °C for 45 min (stage II). In stage II, the measuredtemperature for the sample and SiP2O7 was 600and 950 °C, respectively. Samples were then naturally cooledto room temperature under argon flow.The intercalation process was performedat 1 atm in a single-zone furnace under argon flow. Two kinds of sampleswere used in this study: transferred CVD graphene (Graphenea) ontocleaned Ge(110) substrates (MTI Corp.) and 2D indium samples intercalatedat graphene-SiC(0001) heterointerface, as reported by Rajabpour etal.hν = 1486.6 eV). Ramanmeasurements were performed on a Renishaw inVia system with a 488nm laser (maximum power at 100 mW). STM was performed in a closed-cycleRHK Pan Scan Freedom SPM system in UHV (∼1 × 10–10 mbar) at a base temperature of 10 K. dI/dV measurements were taken using a built-in digital lock-inamplifier operating at 1.3156 kHz with a 10 mV excitation amplitude.Moreover, nano-FTIR of the graphene bubbles was performed on a commercialscattering-type scanning near-field optical microscope (s-SNOM) setup(Neaspec Inc.), and the collected spectra were normalized to thatobtained on a silicon sample using the same acquisition parameters.Nano-FTIR of the P2O5 reference sample was performedon a synchrotron infrared light-based nano-spectroscopy (SINS) setup .SEM and EDS were performedon a Thermo Fisher Scios 2 dual-beam microscope. The cross-sectionalsamples of graphene bubbles for TEM analysis were prepared using afocused ion beam (FIB) Zeiss NVision 40 FIB-SEM. The target locationwas extracted from the specimen and attached to a copper FIB gridusing a conventional FIB milling and lift-out procedure. The HAADFimage and STEM-EDS mappings on the cross-section of graphene bubbleswere collected with a Thermo Fisher Scientific Talos 200× operatedat 200 kV. HAADF was performed with a spot size less than 1 nm witha convergence semiangle of 10.5 mrad. Furthermore, XPS spectra anddepth profiles were carried out in a Thermo-Fisher K-Alpha Plus XPSsystem and in a Kratos Axis Ultra DLD system using a monochromaticAl Kα source  calculations, a grid of 5 × 4 ×1 was used. To obtain density of states (DOS), a grid of 9 ×9 × 1 was adopted for best precision. For more information onsimulating the reaction mechanism, see the Supporting Information.DFT calculations wereperformed using the generalized gradient approximation (GGA) withthe Vienna ab initio simulation package. The projector-augmented-wave(PAW) method was used, and Grimme’s DFT+D2 was applied to betteraccount for the van der Waal’s interaction between graphenemonolayer and absorbed species.


Through qualitative analysis of the response mechanism of graphene PTE devices, we find that the electrode-induced doping of graphene channel near the metal-graphene contacts play a key role in the terahertz PTE response. This work provides an effective way to realize high sensitivity terahertz detectors at room temperature. A highly sensitive room-temperature graphene photothermoelectric terahertz detector, with an efficient optical coupling structure of asymmetric logarithmic antenna, was fabricated by planar micro-nano processing technology and two-dimensional material transfer techniques. The designed logarithmic antenna acts as an optical coupling structure to effectively localize the incident terahertz waves at the source end, thus forming a temperature gradient in the device channel and inducing the thermoelectric terahertz response. At zero bias, the device has a high photoresponsivity of 1.54 A/W, a noise equivalent power of 19.8 pW/Hz The terahertz (THz) radiation 0.1–10 THz) is located in the transition area between electronics and optics and includes a wide electromagnetic frequency spectrum between microwaves and infrared, and its optoelectronic hybrid characteristics have a wide range of potential applications, including wireless communication, spectroscopy, material science, sensing and imaging, etc. –10 THz i,3. SincePTE) of PTE detector is the integral of the product of Seebeck coefficient and temperature gradient along the conduction channel of the device. The formula isBT << EF, according to Mott equation, S can also be written asB is Boltzmann constant, e is the electron charge, σ is the electrical conductivity of the material, EF is Electron energy. From Equation (2), S is proportional to the conductivity and the derivative of energy, so the Seebeck coefficient can be regulated by adjusting the Fermi energy level, which is generally divided into gate control and chemical doping. For PTE detectors, if we assume that the Seebeck coefficient varies in a small range along the channel, the asymmetric distribution of temperature is necessary. Photothermoelectric (PTE) detectors are a kind of optical-thermal detector based on the PTE effect. Unlike photoconductive and photovoltaic detectors whose spectral response range is limited by the active semiconductor bandgap used in the detector, PTE detectors are known for their ultra-wideband response operation at room temperature and zero driven bias ,10,11,12Since the discovery of graphene in 2004, researchers have begun to study various 2D materials in depth, and the potential use of these materials as photodetectors in a wide range of electromagnetic spectrum is an important research direction ,17,18,19In this work, a simple antenna coupled asymmetric structure has been used to achieve the excellent device performance of graphene PTE detectors, which can be compared with the state-of-the-art two-dimensional materials terahertz detectors. Furthermore, we propose a qualitative model to analyze the highly sensitive PTE response caused by asymmetric coupling. In particular, we found that the metal-graphene contacts play a key role in the terahertz PTE response. Our results have important guiding value for further optimization of PTE terahertz detectors for two-dimensional materials in the future.2 was used for the construction of graphene PTE detectors. The detectors were fabricated by standard planar micro-nano device processes and two-dimensional material transfer techniques. Firstly, the coupling structure of the metal antenna (5 nm Ti/40 nm Au) was prefabricated by lithography, electron beam evaporation, and lift-off processes. Then, the highly oriented pyrolytic graphite was mechanically dissociated into micron-sized few-layer graphene (~30 nm) using blue film tape, and the graphene flake was accurately transferred to the channel area of the device by using 3D mechanical transfer platform and PDMS-assisted transfer technology. The graphene device is encapsulated with metal leads onto a custom PCB board for subsequent electrical and optical measurement.We used FDTD electromagnetic simulation to simulate and optimize the antenna coupling structure of the detector, which has excellent local field enhancement for far-field terahertz waves. Because high-resistivity silicon has high terahertz transmittance and can effectively reduce the reflection of terahertz waves, a substrate of high-resistivity silicon  covered with 300 nm SiO2. The output port of the multiplier is waveguide-coupled via a standard gain horn antenna, which emits linearly polarized high-frequency radiation into free space. The fundamental frequency microwaves are electrically modulated, and then electrically modulated terahertz radiation is output from the VDI multiplier and irradiated onto the device. In detail, a terahertz beam electrically modulated with a frequency of 1 kHz is irradiated onto the detector, and the generated photocurrent signals are amplified by a low-noise current amplifier (SR570) and then recorded by an oscilloscope (Tektronix MSO2024B). The noise spectrum of the detector is obtained by the spectrum analyzer (Keysight N9322C). The test process of the device was all carried out in room temperature and air environment. The output characteristic curve of the device was firstly measured using a high-precision digital source meter (Keithley 2400). The SMB100A microwave source (110 kHz–20 GHz) was used to drive a VDI solid-state 6-fold frequency multiplier to produce high-frequency radiation (75 GHz–110 GHz) with a power density of 5 mW/cmIn order to enhance the PTE response of graphene to improve the performance of terahertz detection, it is necessary to design an efficient asymmetric optical coupling structure. We designed a logarithmic antenna coupling structure which can make the local enhancement region of terahertz field at the source terminal of the device. A) is an important parameter to evaluate the performance of the detector. Its calculation formula isph is the photocurrent signal of the detector, Pin is the power density of the terahertz beam, and A is the area of the detector. In this measurement, the power density of the terahertz beam is 5 mW/cm2, and the area of the detector is 600 µm × 300 µm (including the antenna). As shown in n is the noise value at the modulation frequency, and RA is the responsivity of the detector. The noise spectrum of detectors usually contains several different main noise sources, including 1/f noise, shot noise and thermal noise (Johnson-Nyquist noise) [1/2 at 105 GHz, showing good detection sensitivity. The performance comparison of the 2D material-based terahertz detectors is shown in In the following, we discuss on the speed of the PTE detector, which is usually defined as the time measured from 10% up to 90% on rising edge of signal as well as the recovery time . As shown in t noise) ,29,30. T1 is the Seebeck coefficient in the metal-doped area, S0 is the Seebeck coefficient of graphene away from the metal electrode, T0 is the metal temperature, T1 and T2 are the temperature at the end of the doped graphene, and Th is the highest temperature point of channeled graphene. According to Equation (5), the electrode-induced doping of graphene channel near the metal-graphene contacts play a key role in the terahertz PTE response. The thermoelectric response of graphene is determined by the electrode-induced doping on graphene and the temperature difference along graphene channel. Due to the good doping of metals to graphene, this ensures the difference of Seebeck coefficients between the doped and undoped regions of graphene. Considering the contribution of Seebeck coefficient difference, the PTE response will be further improved if the signs of the Seebeck coefficient are opposite. Furthermore, the location of the optical coupling antenna in the graphene channel determines the temperature difference required to achieve the graphene PTE response. It can also be seen that when the coupling antenna is located in the middle of the device channel, due to the symmetrical configuration, the temperature difference will be zero, and the PTE response is completely suppressed. As the coupled antenna moves away from the center of the graphene channel, the temperature at the end of the graphene doping region near the coupled antenna will be closer to the maximum temperature point, and the effective temperature difference to drive the PTE response of the graphene will be expanded, thus improving the graphene PTE response. In order to optimize the device performance in the future, we proposed a qualitative theoretical model to analyze the high terahertz sensitivity caused by the asymmetric antenna coupling structure. As shown in 1/2, and a response time of 900 ns. Furthermore, through qualitative analysis of the response mechanism of graphene PTE devices, we find that the electrode-induced doping of graphene channel near the metal-graphene contacts play a key role in the terahertz PTE response. These results show that the optical coupled structure we designed effectively improves the thermalelectric terahertz detection sensitivity, providing an effective way to further improve the performance of two-dimensional materials terahertz detectors in the future.In summary, we achieved sensitive graphene-based PTE terahertz detectors by using an efficient asymmetric antenna coupling structure, which exhibits excellent zero bias detection performance in the frequency range of 75 GHz to 110 GHz. The measurement results show that the graphene device has a zero bias photoresponsivity of 1.54 A/W, a noise equivalent power of 19.8 pW/Hz


Sarcoidosis is a complex inflammatory multisystem disease of unknown etiology that is characterised by epithelioid cell granulomatous lesions affecting various organs, mainly the lungs. In general, sarcoidosis is asymptomatic, but some cases result in severe complications and organ failure. So far, no accurate and validated modelling for clinical and pathohistological manifestations of sarcoidosis is suggested. Moreover, knowledge about disease-specific diagnostic markers for sarcoidosis is scarce. For instance, pulmonary granulomatosis is associated with the upregulated production of proinflammatory molecules: TNF-α, IL-6, CXCL1, CCL2, CCL18, CD163, serum angiotensin-converting enzyme (sACE), lysozyme, neopterin, and serum amyloid A (SAA). Quantum dots (QDs) are widely applied for molecular diagnostics of various diseases. QDs are semiconductor nanoparticles of a few nanometres in size, made from ZnS, CdS, ZnSe, etc., with unique physical and chemical properties that are useful for the labelling and detection in biological experiments. QDs can conjugate with various antibodies or oligonucleotides, allowing for high-sensitivity detection of various targets in organs and cells. Our review describes existing experimental models for sarcoidosis , their advantages and restrictions, as well as the physical properties of quantum dots and their potential applications in the molecular diagnostics of sarcoidosis. The most promising experimental models include mice with TSC2 deletion and an in silico multiscale computational model of sarcoidosis (SarcoidSim), developed using transcriptomics and flow cytometry of human sarcoid biopsies. Both models are most efficient to test different candidate drugs for sarcoidosis. Sarcoidosis is a multisystem inflammatory disease of unclear etiology, most often affecting the lungs (up to 90% of cases) and lymph nodes. Sarcoidosis is characterised by the formation of non-caseous granulomas and ranks first in frequency among granulomatous diseases. Women, as well as workers disproportionately exposed to chemical agents , are at the highest risk of developing sarcoidosis ,2. SarcoMycobacteria and Propionibacteria, as a likely cause of sarcoidosis. This hypothesis is supported by the meta-analyses conducted from 1980 to 2015, confirming the link between sarcoidosis and mycobacteria  [P. acnes in the pathogenesis of sarcoidosis is no less widespread. Endogenous infection caused by symbiotic P. acnes can lead to the formation of granulomas in individuals who are predisposed to a hypersensitive Th1 immune response against an intracellular proliferation of latent P. acnes [Molecular mechanisms behind the development of sarcoidosis are not definitively established. Its pathogenesis is associated with infection-related, genetic, and immune factors. Infection-related factors include microbial agents, such as strains) . In addistrains) . A CD4+ P. acnes . Some HLP. acnes ,12, wherP. acnes .In this regard, the analysis of existing models for sarcoidosis as well as the development of novel disease models are critical to elucidate the disease pathogenesis and to advance diagnostics and therapies . This reBased on the available data on the pathogenesis of sarcoidosis, various in silico, in vitro, and in vivo granulomatous disease models were developed. Sarcoidosis models come in 5 types: BAL cell-based models, human peripheral blood mononuclear (PBMCs) models, investigation of sarcoid biopsies, animal models, and computational models. According to Bernard V. et al., a good model should meet the following criteria: to be isomorphic, homologous, and predictive .Investigations of BAL cells from patients with sarcoidosis are considered as one of the ways to study the molecular mechanisms of the pathogenesis. The method consists in the BAL procedure, extracting viable cells from the obtained liquid and seeding them on a culture medium. Flow cytometry is used to sort various types of cells, for example, CD4+, CD8+ lymphocytes, or macrophages. Using this method Wiken et al. (2010) demonstrated the absence of the differences between M1/M2 polarisation of alveolar macrophages in patients with and without sarcoidosis. In the same study, the expression of toll-like TLR-2 mRNA in BAL cell culture was reduced 2-fold in patients with sarcoidosis and reduced 4-fold in Lofgren’s syndrome compared to healthy controls .p < 0.05). The proportion of CD163+ cells was elevated in the group of ILD, but without significance (43.6% vs. 34.6%). At the same time, there was no predominance of M1 or M2 macrophage phenotype among BAL cells obtained from patients with interstitial lung diseases: hypersensitive pneumonitis, nonspecific interstitial pneumonia, and idiopathic pulmonary fibrosis [In addition, it is possible to study BAL cells by immunocytochemistry . Phenotyfibrosis .In sarcoidosis, BAL cells with the expression of the TWIST1 transcription factor—which is a gene associated with the macrophage phenotype—is a 5,1-fold increase comparing healthy donors. The BAL obtained from patients with sarcoidosis demonstrated relative mRNA expression of IFNγ (7-fold), IL18R1 (5-fold), IL12Rb2 (6-fold), STAT1 (2-fold), STAT4 (3-fold), CXCL11 (4-fold), CXCL10 (3-fold), CXCL9 (5-fold), and CCL5 (6-fold), associated with M1 macrophage polarisation. TWIST1 protein synthesis in BAL cells increased with the addition of lipopolysaccharide (LPS) and TNF-α, according to the immunofluorescence assay (quantitative data were not represented) [It is believed that bone marrow mesenchymal stromal cells (CM-MSCs) can “reprogram” various types of macrophages for an anti-inflammatory phenotype. BAL cells (70–94% of macrophages) from patients with sarcoidosis and healthy donors were cultured with CM-MSCs and stimulated LPS. After that, the content of IL-10 and TNF-α in cultures was determined. In cells obtained from patients with sarcoidosis, a 20-fold decrease in the concentration of TNF-α—which characterises the pro-inflammatory phenotype M1—and a 50-fold increase in the concentration of IL-10—a marker of the anti-inflammatory phenotype M2—were revealed in comparison with cultures without adding BM-MSCs. BAL from healthy donors did not demonstrate changes of cytokine synthesis during the addition of BM-MSCs .The TLRs are a family of immune recognition molecules that initiate the production of proinflammatory cytokines in response to the molecular patterns of pathogens. Cytokines TNF-α and IL-6 are possibly involved in the granuloma formation. Gabrilovich et al. 2013) showed that the synthesis of proinflammatory cytokines TNF-α and IL-6 increased in BAL cells in response to the TLR-2—lipoprotein M. tuberculosis (LpqH) stimulation. Stimulation of these LPS cells did not affect the production of cytokines by BAL cells in sarcoidosis. TLR-2 is capable of forming heterodimers with TLR-1 and TLR-6: TLR-2/1 and TLR-2/6, respectively. To characterise the mechanisms of TLR-2 hypersensitivity in sarcoidosis, the response of BAL cells to ligand stimulation of these heterodimers was analysed. For TLR-2/1, such a ligand is the lipopeptide Pam-3-Cys-SKKKK (Pam-3-Cys). For TLR-2/6, the peptide FSL-1 is obtained from the lipoprotein M. salivarum LP44 (MALP). Stimulation of BAL cells obtained from patients with sarcoidosis with Pam-3-Cys did not affect cytokine synthesis. With the action of FSL-1 in sarcoidosis cells compared with healthy donors, a 2-fold decrease in the concentration of TNF-α was observed. Probably, in pulmonary sarcoidosis, there is a violation of the regulation of the local heterodimeric TLR-2 response [13 showedKraaijvanger et al. (2020) proposed a comprehensive review of blood serum and BAL fluid biomarkers produced by the innate and adaptive immunity cells involved in the formation of granulomas in sarcoidosis. According to this model, sarcoidotic granulomas consist of a dense nucleus formed by epithelioid and multinucleated giant cells, and they are surrounded by T-helpers, B-lymphocytes, macrophages, and dendritic cells. During granuloma formation, these immune cells release numerous signalling molecules. Macrophages play a key role in the formation of granulomas by producing angiotensin converting enzyme (sACE), lysozyme, neopterin, CD163, CCL18, and serum amyloid A (SAA). In addition, through antigen presentation, macrophages induce T-cell activation and upregulated expression of the soluble IL-2 receptor (sIL-2R). The BAFF factor is involved in B cell activation and, thus, contributes to granuloma formation .Comparison of gene expression in sarcoidotic BAL cells revealed more than 1500 differentially expressed genes in contrast to healthy subjects . For exaM. tuberculosis immobilized on polystyrene granules was developed. After 7 days, granuloma-like multicellular aggregates were formed in the culture with the presence of macrophages in the centre and lymphocytes on the periphery. In the sarcoidosis and healthy control PBMC, the incubation with PPD granules activated the Th1-immune response. But, in the culture of PBMC from patients with sarcoidosis, the concentration of IFN—γ was less markedly increased 3,5-fold, and the concentration of TNF-α was more pronounced (4-fold) compared to the control cultures. It is obvious that granules coated with the human serum albumin (HSA) caused selective reaction of monocytes/macrophages  in PBMC in sarcoidosis, not in the control group. Such nonspecific activation of macrophages in the response to HSA-coated granules indicates the activation of macrophages and T cells in patients with pulmonary sarcoidosis [Recently, a model with PBMC exposed to purified protein derivatives (PPD) of onounced -fold comThe same model was used to demonstrate that macrophages retain the M2 phenotype in sarcoidotic PBMC cultures. This phenotype was determined by gene expression and CD163 protein receptor immuno-staining in PPD granules culture, in contrast to uncoated granules. In addition, analysis of the mRNA expression profile demonstrated that 1274 differentially expressed genes were detected in PBMCs obtained from patients with sarcoidosis after PPD stimulation. In a group of patients with sarcoidosis, IL-13 cytokine was reported to regulate differential gene expression. M2 polarisation of macrophages was probably caused by the STAT6 factor activation by IL-13 .M. tuberculosis, while the immunophenotype of their granulomas is similar to that of clinical sarcoidosis. Comparison of PBMCs’ transcriptomes in sarcoidosis and in healthy controls revealed 270 differentially expressed genes involved in IL-1, IL-6, and IL-8 signalling pathways; mononuclear migration; and regulation of cellular LPS response [A modification of the described model suggests using microparticles obtained from the cell wall of Mycobacterium abscessus (MAB), instead of PPD granules, for PBMC culture. The granuloma is observed after 72 h of exposure. MAB-stimulated granulomas have Th1 and Th17 inflammatory profiles dominated by IL-7 (2-fold), IL-7R (2-fold), and IFN-γ expression . Notably, when exposed to α-melanocyte stimulating hormone (𝛼-MSH), the model produced an anti-inflammatory effect with reduced IL-7, IL-7R, and IFN-γ expression. This effect is linked to the CREB transcription factor activation . Here, wresponse . In geneThe newest class of in vitro sarcoidosis models is based on pathogenic environmental factors, associated with the release of carbon nanoparticles into the ambient environment, such as fuel combustion, industrial manufacturing, or burning fires. Individuals exposed to such nanoparticles are at risk of developing chronic lung diseases, including sarcoidosis and fibrosis . An expeThe scheme of the use of human cells in the sarcoidosis modelling is in Animals, except horses, do not develop sarcoidosis, which complicates the development of experimental animal models of the disease ,28. CurrM. tuberculosis whole cell lysate in complete Freund’s adjuvant or recombinant mKatG protein  in incomplete Freund’s adjuvant. Two weeks later, the animals were injected with mKatG-coated sepharose granules via intratracheal instillation. Granulomatous response to mKatG granules was associated with the proliferation of antigen-specific T cells and the production of IFN-γ. Chen et al. (2010) used this model to demonstrate that chronic exposure to SAA exacerbates persistent experimental granulomatous inflammation in animals with prior mKatG sensitisation, which is probably triggered by TLR-2 [M. tuberculosis and its antigens to induce granuloma.One of the approaches is preliminary sensitisation of rodents to candidate antigens, followed by their intrapulmonary administration. Lewis rats and C57BL/6 mice were immunised using intraperitoneal injection of either by TLR-2 . The modMycobacterium sodA (superoxide dismutase A) and sodA-coated granules. Granulomas concentrated around the granules, and an increased CD4+ immune response and elevated levels of IL-2 and IFN-γ in the BAL were detected in such animals  sensitised C57BL/6 mice using P. acnes lysate with incomplete Freund adjuvant and simultaneous administration of dendritic cells two times, with a two-week timespan between sensitisations. After a week, granulomas were identified in the lung. If dendritic cells were incubated with P. acnes prior to administration, granulomas were larger in size and number. The advantage of this model is explained by significant similarity in cellular composition of granulomas and sarcoidotic granulomas exhibiting lymphocytes, epithelioid cells, and multinucleated giant cells. In BAL cells from animals, the levels of CXCL9 and CXCL10 as well as Th1-expressed cytokines and chemokines, including TNF-α and IFN-γ, were increased [P. acnes with complete Freund adjuvant at a two-week interval. Next, CD4+ T cells were isolated from the lymph nodes of immunised animals and injected into the tail vein of mice without immunisation. Two weeks after intravenous administration of T cells in mice, there was an aggregation of epithelioid and mononuclear cells and a 2-fold increase in ratio of CD4+/CD8+ observed in the BAL and peribronchial lymph nodes of mice with challenge of P. acnes in CFA comparing mice with only CFA administration. The increased number of CD4+ T lymphocytes expressed IFN-γ, which is similar to the histopathological characteristics in sarcoidosis [Alongside with Mycobacterium, ncreased . A diffecoidosis .P. acnes immunisation model is compatible with the lung histopathology in patients with sarcoidosis [Granulomas were predominantly located in subpleural, peribronchial, and perivascular regions of the lungs and featured antigen-presenting cells in the centre and CD4+ T lymphocytes in the periphery. CD4+ T cells expressed IFN-γ but not IL-4, while the CD4+ to CD8+ lymphocyte ratio was increased in BAL. This coidosis .P. acnes isolated from sarcoidosis patients, instead of heat-killed P. acnes, promotes formation of larger granulomas. Interestingly, mice with mutations in the Myd88 and Cyb genes showed increased P. acnes persistence in the lungs [Werner et al. (2017) revealed that injection of he lungs .Barna et al. investigated the role of MWCNT carbon nanoparticles in mice with a deficiency of genetic factors associated with human sarcoidosis. This may contribute to the development of immunoassociated lung granuloma . In miceThe deficiency of ABC-transporter of alveolar macrophages ABCG1 is also associated with granulomatous inflammation in the lungs . RelativThus, a large number of signalling molecules are involved in the pathogenesis of sarcoidosis, which may be markers of the disease. The main ones are listed in There is a variation of the MWCNT model, when ESAT-6, a peptide of 15 amino acid residues and a component of mycobacterial antigen, is injected into animals together with MWCNT. For 60 days after simultaneous administration of ESAT-6 peptide with MWCNT, an increase in granulomatous lesion and pulmonary fibrosis was revealed compared with the administration of MWCNT . IncreasGenetic modifications in the study of sarcoidosis are used as an additional method, in the case of MWCNT for example, when their effects were studied in mice with knockout of various genes involved in the pathogenesis. The deletion of the Tsc2 gene makes it possible to obtain manifestations of sarcoidosis in mice. TCS2 (tuberin) is a tumour suppressor that normally suppresses the activation of the mTORC1 complex, a negative regulator of autophagy. Deletion of Tsc2 leads to the activation of mTORC1 and induced hypertrophy, proliferation, and granulomatous inflammation in mice in vivo. Macrophages with Tsc2 gene expression deficiency form granuloma-like structures . HoweverThus, the most used common models of granulomatous lung lesions in animals include intraperitoneal or subcutaneous sensitisation with bacterial components, as well as the introduction of foreign particles—multi-walled carbon nanotubes, which is a reflection of the bacterial theory of sarcoidosis and the theory of immune response to environmental components . In addiBased on stochastic Petri nets , a two-cMathematical modelling can be used to evaluate the interactions of immune cells and inflammatory mediators during granuloma formation. The targets of therapeutic agents identified in this way can be tested in in vitro and in vivo models.The summary of gene expression and protein synthesis used in experimental modelling of sarcoidosis is in Diagnosis of sarcoidosis is difficult due to the lack of uniform criteria and specific biological markers of the disease. The diagnosis is based on computed tomography data and biopsy of the affected tissues; one of the conditions is the exclusion of alternative causes with examination for tuberculosis, fungal infections, etc. . A long Such methods include the visualisation of tissues and organs with the registration of autofluorescence or specific staining of cells and tissues with contrasting agents . SubstanQuantum dots (QDs) are a class of fluorophores, which are semiconductor nanocrystals with a size of 1–15 nm. According to the chemical composition, 12 classes of QDs are distinguished in accordance with the position of their constituent elements in the Periodic Table of Mendeleev . ClassifDue to small size, QDs obey the laws of quantum mechanics and have special physicochemical properties and optical characteristics that distinguish them from other fluorophores used in biomedical research. QDs have a high molar extinction coefficient, a wide absorption spectrum, a narrow and symmetrical fluorescence spectrum, and a high resistance to photodegradation. A wide range of excitation makes it possible to use a mixture of several QDs in the study. The physicochemical properties of QDs depend on the method of their synthesis, which can be divided into four groups: colloidal synthesis, synthesis based on biomatrix, electrochemical assembly, and biogenic synthesis . DynamicBiomedical applications of QDs currently include in vitro diagnostics, visualization of living cells, use in photodynamic therapy, and drug delivery.Due to the unique optical properties of QDs, it became possible to modify the method of immunohistochemistry (IHC). Standard chromogens that allow the visualisation of the immunohistochemical reaction have high photosensitivity and the ability of nonspecific binding, which can lead to the background staining of drugs. Some chromogens  have high toxicity and carcinogenicity ,58. The A protocol for the prognostic diagnosis of triple-negative breast cancer was proposed based on the visualisation of the topoisomerase 2 alpha (TOP2A) marker using QDs . MultiplZhao et al. created “affibodic” QDS (AF-QDs) for the human epidermal growth factor receptor type 2 (HER2) in human lung tumour cells . In addiChen et al. (2009) evaluated the ability of QD-IHC to visualise caveolin-1 and PCNA in a lung cancer tissue micromatrix compared to traditional IHC. It was demonstrated that both methods revealed the studied markers in both maligned and normal lung tissue. However, the sensitivity of QD-IHC was higher than IHC. The detection level of caveolin-1 and PCNA using QD-IHC was 57% 40/70 samples) and 86% (60/70 samples), respectively, which was higher than the detection level of markers using IHC (47% (33/70 samples) and 77% (54/70 samples), respectively. Less pronounced background staining was observed with QD-IHC than with IHC [ samples Another type of semiconductor nanocrystals—rod-like semiconductor nanocrystals, QRs, injected intramuscularly into live mice—can be visualized using confocal microscopy in formalin-fixed tissue sections filled with paraffin. Despite the small number of QRs injected (25 picomoles), they were clearly visualised in muscle sections after 24 h, and their fluorescent signal was stronger than that of QDs CdSe/ZnS functionalised in a similar way. In addition, in the case of QRs, the signal on the slice was preserved even 21 days after injection. This indicates the need to apply various modifications of semiconductor nanocrystals depending on the type of experiment .In addition to tissue staining, the use of QDs is possible in cell culture. Ornberg and Liu (2007) published a detailed protocol for the visualisation of nuclear, cytoplasmic, and membrane compartments in a number of cell cultures using commercial secondary antibodies conjugated with QDs. Various variants of fixation and permeabilization, as well as features of cell smear conclusion were described .Taking into account the possibility of multichannel imaging, IHC with QD looks like a promising method for the study of the molecular aspects of the development of granulomatous inflammation in sarcoidosis; however, such studies have not yet been conducted.In addition, QDs are used to visualise cellular components. Due to their small size, QDs are captured by cells during incubation, allowing for visualisation using a fluorescent microscope. Once conjugated with tissue-specific ligand molecules, QDs can be used to visualise organs and tissues .A number of works are devoted to the visualisation of lung tissue in inflammation, chronic obstructive pulmonary disease, and vascular lung pathologies in mouse models . Saitoh Using QDs, it is possible to modify the fluorescence-activated cell sorting (FACS) method, a flow cytofluorimetry technology used to evaluate the functioning of drug delivery systems, isolate individual cell populations, detect cell markers, and map immune cells . For exaQuantum dots seem to be a promising method not only for targeted drug delivery but also for the diagnosis of various diseases. The combination of IHC and QDs may be a promising method for the diagnosis of sarcoidosis. Taking into account the fact that immune cells involved in the formation of granuloma in sarcoidosis form a certain secretory phenotype, it is possible to use IHC with QDs to simultaneously identify several biomarkers in samples of damaged tissue in order to more accurately identify the molecular and morphological characteristics of cells. The cytotoxicity of QDs is not a problem here, since the study is carried out on a fixed biomaterial, but it remains an invasive procedure, which may be a disadvantage of this method. However, in addition to the standard histological examination, the use of IHC with QDs can significantly increase the accuracy of differential diagnosis. It also seems possible to use a modification of the FACS method with QDs to sort BAL cells from patients with sarcoidosis .Drobintseva et al. (2015) studied the cytotoxicity of some QDs and their effect on the morphology of breast carcinoma cells ZR-75-1 and normal human mononuclears. QD ZnSe:Mn/MPC (mercaptopropionic acid) did not affect cell morphology during 48 h of incubation, even in high concentrations (up to 17.5 mM/mL). However, Qds CdSe caused apoptosis of 20% of cells after 18 h of the incubation . Thus, iThus, the reduction of cytotoxicity of QDs and studies directed to their application in various experimental models of sarcoidosis are a promising area for the development of molecular medicine. It can be concluded that, despite the absence of a generally recognised model of sarcoidosis, studies of the occurrence and development of granulomatous inflammation in various models are being successfully conducted.The search for optimal models of sarcoidosis is important for the diagnosis and development of effective therapy for this disease. Currently, five main models of sarcoidosis are described: BAL cell-based models, PBMC, studies of biopsies of damaged tissue, animal models, and computer models. Sarcoidosis modelling is carried out in vitro using human lung cells and in vivo in mice with the experimentally induced fibrosis and lung granulomas, as well as in animals with impaired expression of the Tcs2 gene—an activator of the mTOR signalling pathway. In most of the described models, it is possible to evaluate the expression of genes and synthesis of proteins involved in the pathogenesis of sarcoidosis.The quantum dot method is promising for investigations of the molecular aspects of the granulomatous inflammation in sarcoidosis, however, such studies were not yet conducted. In addition, QDs have cytotoxic properties, which impose additional restrictions on this type of research.This review indicates the need for the optimal combination of available methods for diagnosing and evaluating the molecular mechanisms of the pathogenesis of sarcoidosis in silico, in vitro, and in vivo. Such investigations are an actual task of molecular medicine.


This paper builds on the context and recent progress on the control, reproducibility, and limitations of using graphene and graphene-related materials (GRMs) in biomedical applications. The review describes the human hazard assessment of GRMs in in vitro and in vivo studies, highlights the composition–structure–activity relationships that cause toxicity for these substances, and identifies the key parameters that determine the activation of their biological effects. GRMs are designed to offer the advantage of facilitating unique biomedical applications that impact different techniques in medicine, especially in neuroscience. Due to the increasing utilization of GRMs, there is a need to comprehensively assess the potential impact of these materials on human health. Various outcomes associated with GRMs, including biocompatibility, biodegradability, beneficial effects on cell proliferation, differentiation rates, apoptosis, necrosis, autophagy, oxidative stress, physical destruction, DNA damage, and inflammatory responses, have led to an increasing interest in these regenerative nanostructured materials. Considering the existence of graphene-related nanomaterials with different physicochemical properties, the materials are expected to exhibit unique modes of interactions with biomolecules, cells, and tissues depending on their size, chemical composition, and hydrophil-to-hydrophobe ratio. Understanding such interactions is crucial from two perspectives, namely, from the perspectives of their toxicity and biological uses. The main aim of this study is to assess and tune the diverse properties that must be considered when planning biomedical applications. These properties include flexibility, transparency, surface chemistry (hydrophil–hydrophobe ratio), thermoelectrical conductibility, loading and release capacity, and biocompatibility. The interdisciplinary field of tissue engineering (TE) comprises the use of biomaterials, biological active agents, including cells, and engineering to restore or develop biological substitutes for various tissues in the body . AssemblCells may be grown in two-dimensional (2D) or three-dimensional (3D) cultures. Generally, 2D cultures comprise the growth of cells on a flat surface, such as a flask or petri dish. This method is commonly used in research given its cost-effectiveness, feasibility, and ease of observation when analyzing cells; however, it lacks the capacity to accurately resemble in vivo cellular environments. On the other hand, 3D cultures, which may be performed using the hanging-drop method, microfluidic platforms, or scaffolds, allow for interactions between cells and the extracellular matrix (ECM) to take place . As suchScaffolds are structures that are used in both in vitro and in vivo TE applications and serve as support to ensure the growth of the ECM, cell adhesion, and subsequent tissue growth . ScaffolThe development of scaffolds that can encourage neural tissue regeneration has generated much interest for GRMs. Recent studies have demonstrated that GRMs encourage the adhesion, proliferation, and differentiation of various cells, including embryonic stem cells (ESCs), neural stem cells (NSCs), mesenchymal stem cells (MSCs), and induce pluripotent stem cells. Many researchers have found that scaffolds based on conductive materials can boost NSC proliferation and differentiation into neuronal or glial cell lineages . GRMs arIn 2015, Jakus et al. demonstrated that human mesenchymal stem cell (hMSC) adhesion, viability, proliferation, and neurogenic differentiation may be supported by 3D printable graphene (3DG) . These pMolecularly changing the surfaces of neural probes with anti-inflammatory agents, adhesion of proteins, and bioactive chemicals comprises one method of regulating the inflammatory process and improving neural electrode incorporation into brain tissue . HyaluroA significant challenge in using neural electrodes for recording brain activity both in vitro and in vivo is developing novel materials that produce seamless neural interfaces with high sensitivity. Another issue that has driven the creation of innovative materials is ensuring long-term stability. By considerably boosting the signal-to-noise ratio, electroactive nanomaterials such as silicon nanowires , carbon The ECM is a critical component of all types of tissues and serves as a natural scaffold for cells to adhere to and differentiate. The ECM, therefore, contributes greatly to the homeostasis and morphogenesis of tissues. In 3D TE, scaffolds may be developed using various polymeric or non-polymeric materials to mimic the ECM of target tissues. When selecting biomaterials for scaffolds, it is important to choose a material with properties that are suitable for the target tissue and cell types being used as the properties of the material should mimic those of the ECM of the target tissue. This will ensure that the material will contribute to obtaining the desired results.The properties of various scaffolds can differ in terms of structure, rigidity, strength, pore size, and the concentrations of the polymers used . The comBiomaterial-related scaffolds provide porous networks, mechanical and structural support, shape, and hierarchical structures with hydrophilic properties to allow for cell attachment, cell–cell communication, proliferation, and differentiation for tissue regeneration in TE. To date, most synthetic biomaterials for TE applications may be generated from lactic acid, caprolactone, or glycolide monomers, which can form poly(L-lactide), polycaprolactone, and poly(L-glycolic acid) materials or copolymers .Chitosan, alginate, starch, collagen, HA, cellulose, fibrin, silk, and their derivatives may also be utilized to build scaffolds . To manaRecent studies have assessed the potential of silkworm silk as a unique biomaterial that may address many of the shortcomings of the current materials used in TE scaffolds. The natural protein, silk fibroin (SF), which is obtained from silkworm silk, has been studied as a potential biopolymer for TE due to its biocompatibility, tunable biodegradation, minimal immunogenicity, adaptability to different forms , excellent mechanical strength, and ease of accessibility ,25. SF cHA, a polymer that is composed of linear glycosaminoglycan and is abundant in the ECM, has been shown to impact cell signaling pathways, which is critical in TE. Clinical trials have further shown that HA is useful in treating osteoarthritis, and the US Food and Drug Administration has approved HA as a biomaterial for human use .Stem cells are usually grown in vitro in a monolayer on a flat plastic substrate. This method, however, cannot fully replicate the characteristics of cells in their native microenvironments. Thus, it is important to offer an adequate microenvironment for developing functional tissues by creating biomimetic scaffolds that are analogous to stem cell niches . ConductTo date, various biological materials made of natural or synthetic materials have been used for peripheral nerve regeneration, including HA, SF, polypropylene alcohol, sodium alginate, gelatin, chitosan, and polyacrylamide (PAM) ,37,38,39Due to their excellent biocompatibility, ability to retain water, and drug-delivery capabilities, hydrogels have recently become widely used in TE including nerve regeneration . For exaDue to their excellent biocompatibility, PAM hydrogels are frequently used in the field of peripheral nerve regeneration . Pure PAOne reason why graphene has drawn the interest of scientists worldwide is its advantage to be involved in a significant number of applications due to its unique characteristics . It has 2 hybridization, which makes it electroconductive  expression . GraphenTE has allowed for improved diagnostic and therapeutic techniques. Tissue and organ transplantation methods may also be improved through TE with various biomaterials. Biomaterials have been proven to be useful in developing biomimetic tissue constructs; however, different host tissues have different physical, chemical, and electrical properties, which individual biomaterials may not be able to replicate independently. Developing composite or hybrid materials is therefore necessary to ensure each property of a tissue is mimicked effectively . Graphenl-lysine, can control the electrical resistance of the materials and subsequently facilitate the adhesion of neural cells and outgrowth of neurites [Lu et al. demonstrated that GO can interact with DNA oligonucleotides via multiple interactions and thus could be used to protect them from enzymatic cleavage while delivering the oligonucleotides into target cells . Tang etneurites .Various methods may be used to characterize 3D graphene-related scaffolds. For example, the morphology of a graphene-related scaffold may be determined using transmission electron microscopy or atomic force microscopy imaging ,110. RamGraphene has the capacity to simulate the complex signaling systems in the nervous system—neurons connect electrical messages using chemical signals, such as acetylcholine —given it2+ oscillations in the neurons doubled in comparison with that in the control group. These findings suggest that GRMs may encourage the growth of neural networks and improved brain circuits. Liu et al. developed an implanted graphene-related brain electrode to track electrophysiological and neurochemical signaling in vivo [2O3 nanocomposite was developed. This rGO-modified electrode provided increased electron transport between tissue and electrode surfaces, lower detection limits, and greater sensitivity to hydrogen peroxide than standard gold electrodes did. Feng et al. also exhibited the use of soft nanofibers based on graphene for the monitoring of brain activity [3 plasma treatment prior to the reduction in graphene oxide nanofibers (GO-NFs) to produce the G-NFs. The ultrathin graphene shells of the G-NFs, which completely enveloped the nanofiber surfaces, resulted in high electrical conductivity and excellent flexibility. Thus, neurons on the G-NFs generated more neurite branches and expanded faster than neurons on the graphene films and tissue culture plates did. Neurons on the G-NFs also exhibited elevated levels of class III beta tubulin (TUJ1) and microtubule-associated protein 2 (MAP2) due to the geometric cues of the G-NFs, which closely mirrored nanofibers and mature neuronal markers. By applying electrical stimulation on neurons through G-NFs (referred to as G-NFs/ES), a growth rate that was noticeably boosted and approximately twice as fast as that of neurons on the G-NFs was identified. The activation of the calmodulin-kinase pathways by Ca2+ influx, which is driven by the depolarizing effect of the voltage-gated Ca2+ channels induced by G-NFs and increases intracellular Ca2+ concentrations, may have been the reason for this stimulation. Furthermore, the 3D G-NF nanofibers had a considerable impact on the development and growth of motor neurons. The G-NFs provided an anisotropic electron transfer at the points where the neurons made contact, which could have led to localized electrical stimulation.GRMs have been utilized in brain interfaces and electrical recordings due to their electrical properties ,120. A u in vivo . To measactivity . NegativNumerous studies have highlighted the use of graphene-related scaffolds in facilitating the adhesion and differentiation of neural cells ,126. NerThe “gold standard” for healing damaged nerves or gaps in clinical settings is autologous nerve grafting with sutures. Long defects or gaps are challenging to fix due to the slow rate of axon regeneration (approximately 2–5 mm/day). A sufficient supporting bridge, also known as a conduit, is therefore needed to reconnect damaged nerves .In a study by Hong et al. , a 2D gr2/g) and 3D porous structures. Additionally, their interconnected gaps improved cellular communication and allowed for efficient nutritional mass transfer for cells. Furthermore, the 3D-GFs displayed microscale topographic features, such as curvatures or anisotropic microstructures, which was in contrast with the features of 2D GRMs. These findings indicate that GFs may facilitate enhanced neuronal differentiation.Li et al.  further Despite the fact that GRMs have been extensively used to create films  or 3D scJakus et al. used graphene and poly(lactide-co-glycolide) (PLGA) to generate a bioink, which they then utilized to 3D print a nerve conduit with a specific size . They deIn a study by Feng et al. on the influence of GO and rGO membranes on the differentiation of adipose stem cells (ADSCs) into nerve cells, it was discovered that GO had a stronger effect on the ability of ADSCs to differentiate into neurons after 7 days of culture . One of Another possible treatment option for neurodegenerative illnesses involves the growth of NSCs, according to Huang et al., who embedded NSCs in hydrogels that were made by combining graphene or GO with polyurethane (PU) using 3D bioprinting . The visIn a study by Niu et al. , rat indIn another study by Magaz et al. , electroIn order to create a GO/antheraea pernyi silk fibroin (ApF)/poly(L-lactic acid-co-caprolactone) (PLCL) scaffold, Wang et al. coated GO onto an electrospun ApF/PLCL composite scaffold . FollowiSince graphene is a multifunctional carbon nanostructure, it is not infrequent for it to be utilized in cancer treatments and therapies. QDs, other carbon nanomaterials, GO, and graphene itself are usually involved in processes such as tumor screening, chemotherapies, and photothermal and photodynamic therapies . GraphenThe surface of graphene has been found to exhibit covalent and noncovalent activities, which are of utmost importance for its interactions with cancer cells, tissues, and anticancer drugs . VariousAcidic pH and hydrogen peroxide are two of the features that play a vital role in the activities of tumor microenvironments. Lin et al.  showed tGRMs are attractive for drug delivery and gene delivery especially because their surface can be tuned easily from highly hydrophobic to highly hydrophilic . A proper functionalization can lead to a proper hydrophyl-hydrophobe ratio and thus the delivery rate can be adapted according to the needs. Moreover, GRMs are able to absorb visible and infrared light and thus photothermia can be generated with the main aim to act as a triggering factor in delivery. These findings highlight graphene as an ideal platform for gene and drug-delivery technologies . Gene therapy is a medical approach that either treats or prevents disease by targeting genetically and adversely altered genes through various nanocarriers, such as graphene . Lo et aGO has also been used as a nanocarrier for methotrexate, a drug against cancer cells, to test if the toxicity of the drug can be enhanced in tumors. Abdelhamid and Hussein  tested tIt is also critical to consider the antimicrobial and antibacterial activities of graphene to achieve an in-depth understanding of its mechanisms and functions for biomedical applications. Biofilm formation is one of the most common ways to study a microorganism colony . HoweverE. coli. The results revealed the activity of E. coli was restrained and high levels of antimicrobial efficacy were achieved on the graphene and GO aluminum plates. Additionally, graphene nanowalls have been used as coatings for stainless steel-related materials and exhibited high activity against Gram-positive and -negative bacteria. The bacterial membranes appeared to be damaged following their exposure to the sharp edges of the graphene nanowalls [Various microbes have been identified for use in medical devices such as ventilators, urinary catheters, and dentistry devices. Developing devices with antimicrobial coatings is therefore necessary to diminish contamination levels. GRMs can be utilized to create suitable nanomaterial layers or device coatings to eliminate microorganisms . Graphenanowalls . Along with the intrinsic antimicrobial activity of the GRMs, more complex formulations based on GRMs and metal and metal-oxide nanoparticles, polyelectrolytes or antimicrobial agents were proposed. Certainly, the GRMs/Ag(/Polymer) or GRMs/ZnO(/Polymer) binary(/ternary) systems are some of the mostly studied GRM-based antimicrobial supports . A speciSome of the most important applications of the GRMs are presented in It has further been found that the high surface area of graphene in conjunction with its high chemical stability  enhancesNeuroMem, a neurodevice that exploits graphene to mirror the activity of synapses in the brain, is an extraordinary and promising development for future practices in the field. It is a low-cost memristor device that not only portrays behavioral and neurological data but also provides access to memories through enhanced neuroimaging . GraphenGraphene has been given the title “material of the future” and is increasingly being introduced in all the fields within science. However, it is necessary to consider the potential toxic effects of graphene in animal and human health. Over the years, environmental and biological safety concerns have been raised about the use and risks of graphene. Although graphene appears advantageous due to its abovementioned properties, studies have shown that it is associated with cellular apoptosis, necrosis, autophagy, cytoskeletal disorders, organelle and protein dysfunction, oxidative stress, physical destruction, DNA lesions, and inflammatory responses ,177. Graphene can also appear noxious if it is conglomerated in inordinate amounts in tissues and organs , such asThe type and quality of graphene are significant factors that must be considered in biomedical applications since they can affect mammalian cells and lead to cell membrane lesions and, consequently, apoptosis. These issues comprise some of the major challenges in the commercialization of graphene. There is therefore a need to develop GRMs with exceptional quality and in large amounts with little cost . Despite the abovementioned challenges, the focal point in developing GRMs is controlling the size of graphene products, which will allow for toxicity to be diminished in animals and humans . In a stThe toxicity of graphene can also be reduced by forming polymer nanocomposite molecules with GRMs. The materials can be constrained through the polymer matrix nanocomposites, which further restricts their permeability. Enriched oxygen graphene molecules can further diminish the hydrophobicity of polymeric matrices to assist in cell adhesion and expansion . By elucidating the mechanisms of graphene and considering the current comprehension of its functionalities, it can be assumed that not all GRMs can necessarily be characterized as hazardous. Likewise, not all of the materials can be characterized as harmless . NumerouDespite there being many studies that demonstrate the promising characteristics of graphene in scaffolds for nerve TE, implants, and therapeutic drug-delivery vehicles, several studies have shown that graphene can exert toxicity in the host or to cells ,182,183.Another limitation of using graphene nanoparticles in biomedical applications involves their shapes, which typically comprise sharp edges. These edges have been found to be damaging to cell membranes as they can penetrate the membranes edge-first, especially when the graphene particles are of a small size . SignifiOther studies have shown that graphene itself can be non-biodegradable, which can lead to toxicity and hinder effective tissue regeneration . BiodegrGiven the potential of using graphene in biomedical applications, many studies have aimed to determine methods to reduce its toxicity and improve its biocompatibility. Although graphene does not degrade on its own, its carboxylate derivatives can degrade under suitable conditions . StudiesGraphene has also been demonstrated to be hazardous in vivo in animal experiments. For example, one study found that after injecting GO into the lungs and liver, GO aggregated in a dose-dependent manner . These nFollowing functionalization with PEGylation, graphene has been found to act differently in vivo and be far more biocompatible. PEGylated graphene with a diameter between 10 and 50 nm has been found to exhibit low levels of cytotoxicity over the course of 1 month. PEGylated graphene has also been found to accumulate and then be gradually removed over time . The decAnother study examining the impact of GO on medical devices examined the biocompatibility of GO after the implantation of subcutaneous and peritoneal tissue at two oxygenation levels. The findings revealed an inflammatory response that resembled a usual reaction to a foreign body. GO (20 mg/kg) was injected into several tissues with varied C-to-O ratios. Flow cytometry was used to measure the number of inflammatory cells. The macrophage levels were lower in the GO-treated groups than in the control group during the course of a 2-week monitoring period. Notably, larger monocyte numbers and a stronger pro-inflammatory milieu were linked to higher GO oxidation levels. The variables that determine graphene-related toxicity are therefore the dose of graphene used, its size, the treatment period, and the number of layers of graphene. It has also been proposed that the surface characteristics of graphene and its derivatives, such as the functional groups and their chemical structures, are directly connected to the toxicity of the materials. Cytotoxicity may, however, be reduced by crosslinking the derivatives with biocompatible materials or controlling the sizes of the compounds through synthetic methods . Graphene-related products can further damage cells and induce oxidative stress. Consequently, while functionalizing graphene and its derivatives, it is crucial to consider their purity. Overall, given that GRMs come in various sizes and shapes and can be produced through various methods, determining and minimizing the toxicity of the materials for clinical usage is essential.Overall, studies in the field have produced conflicting results. Many studies have demonstrated the cytotoxicity of graphene nanomaterials in TE applications, whereas others have indicated that they are biocompatible. These inconsistencies may be due to several causes, including the cell types being assessed and their genetic background, experimental techniques and parameters, culture conditions for in vitro experiments, the derivatives and forms of graphene being used, and their physicochemical properties. Further studies are therefore necessary to understand the correlation between these factors and graphene-related cytotoxicity. A larger number of methods to functionalize graphene for use in specific TE applications, such as in neurodevices, targeted drug delivery, implants, DNA sensing, and scaffolds for tissue regeneration, are also needed given the numerous factors that can influence the cytotoxicity and efficacy of graphene-based and -coated materials and devices. Moreover, many studies have assessed the toxicity of graphene and its derivatives in lung or liver cells, but few have studied the cytotoxic effects of graphene on neural cells. Elucidating these issues and further studying the impact of graphene on neural cells will allow for in vivo applications to be more successful, which may eventually lead to human clinical trials taking place and, finally, the commercial and biomedical use of GRMs and devices in nerve TE.