We report the formationof nanobubbles on graphene with a radiusof the order of 1 nm, using ultralow energy implantation of noblegas ions  into graphene grown on a Pt(111) surface. Weshow that the universal scaling of the aspect ratio, which has previouslybeen established for larger bubbles, breaks down when the bubble radiusapproaches 1 nm, resulting in much larger aspect ratios. Moreover,we observe that the bubble stability and aspect ratio depend on thesubstrate onto which the graphene is grown (bubbles are stable forPt but not for Cu) and trapped element. We interpret these dependenciesin terms of the atomic compressibility of the noble gas as well asof the adhesion energies between graphene, the substrate, and trappedatoms. Delving into the physicalmechanisms that determine the stability and shape of these subnanometerbubbles reveals that they constitute a fundamentally different regime,exhibiting an extreme aspect ratio, tensile strain, and pressure.The unique properties of this subnanometer regime open an unexploredground for applications of nanobubbles in 2D materials.5 Inthis regime, bubbles have been observed on various substrates  with a variety of trapped substances,15 which do not appear to significantly affect the bubblestability.6 The substrate and trapped substance do affectkey properties, such as shape (in particular the aspect ratio) andthe pressure inside the bubble.6 This dependence is largelydetermined by the balance of the adhesion energies: between grapheneand the substrate (γGS), between the substrate andthe trapped substance (γSb), and between the grapheneand the trapped substance (γGb).5 A particularly striking feature demonstrated for nanobubblesin the few nm regime and larger is that the aspect ratio exhibitsuniversal scalinghmax isthe bubble maximum height, R is the bubble radiusat the base, c1 is a constant (0.7), and Y is the Young modulus.5 Here,by combining scanning tunneling microscopy (STM) measurements withmolecular dynamics (MD) simulations and density functional theory(DFT) calculations, we show that this universal scaling breaks downat small R (near 1 nm and below). We also observethat the bubble stability is strongly dependent on the substrate.We interpret these dependencies in terms of the role of the atomiccompressibility of the noble gases as well as of the adhesion energies. Moreover, these nanobubbles are found to induce high levels ofstrain (of the order of 10%) on the overlaying graphene and are predictedby our MD simulations to hold the noble gas atoms under extreme pressures(exceeding 30 GPa).Theproperties of graphene bubbles with a radius of a few nm andlarger are relatively well understood on the basis of elasticity theoryas well as graphene’s elastic properties and its van der Waals(vdW) attraction to the substrate.17 Nanobubbles are formedby implanting noble gas ions , with a kinetic energyof 25 eV, with perpendicular incidence with respect to the surface.Bubbles were found to only form for graphene on Pt(111) . In the following,we will focus on Pt(111) and return to Cu(111) further below whendiscussing how the bubble stability depends on the substrate. Ionimplantation has been previously used to form graphene nanobubblesof noble gases.15 In contrast to the previous studies, where ion beams with energiesof 500 eV and higher were used, our approach is based on ultralowenergy (ULE) ion implantation. Such low energies are crucial to minimizeirradiation-induced damage. Based on our MD simulations , we selected 25 eV  as sufficiently high for a significant fraction of theions to be transmitted through the graphene layer but sufficientlylow to minimize carbon atom displacements . While ULE ion implantation has been previouslyused for doping of graphene  where vacanciesare required , such defects must be avoided in the context of thepresent work so that the intrinsic elastic properties of grapheneare maintained. The graphene bubbles observed in our samples are identifiedas nanometer-scale protrusions on the surface of graphene (grown onPt(111), implanted with the noble gases) as shown in the STM topographiesin Supporting Information). The high structural order of theirradiated surfaces is supported by our atomic-resolution STM measurementson the as-implanted surfaces (Supporting Information). Indeed, Raman spectroscopy measurements show some degree of disorder. Since the selected implantationenergy is below the threshold for vacancy formation , this disorder is likely associatedwith the breaking of C–C bonds without the production of Cvacancies and likely resulting in locally enhanced interaction ofthe Pt atoms at the interface, leading to the subtle defect featuresobserved by STM.Our samples consist of epitaxial graphene grown by chemical vapordeposition (CVD) on epitaxial Pt(111) and Cu(111) thin films grownon sapphire(0001) substrates. Pt(111) 1, not fo Pt(111) S1. In th Pt(111) S1. In th Pt(111) S1. In th Pt(111) S1. In th Pt(111) S1. In th Pt(111) S1. In th Pt(111) S1. In thR values (>1 nm), the aspectratiotends to converge to a constant value of about 0.2, which is in agreementwith the universal scaling previously observed for bubbles with aradius of few nm and larger.5 However,as R approaches the subnanometer regime, the universalscaling breaks down, showing an increase in the aspect ratio and approaching1 for Ne bubbles. From the experimental data in hmax0  andan average value for The radius and aspectratio of each bubble, for the different elements, are plotted in 5 However, as described in the Supporting Information, the effect is still negligible for the bubbles described here andis in fact in the opposite direction (decreases the aspect ratio).The observed breakdown of the universal scaling must therefore originatefrom a different mechanism, namely the existence of a minimum valuefor hmax (hmax0), correspondingto one atomic layer of the trapped gas atoms. As R approaches this regime, hmax becomesa constant value (hmax0), and consequently, hmax/R transits into a ∼1/R dependence. This is illustrated in hmax/R data with the function(hmax/R = c/R), where c (around 3 to 4 Å)is comparable to hmax0. This ∼1/R fit crossesthe value corresponding to hmax/R becomes constant, given by Bending rigidity  over the sample surface. Such nonhomogeneitycan result from the varying (relative) orientation of the grapheneand Pt lattices  as well as possible local variationsin graphene–Pt adhesion due to the subtle graphene disorderobserved in the STM and Raman data, discussed above. Although thispossible effect of subtle disorder on the graphene–substrateadhesion may also play a role in the stability of the bubbles, itdoes not appear to be a dominant effect, since our MD simulationsreproduce well the stability for Pt and instability for Cu withouttaking into account this disorder.This behavior iswell reproduced by our MD simulations of bubbleswith a varying number of trapped atoms  blurs out these differences.Nevertheless, the data strongly suggest that this quantity does dependon the trapped element. Such a scenario can be understood as due toa variation in γ, that is, higher for Ne (γ ≈ 0.13eV/Å2) and for He (∼0.08 eV/Å2) than for Ar (∼0.02 eV/Å2). Taking γGS = 0.25 eV/Å2 for graphene on Pt23 implies that γSb + γGb is of the order of γGS for Ar (giving γ= 0.02 eV/Å2) but significantly smaller for He andNe. In other words, in the bubble configuration, the interaction (ofvdW nature) of the gas atoms with the Pt surface or with the graphenelayer appears to be more repulsive for Ne and He compared to Ar.Letus first consider the R regime,where the Ne bubbles clearly reach higher hmax values than for He and Ar bubbles  for Ne than for He andAr (rvdW), which increasesfrom He, to Ne, to Ar  and only slightly higher forNe (3.5 and 3.16 Å), it is significantly smaller for Ar (3.1and 3.88 Å). This is indeed consistent with the fact that Aris the most compressible of the three elements, followed by He andNe . This is indeed consistentwith our MD simulations. The average z-motion amplitude(averaged over time and over the trapped atoms) obtained from theMD simulations (z⟩in hmax0 to be larger than the (compressed) vdW diameternoble gas atoms.A similar trend is observed in the low- bubbles 2 and hmae andAr 1. Thisie, to Ar 1. Since,e andNe ). In addulations 3, for thns z⟩in 1 is indediameter4b, that diameter4a, by anAsurface) as well as the (projected)area of the base of the bubble (Aprojected). Asurface is the area of the strainedgraphene region, whereas Aprojected wouldbe the area of that region if the bubble would not exist. The tensilestrain can then be estimated as R ≈ 1 nm, we obtain ϵA values of the orderof 10%. More details are provided as Supporting Information. Regarding pressure, according to the general understandingof surface-induced pressure in solids, it scales with the ratio ofsurface area to the volume of the solid phase.24 For the bubbles under consideration here, as R decreases and the atoms inside the bubbles become more monolayer-like,the surface-to-volume ratio (∼Δz–1) increases dramatically, since Δz → 0. One can therefore expect the pressure to also increasedramatically in the limit of small R. Our MD calculationsshow exactly that  is the trace of the virial stress tensor,and Vb is the volume available to thegas atoms. We note that this method, based on the virial stress tensor,is more general and more appropriate in the present case comparedto other methods based on membrane theory and plate theory. The lattermethods are based on elasticity theory, which is valid in the large-bubblelimit, but tends to overestimate the pressure for small bubbles.6 At such high pressures, at room temperature,these noble gases are expected to be in a solid phase or near theirmelting transition, which is around 10 GPa for He,26 5 GPa for Ne,27 and 1.5 GPafor Ar.28 Considering the pressures estimatedhere (R < 1 nm). Our MD simulations are indeed consistent with this expectation(cf. the videos provided as Supporting Information), showing ratherstable ordered atomic arrangements for Ne (Videos V1 and V2) and Ar (V3 and V4) and more disorderedand dynamic arrangements for He (V5 and V6).Thebreakdown of the universal scaling, leading to extreme aspectratios, is likely to be associated with other unusual physical propertiesin these subnanometer bubbles. Although studying such properties indetail is beyond the scope of this Letter, it is worthwhile discussingstrain and pressure as examples. The tensile strain induced on grapheneby the underlying trapped atoms can be estimated from our STM measurementsas follows. From the STM topography of a bubble, one can determinethe surface area of the graphene layer that wraps the three-dimensionalbubble  are immobilized as intercalated species.The remainder is likely to escape via graphene defects .This bubble instability for graphene on Cu is confirmed in our MDsimulations (cf. the videos provided as Supporting Information): Ifa bubble configuration (similar to those in Pt—videos V7 and V8) is givenas the initial state, the time evolution shows graphene peeling offthe Cu surface, resulting in the dispersion of the trapped gas atoms(V9 and V10). This instability can be easily understood as due tothe much weaker adhesion of graphene to Cu (γGS =0.045 eV/Å229) comparedto Pt (0.251 eV/Å223),that is, the Cu–graphene binding is too weak to sustain thehigh pressures associated with the bubbles. In order to assess ifthe gas–metal adhesion (γSb) also plays arole in this stability difference, we used DFT to calculate the adsorptionenergy and the adsorption distance of isolated He, Ne, and Ar atomson Pt(111) and Cu(111) surfaces (Table S4). Although the adsorption energies are indeed larger for Pt thanfor Cu when comparing the gas elements one by one, it still does notexplain the observed difference in stability. For example, the adsorptionenergies of Ar on Cu (for which bubbles are not stable) are largerthan those of He on Pt (for which stable bubbles are observed). Wetherefore conclude that bubbles are not stable on flat Cu terracesdue to the much weaker adhesion of graphene to Cu as compared to Pt.As mentioned above, unlike for Pt, bubblesare not observed onCu flat terraces. It appears that only the atoms that are trappedin defects , which expands evenfurther the range of possible applications. In particular, since thebubble formation is based on ion implantation, our approach is compatiblewith virtually any implanted element, 2D material, and substrate.To conclude, using ULE implantation of noble gas ions , we produced nanobubbles on graphene with varying radius,from few nm down to subnanometer scales. These nanobubbles are stablefor graphene on Pt but not for graphene on Cu. While the bubble aspectratio behaves differently for the different elements, the universalscaling behavior (that was previously established for larger bubbles)breaks down in all three cases, for a bubble radius around 1 nm, asthe bubble height approaches a minimum corresponding to about an atomicmonolayer. We interpret the observed dependencies on the substrateand trapped element in terms of the adhesion energies between thethree constituents: graphene, the substrate, and the trapped noblegas element. Moreover, these nanobubbles are found to induce highlevels of strain (of the order of 10%) on the overlaying graphene.In addition to providing insight on the spatial distribution of thetrapped atoms and its relation to the bubble morphology and stability,molecular dynamics calculations also allowed us to estimate the vdWpressure inside the bubbles, exceeding 30 GPa for the smallest bubbles.These remarkably high strains and pressures illustrate the uniquecharacteristics of this subnanometer bubble regime  compared to the previously studied(larger) nanobubbles. These unique properties offer new opportunities,for example, to study physical states of matter and chemical reactionsunder high (vdW) pressure or electronic phenomena associated withstrain-induced pseudomagnetic fields in graphene. Since the behaviorreported here is largely determined by the adsorption energies betweenthe three constituents ,one can expect similar behavior for other 2D materials (e.g., transitionmetal dichalcogenides such as MoS


Diagnosis of interstitial lung diseases (ILD) is difficult to perform. Extracellular vesicles (EVs) facilitate cell-to-cell communication, and they are released by a variety of cells. Our goal aimed to investigate EV markers in bronchoalveolar lavage (BAL) from idiopathic pulmonary fibrosis (IPF), sarcoidosis and hypersensitivity pneumonitis (HP) cohorts. ILD patients followed at Siena, Barcelona and Foggia University Hospitals were enrolled. BAL supernatants were used to isolate the EVs. They were characterized by flow cytometry assay through MACSPlex Exsome KIT. The majority of alveolar EV markers were related to the fibrotic damage. CD56, CD105, CD142, CD31 and CD49e were exclusively expressed by alveolar samples from IPF patients, while HP showed only CD86 and CD24. Some EV markers were common between HP and sarcoidosis . Principal component analysis distinguished the three groups based on EV markers with total variance of 60.08%. This study has demonstrated the validity of the flow cytometric method to phenotype and characterize EV surface markers in BAL samples. The two granulomatous diseases, sarcoidosis and HP, cohorts shared alveolar EV markers not revealed in IPF patients. Our findings demonstrated the viability of the alveolar compartment allowing identification of lung-specific markers for IPF and HP. Among the non-neoplastic lung diseases, interstitial lung diseases (ILD) represent a wide and heterogeneous group of more than 200 distinct clinical entities, characterized by the specific involvement of lung interstitium that may lead to pulmonary fibrosis .In the case of ILD, identifying the disease in a precise and timely manner can often be challenging for many reasons: pathophysiological disease mechanisms are inherently complex, classification systems and definitions are constantly evolving, overlapping conditions are present within the lung and multiple pathological entities may coexist in the same lung . As a reExtracellular vesicles (EVs) facilitate cell-to-cell communication, which plays a vital role in nearly all physiological and metabolic activities . As a geThe endothelium of the lung contributes significantly to EV circulation due to its high vascular density . In addiOur research group has recently conducted EV analyses in peripheral blood of patients with idiopathic pulmonary fibrosis (IPF) using flow cytometry and showing an altered exosome profile . These fThe present study aimed to compare 37 exosomal surface markers through flow cytometry in BAL from patients affected by IPF, sarcoidosis and hypersensitivity pneumonitis (HP), enrolled at Siena Referral Centre for rare lung diseases. To corroborate these findings, a validation cohort was enrolled from two referral centers for ILDs (Barcelona and Foggia).p = 0.0312), while sarcoidosis patients were younger (p = 0.0215) and with a female predominance than IPF and HP (p = 0.0121). According to respiratory functional assessment at baseline, we observed a moderate reduction of DLCO percentages associated with mild restrictive impairment of lung volumes in IPF patients compared with HP and sarcoidosis (p = 0.0399). Demographic, clinical and immunological data of study and validation cohorts are reported in p = 0.860). CD133/1, SSEA, CD14 and CD2 were differently expressed in BAL samples from the Siena and Foggia cohorts than the Barcelona cohort (p = 0.0455). The expression of CD9, CD63 and CD81 were similar in both cohorts (p = 0.905). From comparative analysis, the expression of EV surface markers was similar in the cohort and validation cohort , indicating a risk of erroneously rejecting the null hypothesis lower than 0.01%.MANOVA analysis between IPF, HP and sarcoidosis associated lambda (0.174) with a Assessing AHC analysis , the HarPCA plots  were perHeat map analysis a showed In the present multicenter study, 37 EV surface markers were detected in the alveolar compartment of ILD patients at diagnosis for the first time. IPF, HP and sarcoidosis patients were sampled for BALs and EVs were phenotyped by flow cytometric analysis. The majority of markers identified on EV surfaces were related to the fibrotic damage on alveolar epithelial cells I and II as well as endothelial cells and fibroblasts, as reported in IPF etiopathogenesis is still unknown. A dysfunctional epithelium can be ascribed to genetics and epigenetics . As a reIt has been hypothesized that HP and IPF have distinct lung fibrosis phenotypes, one being a post-inflammation-induced fibrosis, whereas the second is more related to tissue remodeling and repair ,37.The expression of a number of cell- and inflammation-related markers in our study supports the hypothesis that inflammation plays a significant role in the pathogenesis of HP and sarcoidosis.In HP, T-lymphocyte markers, CD3, were higher than in sarcoidosis and IPF. In BAL samples from sarcoidosis patients, however, the highest expression of CD4 and CD8 was associated with alveolar lymphocytosis, indicating that the disease was active at the time of diagnosis. Sarcoidosis patients have demonstrated increased inflammatory responses in circulating blood monocytes . It is lAlveolar samples from HP patients showed the highest expression of several B-lymphocytes markers, including CD40 (extracellular positivity in lymphoid tissues ) and CD2There are both T and B cells in the lung parenchyma, and their organization into B lymphoid-rich tertiary lymphoid tissues is a hallmark of HP that is closely related to the level of airway inflammation . StimulaIt is important to emphasize that even though our study provides a relevant contribution by highlighting the expression markers of EVs in BAL samples, it has some limitations. First, we did not include other fibrotic ILD in our study that may show a similar clinical course to IPF or HP. Second, it is important to validate our findings in other ILD cohorts from different geographical areas. In addition, the analysis of EV surface markers in different biological samples will be of interest. A total of 83  patients from the rare lung diseases referral center of the University of Siena were consecutively enrolled in the study. A multidisciplinary discussion confirmed the diagnosis of IPF and fibrotic HP in seventeen  and twenty-four  patients, respectively, according to the American Thoracic Society/European Respiratory Society (ATS/ERS) guidelines . InfectiThis study was approved by the regional ethical review board of Siena (C.E.A.V.S.E. Markerlung 17431) and complied with the Helsinki Declaration. All patients provided written informed consent prior to participating in the study. Healthy donors were not enrolled for ethical reasons.Twenty-one patients  referred to ILD clinic at Hospital de la Santa Creu i Sant Pau (Barcelona) were retrospectively enrolled. Eleven patients were affected by IPF, 5 by sarcoidosis and 5 by HP. Forty-four patients  followed at ILD referral center of Foggia University Hospital were consecutively and prospectively enrolled. Thirty-three patients had diagnoses of IPF, 5 of sarcoidosis and 6 of HP. All diagnoses were made according to ATS/ERS international guidelines based on multidisciplinary discussion.Pulmonary function tests (PFT) were performed in all patients for diagnostic purposes. Forced expiratory volume in the first second (FEV1), forced vital capacity (FVC) and diffusing capacity of the lung for carbon monoxide (DLCO) were recorded. They were performed according to ATS/ERS standards  using a Patients were treated with fentanyl 100 mcg and midazolam (3–5 mg) i.v. 15–30 min prior to bronchoscopy. Lidocaine was administered to the larynx and bronchi for topical anesthesia. A Pentax bronchoscope EB15-J10  was inserted through the mouth to avoid blood contamination. Physiological saline with 3 × 50 mL solution was instilled in the middle or lingual lobe. Ten to twenty mL of a pooled BAL sample were collected, as recommended by international BAL Task Force Group guidelines for BAL cellular analysis . BAL samg for 10 min and then the supernatant was stored at −80 °C. According to the manufacturer’s instruction  [g for 30 min at room temperature (RT), then 110 μL of each sample was aspirated and centrifuged at 10,000× g for 45 min at RT. EVs were characterized by MACSPlex Exosome Capture  beads and incubated overnight using an orbital shaker (450 rpm). Then, 500 μL of MACSPlex Buffer was added in each tube and centrifuged at RT at 300× g for 5 min. Accordingly, each sample was then incubated with CD9, CD63 and CD81 for 1 h at RT protected from light on the orbital shaker. The washing step was repeated and EVs from Siena and Foggia University Hospitals were characterized by FACSLyric (BD Biosciences) and analyzed by Kaluza software 2.1 , while EVs isolated at Barcelona Hospital were characterized using a MACSQuant Analyzer 16 Flow Cytometer . According to literature data, the results from the two flow cytometers are accurate and comparable [BAL samples were processed as follow: the samples were centrifuged at 1500× Germany) , 120 μL mparable ,54. The The results were expressed as means and standard deviations (SD). One-way ANOVA non-parametric test  and Dunn test were performed for multiple comparisons. The chi-squared test was used for categorical variables. MANOVA analysis was performed to determine any significant effects of IPF, HP and sarcoidosis diagnoses (explanatory variables) considered in interaction or otherwise with 37 EV surface markers (dependent variables). Wilks’ lambda tests were performed, the lower the lambda associated with explanatory variables, the more important the effect of these variables is on the dependent variables’ combination. Agglomerative hierarchical clustering (AHC) was performed to cluster the three groups of diagnoses based on dissimilarities between the median APC  values of 37 EV surface markers. The dendrogram plot was assessed showing the progressive grouping of the data. Unsupervised principal component analysis (PCA) and heat map analysis were applied to surface expression markers in an explorative approach to identify trends in the data by 2D representation of the multi-dimensional data set.p value less than 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 9.4 and XLSTAT2021 software.A The findings of our study have demonstrated the validity of the flow cytometric method to phenotype and characterize EV surface markers in BAL samples. In addition, the viability of the alveolar compartment, as a result of the BAL, was demonstrated, which allowed identifying lung-specific markers for IPF and HP. A further outcome of this study was corroboration of the hypotheses regarding the different fibrotic phenotypes in HP and IPF, where the first is related to cell-mediated inflammation-induced lung disease, while the latter is more often attributed to tissue repair and remodeling. The EV markers identified in BAL samples from IPF patients were not revealed in the HP cohort, most of whom were shared with sarcoidosis as two granulomatous ILD.


The devices show unique metaplasticity, a useful feature for generalizable deep neural networks, and we demonstrate that metaplastic BLASTs outperform ideal linear synapses in classic image classification tasks. With switching energy well below the 1 fJ energy estimated per biological synapse, the proposed devices are powerful candidates for bio-interfaced online learning, bridging the gap between artificial and biological neural networks.CMOS-based computing systems that employ the von Neumann architecture are relatively limited when it comes to parallel data storage and processing. In contrast, the human brain is a living computational signal processing unit that operates with extreme parallelism and energy efficiency. Although numerous neuromorphic electronic devices have emerged in the last decade, most of them are rigid or contain materials that are toxic to biological systems. In this work, we report on biocompatible bilayer graphene-based artificial synaptic transistors (BLAST) capable of mimicking synaptic behavior. The BLAST devices leverage a dry ion-selective membrane, enabling long-term potentiation, with ~50 aJ/µm Designing biocompatible and flexible electronic devices for neuromrophic applications remains a challenge. Here, Kireev et al. propose graphene-based artificial synaptic transistors with low-energy switching, long-term potentiation, and metaplasticity for future bio-interfaced neural networks. A promising approach for overcoming the power and latency shortfalls of traditional computing is through massively parallel neuromorphic systems3. A wide variety of devices have been proposed to build such systems, from mature technologies such as metal-oxide5 and phase change memories7 to emerging devices such as electrochemical8 and magnetic memories12. Most of these systems, however, employ rigid materials, making them less suited for direct integration with biological matter. Direct interfacing of artificial neuromorphic systems with biological living neurons is a highly ambitious goal, which in the long term may lead to effective brain implants and creation of artificial tissue. There have recently been device innovations to this end utilizing organic materials such as maltose-ascorbic acid13, zein14, PVA15, and cellulose16 but with various deficiencies in synaptic performance. Two-dimensional (2D) materials are a promising material class for bioelectronics18 and neuromorphics21 due to their unique electronic properties and atomically thin structure, allowing for imperceptible interfacing with tissue. However, existing 2D-material based neuromorphic systems often employ elements that would induce toxicity when interacting with biological systems, e.g., involving elements like Li+ ionic carriers23 which directly impact the nervous system24. Other 2D carbon-based systems have non-ideal conductance responses that negatively impact performance in neuromorphic applications27. As a result, new device innovations are needed for future neuromorphic computing solutions that can directly integrate with biological tissue.As the world becomes more interconnected and data-driven, effective deployment of data-intensive computation methods becomes more critical. Large and complex data structures require constant extrapolation, interpolation, and classification, which are ill-suited for memory-constrained von Neumann architectures28 with mobile islands of positively charged water/proton clusters  at the Nafion-graphene interface, yielding high-precision conductance states for synaptic operation in artificial neural networks. Altogether, the proposed devices feature favorable synaptic characteristics and energy efficiency down to 50 aJ/µm2. We evaluate this behavior through neuromorphic simulations on several classification tasks using a prototypical neural network and show that the BLAST devices feature metaplasticity that allows online learning performance exceeding ideal linear, numerical synapse results.The biocompatible graphene-based artificial synaptic transistors (BLAST) introduced in this work are a combination of two flexible, soft, and biocompatible elements: Nafion and graphene. Nafion plays the role of a solid polymeric electrolyte made of a negatively charged polysulfonated backboneers Fig. . When Na2 in area) and microscale . The mBLASTs were fabricated using 180 µm thick Nafion-117, adhesive conductive gold tape to form source and drain electrodes, and graphene electronic tattoos32 supported by 200 nm thick PMMA (details in Methods). It is important to note here that both graphene and Nafion are known to be biocompatible substrates for cellular interfaces. Since a few decades of its discovery, Nafion has been studied for biocompatibility as a bare substrate and has also been widely accepted as a coating material for numerous in vivo biosensing applications35. Graphene, despite the original claims of cytotoxicity, has gained trust in the biomedical research community as a highly biocompatible substrate. Unlike the nm-sized flakes of exfoliated graphene, CVD-grown monolayer graphene supported by a substrate is known to not alter any cellular function36, and often serves as an advantageous substrate for cell growth39. To make mBLASTs, we leverage the previously developed large-scale graphene electronic tattoos (GETs) that are transferred on top of the Nafion, self-adhering and forming a tight interface between the graphene and Nafion. The water-assisted transfer, though rapid (<10 s), leads to hydration of the Nafion membrane; hence the used Nafion cannot be considered completely dry, leading to relatively increased mobility of proton clusters29. The fabricated devices, as shown in Fig. 2. In a separate experiment, we varied the area of mBLAST devices and recorded the change in properties. The gold electrode used to apply synaptic gate potential through the Nafion typically outsizes the graphene channel itself to ensure that the whole graphene-Nafion interface is used effectively. The device schematics and cross-section can be seen in Fig. In this work, two kinds of BLAST devices were fabricated: macroscale  current pulses cause the semi-mobile positively charged proton-concentrated clusters within Nafion to move toward (away) from the graphene interface, shifting the interface capacitance and affecting the electrical double layer, which consecutively affects the charge carriers’ density and graphene channel conductance. To corroborate the hypothesis, we gathered transfer curves of the BLAST devices, shown in Fig. entclass1pt{minima43. Since the Nafion is not fully hydrated and there is no proton source at the gate, there are no freely moving protons to deploy30. Hence, the observed effect is related to the hybrid interface between graphene and Nafion. We hypothesize that the proton clusters migrate toward the graphene-Nafion interface, forming an electrical double layer and either hole- or electron-doping graphene, a similar mechanism to that seen in supercapacitors with carbon-based electrodes44. Our observed surface effect requires far fewer protons to accomplish a conductance change than the bulk electrochemical reaction in PEDOT:PSS due to the atomically thin nature of graphene. Coupled with the slow kinetics of the proton cluster movement within the dry Nafion matrix, long-term potentiation can be explained. It should be noted that voltage pulses can also be used in place of current pulses with the addition of a switch at the gate to open the circuit when a pulse is not being delivered, a common technique to enhance state retention in electrochemical devices45. In the case of the BLASTs, current pulses are used due to ease of use in the experimental setup and to promote the linearity and symmetry of synaptic response because the amount of charge delivered to the device mediates the change in conductance. These characteristics will be described in the subsequent section.Figure 46. The conductance evolution behavior of the mBLAST device is linear and symmetric, indicating the potential use in online learning applications. This is evaluated by applying a ramp test: a series of repeated positive and negative spikes cycling the device across its dynamic range. Linearity and symmetry are calculated by collapsing 50 repeated ramping cycles into a single ramp  cannot be concluded due to the appearance of two area-dependent clusters, a general decrease in conductance change as area increases is evident. This is corroborated when only considering devices with channel area larger than 1 mm2  is performed for more than ten million cycles see Fig. . No deviamp Fig. , the per47. In this work, we report on long retention times exceeding 400 seconds (s), shown in Fig. 49; however, when working with biologically relevant systems, the average frequency of events is <1 Hz. Hence, 400 s retention time is adequate. Additionally, due to the dependence of Nafion ion conductivity on hydration, there is a possibility to tune the retention by controlling the moisture level in the Nafion membrane50. The dominance of short-term potentiation depends on the conductance regime of the device, where changes in conductance are primarily short-term when the conductance is near maximum and minimum , and to then transfer the weights to memory with long retention time for inference when training is finished55. As a result, the targeted application of the BLASTs is for neural network training.Retention time is another essential figure of merit for synaptic transistors and many neuromorphic computation systemssee Fig. , the dev2 to ~100 mm2 were fabricated and tested by applying a set current pulse of 1 µA amplitude, 10 ms duration and recording the change in conductance. The calculation of charge modulation through current spike and energy per spike calculation are given in Supplementary Note 2 in energy dissipation, but the results still indicate that device scaling still greatly benefits the devices. These results still exceed or match comparable organic and 2D devices at a competitive speed  graphs presented in Fig. 47, we show that such asymmetry of experimental data can yield algorithmic benefits in compute-limited situations.Although the aforementioned mBLASTs are biocompatible, feature superior energy efficiency, and have outstanding neuromorphic plasticity characteristics, the devices are still rather large. In order to implement more complex neuromorphic functionalities, the device sizes must be scaled down, which we achieved by building microscale devices (µBLASTs). The µBLASTs were made using UV-lithography with HD-8820 passivated graphene field-effect transistors  University of California-Irvine Human Activity Recognition (UCI-HAR)59 dataset for movement classification from biometric sensor data, and (3) Fashion-MNIST60 clothing article classification. The mBLAST devices were applied on MNIST and UCI-HAR, while the performance of the µBLASTs was evaluated on UCI-HAR and the more complex Fashion-MNIST datasets. Training is accomplished by simulating a small multilayer perceptron with two synaptic weight layers L1 and L2 using CrossSim61, a physics-rich neuromorphic crossbar simulator. See Methods for more details on the training simulation setup.After analyzing the device characteristics of the mBLASTs and µBLASTs, the experimental conductance update characteristics are used to simulate online learning neuromorphic tasks. The experimental device data is used to construct LUTs that can be directly sampled by the crossbar simulator, allowing the simulation to account for linearity, symmetry, and write noise directly from the experimental results. The following three classification tasks were chosen: (1) Modified National Institute of Standards and Technology (MNIST)62 updates  and one trained using µBLASTs. The representative distributions for the two neural network layers are plotted in in Fig. 2) at >10 kHz speeds, competitive with other 2D materials-based devices and allowing the potential for the interfacing of biological and artificial synapses. Neural network simulations show that the low read and write noise, coupled with the linear and symmetric synaptic characteristics of the mBLASTs allow for near-ideal classification. More importantly, the µBLASTs are demonstrated to be metaplastic synapses that realize weight normalization, a weight regularization algorithm used to train generalizable neural networks. This allows the performance of µBLAST networks to exceed the classification accuracy achieved by ideal linear numeric weights on difficult tasks. These characteristics make BLAST devices promising candidates in the intersection of bioelectronics and neuromorphic computing.In conclusion, we have demonstrated a novel solid electrolyte gated graphene device with unique artificial synapse behavior. We experimentally revealed that these bio-compatible devices operate at low energy density (<50 aJ/µm2 square was then cut out and spin-coated with PMMA (PMMA 950 A4) to yield a ~200 nm thick layer. To do so, the copper/graphene stack must be placed on a silicon wafer with the graphene facing up. Kapton tape is then used to secure all sides of the copper/graphene stack to the wafer such that a watertight seal is formed under the copper to prevent PMMA from leaking underneath the stack. Afterwards, PMMA (950 A4) is spin-coated onto the stack at ~2500 rpm for 60 s. The sample is then baked on a hotplate at 200 °C for 15–20 min before it is ready for etching.The graphene electronic tattoos were fabricated starting with monolayer CVD grown large-scale graphene on copper, purchased from Grolltex. A~2 × 2 in4)2S2O8, 0.1 M) to etch away the copper. The PMMA/graphene film is then cleaned in a series of water washing steps and then transferred onto a temporary tattoo paper. The graphene/PMMA/tattoo paper is then dried and cut into arbitrary desired locations. In order to transfer the graphene/PMMA onto an arbitrary surface, it is first soaked in water for ~5–10 min.The PMMA/graphene/Cu foil is then placed into ammonium persulfate  film. After preparing the gold EVA gate, it is brought in contact with a piece of Nafion-117 of the desired dimension . This step is performed on a hotplate (at ~150 °C) for no longer than 15 s. Following the application of the Nafion strip, adhesive gold contacts are placed 3–10 mm apart from each other, perpendicularly from the gate, on top of the Nafion to form the source and drain terminals. The GETs (~5–15 cm length and 3–10 mm width) are prepared for transfer onto the BLAST device starting by soaking in DI water for 5–10 min. The graphene should show signs of slight separation from the tattoo paper when sufficiently soaked. The GET can then be placed on top of the Nafion and transferred onto the top of the device such that the graphene contacts both gold source-drain contacts and forms a channel across the Nafion.CVD grown graphene was covered with a 200 nm thin layer of PMMA for transfer. After etching copper in ammonium persulfate (see above), the graphene was transferred onto the wafer with pre-fabricated Au/Ti (10/50 nm thick) markers. A photoresist was used to protect the graphene channel areas during exposure to oxygen plasma. The stack of 10 nm Ti and 90 nm Au was e-beam assisted evaporated on the wafer through a pre-defined structure of lift-off resists to form source and drain electrical connections. Photostructurable polyimide, HD8820 was used in the last step to form the passivation. Spin- coated at 5000 rpm, exposed at i-line UV light, developed in 0.26% TMAH, and hard baked at 350 °C, the polyimide forms a 3 µm thick passivation. The devices were then diced and spin-coated with the liquid Nafion-117 containing solution (Sigma–Aldrich) three times , forming a 666.7 ± 28.9 µm thick layer.The three-terminal devices were measured using high-precision source/measure unit Agilent 2902B. One of the SMUs is used to apply 0.1 V of the drain-source potential, while the gate is used in the current-pulsing mode to apply conductance changes.Two pulse train tests were used to characterize conductance change due to periodic pulsing. This was done by sending either 50 or 250 consecutive write current pulses with corresponding widths and amplitudes of 100 µs and 100 µA, 1 ms and 10 µA, 10 ms and 1 µA, and 100 ms and 100 nA. All pulse trains were conducted with both positive and negative pulses. The pulses were followed with a 150–200 s relax to observe conductance retention.A pulse ramp experiment ran multiple sequences of a train of 20 negative pulses followed by a train of 20 positive pulses. The level experiment consisted of an arbitrary sequence of negative and positive current pulses of 10, 20, and 30 µA that were pulsed through the gate every 5 s at a pulse width of 1 ms. Both tests were used to demonstrate distinct and repeatable conductance levels.The temperature dependence test measured macroscale device performance at varying temperatures . The 0° test was performed inside of an insulated Styrofoam container filled with ice. The macro device was lowered onto ice using a petri dish and connected to the Keysight B2902A using wires thin enough as to not disturb the insulating seal. At 25°, 40°, 60°, and 80°, the device was secured to the top of a hotplate using Kapton tape while a thermocouple was used to verify the temperature. The above-mentioned tests were performed at each temperature with a minimum time of 30 min after each temperature change to ensure the device was successfully brought to temperature.61. During a weight update, the ideal backpropagation update is first calculated. To account for synaptic device nonlinearity and write noise, the probabilistic LUT of device updates is queried to sample a conductance change ΔG that depends on the current value of the conductance G of a synapse device. To model device-to-device process variations, each device in the simulated array is randomly assigned to one of four (three) LUTs, each of which is constructed from a different experimentally characterized μBLAST (mBLAST) device. To represent both positive and negative weights, a single weight value is encoded in the difference in conductance between two paired BLAST devices. This is done for two reasons: (1) computing the difference in analog reduces the required dynamic range of the peripheral circuits, (2) using two devices per weight, rather than a fixed analog offset for all devices, avoids coupling the variability and noise in the offset resistor into all the results, which effectively amplifies the variability. When a weight is updated, the conductance of both devices in the pair are updated. The weights are trained using stochastic gradient descent with a batch size of 1, i.e. training samples were shown one at a time. A two-layer MLP was trained for each task, where a sigmoid activation is used after the first layer and a softmax is used after the second layer. The MNIST and Fashion-MNIST networks use a 785 × 300 weight matrix for the first layer and a 301 × 10 weight matrix for the second layer (including bias). The UCI-HAR MLP has a 577 × 200 matrix and a 201 × 6 matrix for the first and second layer, respectively.Neural network training simulation results and synaptic update lookup tables were generated from experimental data using CrossSimSupplementary Information is available and includes comparison table; Supplementary Information


In this study, the HIV-1 P18I10 CTL peptide derived from the V3 loop of HIV-1 gp120 and the T20 anti-fusion peptide of HIV-1 gp41 were inserted into the HPV16 L1 capsid protein to construct chimeric HPV:HIV (L1:P18I10 and L1:T20) VLPs by using the mammalian cell expression system. The HPV:HIV VLPs were purified by chromatography. We demonstrated that the insertion of P18I10 or T20 peptides into the DE loop of HPV16 L1 capsid proteins did not affect in vitro stability, self-assembly and morphology of chimeric HPV:HIV VLPs. Importantly, it did not interfere either with the HIV-1 antibody reactivity targeting sequential and conformational P18I10 and T20 peptides presented on chimeric HPV:HIV VLPs or with the induction of HPV16 L1-specific antibodies in vivo. We observed that chimeric L1:P18I10/L1:T20 VLPs vaccines could induce HPV16- but weak HIV-1-specific antibody responses and elicited HPV16- and HIV-1-specific T-cell responses in BALB/c mice. Moreover, could be a potential booster to increase HIV-specific cellular responses in the heterologous immunization after priming with rBCG.HIVA vaccine. This research work would contribute a step towards the development of the novel chimeric HPV:HIV VLP-based vaccine platform for controlling HPV16 and HIV-1 infection, which is urgently needed in developing and industrialized countries. Human immunodeficiency virus-1 (HIV-1), which causes acquired immunodeficiency syndrome (AIDS), was discovered in the early 1980s, and since then it has become a global epidemic . In spitIn our previous review paper publication regarding design concepts of virus-like particle (VLP)-based HIV-1 vaccines, we mentioned that non-enveloped VLPs, such as papillomavirus VLPs, could play a functional role as delivery vectors to present HIV-1 CTL or neutralizing antibody epitopes ,16. ThisIn this study, we aimed at developing a chimeric VLP-based HPV:HIV vaccine by using human 293F cells, a well-established mammalian cell expression system. The HPV 16 L1 protein acted as the structural vaccine scaffold, and the P18I10 and T20 peptides were selected as HIV-1 immunogens and inserted into the DE loop of HPV16 L1 protein. The immunodominant P18I10 CTL epitope comprising 10 amino acids (residues 311–320: RGPGRAFVTI) is derived from the third variable domain (V3) of the HIV-1 envelope glycoprotein gp120. The P18I10 peptide has been identified as a H-2Dd-restricted MHC class-I molecule to induce cytotoxic T lymphocytes (CTL) responses ,26. The Mycobacterium bovis Bacillus Calmette-Guérin (rBCG) expressing HIV-1 Gag prime and HIV-1 Gag VLP boost may contribute to enhance T-cell immunity [Over the past decade, many different prime-boost formats of VLP-based HIV-1 vaccine have been tested . Althougimmunity ,30. In oimmunity ,32,33. Timmunity . TherefoIn this study, the chimeric HPV:HIV (L1:P18I10 and L1:T20) immunogens were designed and produced by using 293F expression system. The chimeric L1:P18I10 and L1:T20 protein expression was confirmed by immunostaining. The HPV:HIV VLPs were subsequently purified by a 3-step chromatographic method, including cation (CEC), size exclusion (SEC) and heparin affinity (H-AC) chromatography. Then, the in vitro stability, in vitro self-assembly and morphology of purified HPV:HIV VLPs were confirmed by non-reducing SDS-PAGE, molecular mass assay and transmission electron microscopy (TEM), respectively. The sequential and conformational P18I10 and T20 peptides presented on chimeric HPV:HIV VLPs were further characterized by anti-HIV-1 gp120 V3 and 2F5 monoclonal antibodies in vitro by using Western blot and indirect ELISA assay. Finally, the immunogenicity of HPV:HIV VLPs was assessed in BALB/c mice model. We demonstrated that chimeric L1:P18I10 and L1:T20 VLP-based vaccines could induce HPV16- and HIV-1-specific antibody responses and chimeric L1:P18I10 VLPs could induce HPV16- and HIV-1-specific T-cell responses in BALB/c mice. Because the development and manufacturing of an immunogenic HPV:HIV vaccine is still unachievable, this study provided a baseline strategy that may be worth supporting the global efforts to develop novel chimeric VLP-based vaccines for controlling HPV and HIV-1 infections.E.coli-mycobacteriol integrative shuttle vector p2auxo.int. The construction of E. coli/mycobacterial vector expressing HIVA antigen was previously described [2auxo.int was diluted in PBS-Tween20 to 2 × 107 cfu/mL, sonicated to disrupt bacterial clumps and inoculated into the rear food pad or BALB/c mice .Recombinant BCG expressing HIVA immunogen was previously constructed using the escribed ,32,33. BE. coli, M151GlyA , provided by Dr. Pau Ferrer, were cultured in minimal M9-derivative medium , supplemented with glycine (70 μg/mL). The E. coli M151Gly cells were transformed with the p2auxo.HIVA plasmids by electroporation. For this, the E. coli cultures were grown to an optical density of 0.9 at 600 nm, transformed using a Bio-Rad gene pulser electroporator at 2.5 kV, 25 μF, and 200 Ω. The transformed cells were subsequently cultured on M9-D agar plates  without glycine supplementation for selection or with glycine supplementation as a control. The lysine auxotrophic BCG strain, BCGΔlys, kindly provided by W.R. Jacobs Jr., B.R. Bloom, and T. Hsu was transformed with p2auxo.HIVAint plasmid DNA by electroporation. The mycobacteria were cultured in Middlebrook 7H9 broth medium or on Middlebrook agar 7H10 medium supplemented with albumin-dextrose-catalase  containing 0.05% Tween 80. L-lysine monohydrochloride  was dissolved in distilled water and used as a supplement at a final concentration of 40 μg/mL. For transformation, BCG was cultured to an optical density of 1.5 at 600 nm, transformed using a Bio-Rad gene pulser electroporator at 2.5 kV, 25 μF, and 1000 Ω. The transformants were then cultured on ADC-supplemented Middlebrook agar 7H10 medium containing 0.05% Tween 80 without lysine supplementation.Cells of the glycine auxotrophic strain of 2 on an orbital shaker platform rotating at 125 rpm.The 293F cells (gibco), derived from human embryonic kidney (HEK) 293 cells, were cultured in FreeStyle 293 expression medium (gibco) supplemented with 5 mL/L of penicillin-streptomycin (gibco) and incubated in a 37 °C incubator containing a humidified atmosphere of 5% COE. coli DH5α competent cells (Invitrogen) for amplification and extracted by using plasmid Maxi kits . The 293F cells were cultured with 30mL FreeStyle 293 expression medium in a 125mL Erlenmeyer flask  to a density of 1.0 × 106/mL and transiently transfected with L1:P18I10 or L1:T20 pDNAs using the branched polyethylenimine with a MW of 25 kDa (PEI-25K) (Polysciences) at an optimized ratio of DNA to PEI 1:3 (w/w) and DNA to culture medium 1:1 (w/v), according to manufacturer’s instructions [6 cells/mL with approximately 50% viability.The pCDNA3.1 construct contained L1:P18I10 or L1:T20 DNA coding sequences corresponding to chimeric L1:P18I10 and L1:T20 proteins, respectively. The HIV-1 P18I10 CTL peptide (RGPGRAFVTI) or T20 peptide (YTSLIHSLIEESQNQQEKNEQE LLELDKWASLWNWF) were inserted into the DE loop of HPV16 L1 capsid protein. The HPV16 L1 DE loop sequence encoding 130–136 amino acids was replaced with either P18I10I10 or T20 peptide. The L1:P18I10 or L1:T20 DNA coding sequences were modified with Kozak sequence, optimized with human codon, flanked by the restriction enzyme sites of HindIII and XbaI and cloned into pcDNA3.1(+) vector by using GeneArt gene synthesis services . The recombinant plasmid DNA (pDNA) was transformed into ructions . The 293The cells were permeabilized on the glass slide with 100% cold acetone. Subsequently, the fixed cells were probed with anti-HPV16 L1 antibody CAMVIR-1  and captured with anti-mouse IgG-FITC (Sigma). Immune-stained cell monolayers were thoroughly washed with PBS and covered with mounting medium with DAPI (Abcam). The immunofluorescence images were inspected under an inverted microscope at 40× magnification. Transfection efficiency was determined by the ratio of FITC (green)-positive cells to DAPI (blue)-stained cells.8 transfected 293F cells in a 125 mL Erlenmeyer flask (30 mL culture medium/flask) were collected by centrifugation at 1500 rpm for 5 min and washed twice with PBS. Cell pellets were resuspended in lysis buffer formulated with 1% Triton X-100, protease inhibitor (1:100) (Millipore) and Benzonase (25 U/mL) (Millipore). Cell lysates were clarified with 0.45 µm PVDF syringe filter (Millipore). The HPV:HIV (L1:P18I10 and L1:T20) VLP samples were serially purified using cation exchange , size exclusion  and affinity  chromatography. The chromatographic protocols were described in our previous studies [A total of 10 studies ,37 and f studies . The L1  studies .v/v) 2-mercaptoethanol (2-ME) and reacted at room temperature (RT) for 24 h. Samples were separated by 8–16% TGX stain-free protein gels (BIO-RAD). Then, the gels were transferred to PVDF membranes. The membranes were probed with the anti-HPV16 L1 CAMVIR-1 mAb at a dilution of 1:4000. After that, the membranes were incubated with anti-mouse IgG Peroxidase Conjugate  at a dilution of 1:4000. The signal was developed and visualized by chemoluminiscence using Western Blot ECL substrate kit . The blot images were acquired by using Odyssey Fc imaging system.The HPV16 L1, L1:P18I10 and L1:T20 VLPs were mixed with 2× Laemmli sample buffer (BIO-RAD) in the absence or presence of 5%  ultrafiltration devices (SARTORIUS). The HPV16 L1, L1:P18I10 and L1:T20 VLPs with 2-ME treatment were passed through 100 kDa MWCO ultrafiltration devices (Amicon). The retentates were reconstituted to the original volume and collected from the filter device sample reservoir, while the filtrates were collected at the bottom of the centrifuge tube. The L1 signal was measured by using dot blot probed with anti-HPV16 L1 mAb and detected by anti-mouse IgG-peroxidase conjugate (Sigma-Aldrich). Images were acquired using Odyssey Fc Imaging System at a chemiluminescence channel.After charging the carbon-coated copper grids (Sigma-Aldrich) under ultraviolet light for 5 min, commercial HPV16 L1 (Abcam), purified L1:P18I10 and L1:T20 VLPs equilibrated with 20 mM Tris-HCl  were absorbed on grids for 1 min and rinsed three times by miliQ water. The HPV:HIV VLPs were negative-stained with 2% uranyl acetate at pH 4.5 (Sigma-Aldrich) for 1 min. Excess staining agents were removed by Whatman qualitative filter paper (Sigma-Aldrich). Grids were placed in a dehumidifier chamber at least 2 h before observation. Images were acquired using a transmission electron microscope (Tecnai Spirit 120 kV) at magnification SA135K (100 nm) and SA59000 (200 nm), respectively.Equal amounts (500 ng) of HPV16 L1 protein (Abcam), purified L1:P18I10 and L1:T20 VLPs were mixed with 2× Laemmli sample buffer containing 5% 2-ME and boiled at 95 °C for 5 min. Samples were separated by 8–16% TGX Stain-free protein gels and then transferred to a PVDF membrane  using a Semi-Dry transfer device (Bio-Rad). The membrane was blocked with 5% skim milk in TBST. Then, the membranes were probed with the anti-HPV16 L1 CAMVIR-1 mAb at a dilution of 1:4000, anti-HIV-1 gp120 V3 loop mAb  at a dilution of 1:40 and HIV1 gp41 (2F5) mAb  at a dilution of 1:4000, respectively. After that, the membranes were incubated with anti-mouse IgG Peroxidase Conjugate (Sigma-Aldrich) at a dilution of 1:4000. The Western ECL substrate kit (BIO-RAD) was used for signal development. The blot images were acquired by using Odyssey Fc imaging system at a chemiluminescence channel.n = 4 and female n = 4 per group). In groups A and B, BALB/c mice were immunized intramuscularly (i.m.) with 10 μg of L1:P18I10 or L1:T20 VLPs, respectively, by following a homologous prime-boost regime. In the group C, mice were inoculated with 106 cfu of BCG.HIVA2auxo.int intradermally  and boosted with 10 μg of L1:P18I10 VLPs intramuscularly. In the group D, positive control mice were inoculated with Gardasil-9 prime followed by Gardasil-9 boost intramuscularly with 10 μg of HPV16 L1 VLPs. In the group E, negative control mice were immunized twice with PBS buffer. The prime-boost interval was 2 weeks. Mice were sacrificed on day 28. Blood samples were collected from the heart of mice. Sera were recovered by centrifugation and stored at −20 °C for ELISA assay. Murine spleens were removed and pressed individually through a cell strainer  with a 5 mL syringe rubber plunger. Following the removal of red blood cells with ACK lysing buffer (Lonza), splenocytes were washed and resuspended in lymphocyte medium R10 , penicillin-streptomycin, 20 mM HEPES and 15 mM 2-ME) at a concentration of 2 × 107 cells/mL.This is a preliminary proof-of-concept study to demonstrate immunogenicity of HPV:HIV VLPs (L1:P18I10 and L1:T20 VLPs). The dose, administration route and prime-boost interval of our HPV:HIV VLPs referred to previous studies that immunized mice with bovine papillomavirus (BPV):HIV VLPs for inducing antibody responses . Purifie2SO4. The optical density (OD) of each well was measured and recorded at a wavelength of 450 nm by using EL × 800 absorbance microplate reader.To test the HPV16 L1- and HIV-1-specific antibodies binding to chimeric HPV:HIV VLP constructs in vitro, 50 µL of equal concentration (200 ng/mL) of recombinant HPV16 L1 protein , purified L1:P18I10 and L1:T20 VLPs in 50mM carbonate-bicarbonate buffer (pH = 9.6) (Sigma) were 2-fold serially diluted and coated onto the Maxisorb plates (Nunc). The plates were incubated at 4 °C overnight. Plates were blocked with the blocking buffer (5% skim milk in TBST) at 37 °C for at least 2 h. After wash twice with TBST, the VLP-coated plates were incubated with anti-HPV16 L1 CAMVIR-1 mAb at a dilution of 1:8000, 2F5 mAb  at a dilution of 1:8000 and anti-HIV-1 gp120 V3 loop mAb  at a dilution of 1:40 in blocking buffer, respectively, at 37 °C for 2 h. After washing three times with TBST, the plates were incubated with recombinant protein G peroxidase conjugate  at a dilution of 1:4000 in blocking buffer at 37 °C for 1 h. TMB was used to develop the enzyme-linked immunosorbent assay (ELISA) signal and stopped with 50 µL of 2M H2SO4. The OD of each well was measured at a wavelength of 450 nm by using EL × 800 absorbance microplate reader (Biotek).To measure the VLP-induced antibodies in BALB/c mice, the microtiter plates were coated with 50 µL of 2 µg/mL recombinant HPV16 L1 protein , HIV-1 P18I10 peptide , T20 peptide , respectively, with 50 mM carbonate-bicarbonate buffer (pH = 9.6). The plates were incubated at 4 °C overnight. Plates were blocked with the blocking buffer (5% skim milk in TBST) at 37 °C for at least 2 h. At the same time, sera collected from the group A-E immunized mice were diluted with 5% skim milk in TBST at a ratio of 1:50. After wash twice with TBST, the plates were incubated with the diluted sera at 37 °C for 2 h. After washing three times with TBST, the plates were added with recombinant protein G HRP conjugate at a dilution of 1:4000 in blocking buffer and incubated at 37 °C for 1 h. TMB was used to develop the ELISA signal and stopped with 50 µL of 2M H5 fresh splenocytes were added to each well. Subsequently, the cells from groups A and B were stimulated with 2 μg/mL of HPV16 L1 VLPs and HIV-1 P18I10 peptides, respectively. All the samples and controls were plated in duplicate wells. ELISpot assays were incubated for 16 h at 37 °C, 5% CO2. The plates were subsequently washed 5 × with PBS, incubated for 2 h with a biotinylated anti-IFN-γ monoclonal antibody (mAb) diluted in PBS 2% Fetal Calf Serum (FCS) to a final concentration of 2 µg/mL, washed 5 times in PBS, and incubated with the streptavidin-alkaline phosphatase conjugate in PBS 2% FCS. Then, plates were washed 5 times with PBS before incubating with 100 µL of 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) substrate solution . After 5–10 min, the plates were washed with tap water, dried, and the resulting spots counted using an ELISPOT reader . For each animal, the mean of background responses was subtracted individually from all the wells to enable a comparison of the IFN-γ spot forming cells (SFC)/106 between groups. To define positive responses, a threshold was defined as at least five spots per well, and responses exceeding the mean number of spots in negative control wells plus three standard deviations of the negative control wells.The enzyme-linked immune absorbent spot (ELISpot) assay was performed using the commercial murine IFN-γ ELISpot kit , according to the manufacturer’s instructions. The ELISpot plates  were 70% EtOH treated and coated with purified anti-mouse interferon-γ (IFN-γ) capture monoclonal antibody diluted in phosphate-buffered saline (PBS) to a final concentration of 5 µg/mL at 4 °C overnight. Then, 2.5 × 10All statistical analysis was performed using Prism 6 GraphPad software . We used data from experiments carried out over 5 different ELISA plate coating concentration  of recombinant HPV16 L1 protein  and HPV:HIV VLPs. We collected data from 2 groups (HPV16 L1 and HPV:HIV VLPs) and collected three replicates for each coating concentration. The graph in Prism showed the data as a scatterplot showing coating concentration (ng/mL) on the X-axis and OD450 on the Y-axis. Since we hypothesized that our in vitro ELISA data is related in a linear pattern, we performed the linear regression analysis and compared the slopes of the two lines to confirm that data set-1 and data set-2 (antibody reactivity between HPV16 L1 and HPV:HIV VLPs) are different in their actions. We selected 95% confidence intervals alongside the best-fit line. Prism was automatically overlaid a linear regression line on both our data sets and it has plotted a dotted line representing 95% confidence intervals. During the linear regression analysis, the software calculated only the mean Y value of our data set that indicated show the goodness of fit. In addition, Prism gave us a commentary, so we can conclude that the differences between two slopes are extremely significant.n = 4 and female n = 4) in each group. We used Gardasil-9-immunized mice as positive control group and PBS-immunized mice as negative control group. The data of our designed experiments were not matching or pairing. We undertook one-way analysis of variance (ANOVA) to see whether these means were different. We first checked our data fit Gaussian normal distribution. We found that some groups of data in ELISA assay did not past the Gaussian normal distribution test. Therefore, we carried out a non-parametric statistical analysis on these data. By contrast, all groups of data in the ELISPOT assay past the Gaussian normal distribution test. Thus, we preformed a parametric statistical analysis on these data. The significance threshold and confidence level were set to 0.05 . p value showing in general the probability that there are differences between groups. Since our main concern for this experiment is what are the differences between our groups, the multiple comparisons in Prism gave us the outcome of comparison in all groups with every other group.We had 5 sets (ELISA) and 4 sets (ELISPOT) of data collected from mouse immunization experiments. These data sets had an equal number  and approved by local authorities  and Universitat Autònoma de Barcelona Ethics Commitee. The animal experiments strictly conformed to the animal welfare legislation of the Generalitat de Catalunya. All the experiments were approved by the local Research Ethics Committee .The P18I10 peptide from HIV-1 Env third variable domain (V3) loop and T20 peptide from HIV-1 Env membrane‘s proximal external region (MPER) were inserted into to HPV16 L1 DE loop protein to generate chimeric L1:P18I10 and L1:T20 immunogens. The chimeric L1:P18I10 and L1:T20 DNA coding sequence were cloned into pcDNA3.1 (+) plasmid DNA expression vector for transient transfection in 293F cells A. The moSince HPV16 L1 protein C terminal sequence mediates cellular nuclear import machinery during infection , nuclearThe chimeric L1:P18I10 and L1:T20 proteins were produced by using 293F expression system. The capture, intermediate purification, polishing (CiPP) strategy to develop our chromatographic purification protocol is shown in In order to confirm that purified HPV:HIV VLPs displayed similar in vitro stability to HPV16 L1 VLPs, we performed non-reducing SDS-PAGE to evaluate disulfide cross-linking of HPV:HIV capsid proteins A. It is To demonstrate that purified HPV:HIV proteins by chromatography are able to self-assemble to icosahedral particles, we further performed molecular mass analysis under the same reducing condition (5% 2-ME). The commercial HPV16 L1, purified L1:P18I10 and L1T20 proteins without reducing agent treatment were filtered out through 1000 kDa molecular weight cut-off (MWCO) diafiltration devices individually (Transmission electron microscopy (TEM) was used to examine morphologic conformation of HPV16 L1 and HPV:HIV VLPs. The HIV-1 P18I10 and T20 peptides were inserted into DE loops of HPV16 L1 protein, respectively. These commercial HPV16 L1 and chimeric HPV:HIV capsid proteins can spontaneously self-assemble in vitro into integral VLPs in a diameter of approximately 50–60 nm , designated CAMVIR-1, to recognize the highly conserved epitope  of HPV16 L1 protein ,58. A prp < 0.01%). The 2F5 epitope-binding specificity of L1:T20 VLPs was significantly different from HPV16 L1 VLPs (p < 0.01%). These pattern revealed that hydrophobic cellular lipids were not necessary for the binding of 2F5 neutralizing antibodies to HPV:HIV VLPs in vitro. Although the reactivity of anti-HIV-1 gp120 V3 and 2F5 neutralizing antibodies to HPV:HIV VLPs is relatively mild, the binding of anti-HIV-1 gp120 V3 and 2F5 mAb to HPV:HIV VLPs were significantly epitope-specific.Moreover, to determine whether HIV-1 conformational epitopes presented on HPV:HIV VLPs could be identified by the gp120 V3 and 2F5 neutralizing antibodies in vitro, we performed indirect ELISA assay to check the epitope-binding specificity and reactivity. As shown in n = 4) and female (n = 4) mice were evaluated. In the group of L1:P18I10 VLP, L1:T20 VLP and Gardasil-9, anti-HPV16 L1 antibody responses of female mice were on average higher than male mice (p = 0.0041)  B. A veryp = 0.0039). The anti-L1 responses among Gardasil-9, L1:P18I10 and L1:T20 VLP-immunized mice were similar and did not differ significantly (2auxo.int prime and L1:P18I10 VLP boost mice elicited a very low level of anti-L1 IgG. These results suggested that HPV:HIV VLP-immunized mice produced the same level of anti-L1 IgG as Gardasil-9-immunized mice (p = 0.005) (data not shown). In L1:T20 VLP group, a significantly higher antibody response against T20 peptide was detected compared to Gardasil-9 group (p = 0.0083). As expected, anti-T20 titers were undetectable in Gardasil-9, PBS, L1:P18I10 VLP and rBCG.HIVA prime combined with L1:P18I10 VLP boost groups  of HPV16 L1 VLPs used as stimuli. The group of BCG.HIVA2auxo.int prime and L1:P18I10 VLP boost induced higher frequency of IFN-γ secreting splenocytes and significant difference in IFN-γ secretion were observed when compared with mice vaccinated with Gardasil-9 group (p = 0.0103). 3 out of 8 mice (~38%) elicited the highest L1-specific IFN-γ responses (6 splenocytes was observed in BCG.HIVA2auxo.int primed mice compared to mice receiving Gardasil-9 vaccines or L1:P18I10 VLPs. Mice primed with BCG.HIVA and boosted with L1:P18I10 VLPs elicited significantly higher IFN-γ secretion compared with mice vaccinated with L1:P18I10 VLP homologous prime-boost (p = 0.0268). In addition, a significantly higher IFN-γ secretion was observed in mice vaccinated with L1:P18I10 VLPs in comparison with mice receiving Gardasil-9 vaccines (p = 0.0157)  C. As expBoth HPV16 and HIV-1 are sexually transmitted diseases and are currently the focus of many vaccine studies. Although HPV prophylactic vaccines have been commercialized and HIV-1 transmission has been greatly controlled by anti-retroviral treatment (ART) and PrEP, an effective, safe and affordable chimeric HPV:HIV vaccine against both viruses is an urgent need. In this study, (1) we demonstrated that the 293F expression system and the chromatographic purification method could be feasible approaches to produce and purify chimeric L1:P18I10 and L1:T20 VLPs; (2) we confirmed that the insertion of P18I10 or T20 peptides into the DE loop of HPV16 L1 capsid proteins did not affect in vitro stability, self-assembly and morphology of chimeric HPV:HIV VLPs; (3) The sequential and conformational P18I10 or T20 peptides exposed to DE loops of chimeric HPV:HIV VLPs could be detected by anti-HIV-1 gp120 V3 and 2F5 neutralizing antibodies in vitro; (4) The chimeric L1:P18I10 and L1:T20 VLPs could elicit HPV but weak HIV-1-specific binding antibodies in BALB/c mice. Furthermore, the insertion of HIV-1 P18I10 or T20 peptides into HPV16 L1 protein did not affect HPV16 L1-specific antibody induction in vivo; (5) L1:P18I10 VLPs could induce both HPV16 and HIV-1-specific T-cell responses; (6) BCG.HIVA prime and L1:P18I10 VLP boost elicited highest magnitude of IFN-γ producing splenocytes in comparison with L1:P18I10 VLPs homologous prime-boost in BALB/c mice. These finding supported further development of HIV-1 vaccines based on rBCG and chimeric HPV:HIV VLPs. All in all, this study provides a baseline strategy that may be worthy to support the global efforts to develop novel chimeric VLP-based vaccines for controlling HPV and HIV infections.In this study, L1:P18I10 and L1:T20 VLPs could induce the same level of anti-HPV16 L1 binding antibodies as HPV Gardasil-9 vaccine. However, L1:P18I10 and L1:T20 VLPs only elicit low level of P18I10 and T20 epitope-specific HIV binding antibody responses. We hypothesized that might be due because ELISA assay plates were coated with recombinant HPV16 L1 protein, HIV-1 P18I10 peptide and HIV-1 T20 peptide, respectively. The murine anti-L1 antibodies induced by our L1:P18I10 and L1:T20 VLPs might target multiple binding epitopes of L1 protein. By contrast, murine anti-HIV-1 antibodies elicited by our L1:P18I10 and L1:T20 VLPs only target single HIV peptide (P18I10 or T20) on HPV:HIV VLPs. Consequently, the overall maximum OD450 values of anti-HIV-1 binding antibodies were much lower than anti-HPV16 L1 antibodies.The reason that we selected HPV16 L1 capsid protein DE loop as preliminary insertion region of HIV-1 immunogen referred to a previous study that immunized mice with bovine papillomavirus (BPV):HIV VLPs for inducing antibody responses . It is kThe length and site of optimal HIV-1 foreign antigen incorporated into the HPV16 L1 VLPs and the in vitro stability of the resulting chimeric HPV:HIV VLPs should be verified before mice immunization. Our current study have shown that the insertion of P18I10 or T20 peptides into HPV16 L1 protein did not affect in vitro stability, self-assembly and morphology of chimeric HPV:HIV VLPs. These results were in concordance with previous studies indicating that insertion of HIV-1 MPER domain into BPV L1 DE loop sequence did not influence the capacity of BPV L1 capsid protein self-assemble to VLPs . Here, wSelf-assembly is a representative index of in vitro stability of HPV16 L1 VLPs. It is known that pH, ionic strength, temperature  and redoThe P18I10 peptides derived from HIV-1 gp120 V3 loop are presented in HIV-infected cells by major histocompatibility complex (MHC-I) class I molecules . CD8+, cBroader CD8+ T-cell responses against multiple conserved CTL epitopes are beneficial to overcome HIV-1 genetic diversity and escape ,99,100. Currently, our research group is focusing on the development of promising rBCG:HIV vaccines expressing novel HIV-1 T-cell immunogens, such as tHIVconsvX and HIVACAT (HTI) T cell immunogen, to improve HIV-1 variant match and T-cell response breadth. The 2nd-generation HIVconsvX immunogens were designed by redefining the group M conserved regions and utilizes a bivalent mosaic design to maximize the match of potential 9-mer T-cell epitopes in the vaccine to global variants . The HTIThe T20 peptide contains a highly conserved linear epitope 2F5 (ELDKWA). The 2F5 antibody collected from long-term HIV-infected patients was reported to have broadly neutralizing efficacy ,107. The


The advent of graphene opens up the research into two-dimensional (2D) materials, which are considered revolutionary materials. Due to its unique geometric structure, graphene exhibits a series of exotic physical and chemical properties. In addition, single-element-based 2D materials (Xenes) have garnered tremendous interest. At present, 16 kinds of Xenes  have been explored, mainly distributed in the third, fourth, fifth, and sixth main groups. The current methods to prepare monolayers or few-layer 2D materials include epitaxy growth, mechanical exfoliation, and liquid phase exfoliation. Although two Xenes  have not been synthesized due to the limitations of synthetic methods and the stability of Xenes, other Xenes have been successfully created via elaborate artificial design and synthesis. Focusing on elemental 2D materials, this review mainly summarizes the recently reported work about tuning the electronic, optical, mechanical, and chemical properties of Xenes via surface modifications, achieved using controllable approaches  to broaden their applications in various fields, including spintronics, electronics, optoelectronics, superconducting, photovoltaics, sensors, catalysis, and biomedicines. These advances in the surface modification of Xenes have laid a theoretical and experimental foundation for the development of 2D materials and their practical applications in diverse fields. Graphene, initially isolated from graphite via mechanical exfoliation, possesses a layered honeycomb structure and exhibits fascinating electrical and thermal properties . SubsequOver the past few years, many studies have focused on searching for other 2D Xenes with distinctive and exciting properties beyond graphene a 18,19],1918,19]Even though 2D Xenes have demonstrated great potential in numerous applications, their intrinsic properties usually constrain the expansion of these applications. For instance, despite the high mobility, the gapless band structure in graphene restricts its applications in electronic devices. Therefore, finetuning the properties of 2D Xenes plays an essential role in overcoming their intrinsic constraints ,35,36,37The material properties  of 2D Xenes are not only determined by their chemical compositions but are also strongly associated with the atom arrangement in the lattice. Due to the preferred orbital hybridization of various elements in the main group, 2D Xenes have been theoretically predicted or experimentally verified to possess allotropes with diverse crystal lattices . The rep1−ν☐ν; ν represents the vacancy concentration) with various vacancy concentrations and 2D aluminene with various forms Galleneneubstrate .Graphene. The sp2 hybridization of carbon atoms leads to the formation of flat honeycomb graphene with a σ bond between neighboring carbon atoms 118] but of 1.7 K f,g 52]..c of 1.7Silicene. Silicene, the silicon analog of graphene, was first predicted in a theoretical study . . 168]. BB-doping . With a B-doping . In addiB-doping . When axB-doping . MoreoveB-doping .Arsenene. Arsenene mainly has two kinds of allotropic structures, including buckled and puckered . T. T4 clusnochains b,c 24]..4 clustenochains d,e 177]4 clusternochains f, indicaf 30.8 K . Indeed,f 30.8 K . Meanwhif 30.8 K . In addif 30.8 K .Antimonene and bismuthene. The most stable structures of antimonene and bismuthene are α-form (puckered) and β-form (buckled), which are the monolayers of black and gray bulk allotropes, respectively. Monolayer β-Sb and β-Bi possess indirect bandgaps of 2.28 and 0.99 eV, respectively, whereas the direct bandgaps of monolayer α-Sb and α-Bi are 1.18 and 0.36 eV, respectively [ectively ,182,183.ectively ,185,186.Doping with 3d transition metal atoms for antimonene can lead to significant changes in the bandgap and the magnetic moment . For Cr-Selenene and tellurene. Selenene has been predicted to have three allotropic structures, including a 1T-MoS2-like structure (t-Se or α-Se), a tiled helical-chain structure (c-Se), and s square structure (s-Se) [2-like, rectangle, and 2H-MoS2-like structures, respectively [e (s-Se) . Both t-e (s-Se) . Howeverectively . It was ectively . Therefoectively ,196. MeaAs mentioned above, 2D Xenes possess various physical and chemical properties, such as flexibility, layer-dependent semiconducting, high carrier mobility, molecule and light sensitivity, topologically nontrivial band structures, etc.c of 30.8 K [2/graphene hybrids decorated by CdS nanocrystals can act as high-performance photocatalysts for H2 evolution under visible light irradiation [In order to utilize 2D Xenes more effectively, surface modifications become particularly important  to tune f 30.8 K . Iodine-f 30.8 K . Graphenf 30.8 K . MoS2/gradiation . Moreoveadiation . For exaadiation . Polyethadiation . OverallIn total, except graphene, 15 different elemental 2D materials of the main group elements have been experimentally created or theoretically predicted to date. In fact, 14 Xenes, including borophene, graphene, silicene, phosphorene, gallenene, germanene, arsenene, selenene, stanene, antimonene, tellurene, thallene, plumbene, and bismuthene, have been successfully grown on proper substrates using epitaxial methods. Although a lot of research, engineering, and development related to 2D Xenes—most of which were investigated theoretically—has been reported in recent years, experimental studies are still desperately required for the development of synthesis strategies and novel applications. Before realizing the surface modifications of 2D Xenes for the applications of interest, three significant challenges need to be overcome:(i) The synthesis strategies of 2D Xenes must not only ensure reliably large-scale production but also be tailored for the requirements of the application. For example, the applications of electronics and batteries demand low costs and scalable techniques, while plasmonics and spintronics require high fidelity and reproducible techniques. Hence, reliable synthesis approaches play a crucial role in functional design and practical applications.(ii) The strategies to enhance the environmental stability and mitigate the degradation of 2D Xenes must be taken into consideration for certain applications. For instance, an optoelectronic device based on black phosphorene must be concerned with ambient stability, requiring appropriate encapsulation or the passivation of the surface.(iii) Many of the predicted and fascinating properties of 2D Xenes require further efforts to find strategies for their implementation, which may offer opportunities to discover revolutionary technologies. In addition, the exciting and novel properties necessitate not just “one-off” research, but also statistical evaluation to ascertain their viability and accessibility on a commercial scale.Despite the challenges ahead, the exceptional properties of 2D Xenes will significantly impact future applications in various fields. It is hoped that this review will inspire more exhilarating discoveries and applications in the growing family of 2D Xenes.


Azadirachta indica (Neem) extract with an objective to develop new coating materials. The nanomembranes have been synthesized by electrospinning machine over aluminum foil paper using the raw materials PVA grain, SiC, TiO2, Graphene, and neem. The nanomembranes have been characterized by SEM, XRD, FTIR, Surface Roughness, antibacterial, and Cytotoxicity test. FTIR analysis established the presence of PVA and neem indicating the formation of different organic compounds. It also confirmed that no chemical reaction occurred during the synthesis process. The membrane's roughness analysis obtained average roughness values from 1.15 to 3.84. The formation of homogeneous and smooth membranes with the formation of micropores was confirmed by SEM analysis. Miller Indices identified different types of crystal structures in XRD analysis. Antibacterial activity increased with the increase of the percentage of neem confirmed by the antibacterial test. No toxic effects were observed from the membrane during the cytotoxicity test. The obtained data confirmed that the synthesized nanomembrane could be used in different biomedical applications.This paper presents the coating technology on Nano membrane using SiC–TiO2-Graphene with varying percentages of  •Azadirachta indica.Synthesis of Nano membrane using SiC–TiO2-GRAPHENE with varying percentages of •Synthesis by electrospinning machine.•Increased antibacterial activity.•Characterized by SEM, XRD, FTIR, Surface Roughness, Antibacterial and Cytotoxicity test. Nano membrane; Neem; PVA; Antimicrobial activity; Cytotoxicity. Nanotechnology is used widely in a different range of applications, particularly in the field of energy, biotechnology, sensors, and related other areas. Nanotechnology has brought immense creativity and has pushed the limits of our understanding. The most recent technological advances will allow a much wider and more in-depth analysis of Nanoscale materials structures and functions. Nanotechnology and Material Science require integrating and comprehending the physical ideas shown by Biomaterials, nanometer-scale particle technology, and other nanotechnologies. Materials as well as innovative applications combining different areas of nanotechnology are invited. Scientists as well as engineers are brought together from different disciplines such as materials science, forensic engineering, biomedical engineering, chemical engineering, electrical engineering, physics, microtechnology, nanorobots and nanosensors , 2, 3. DNanomembrane can be defined as an organic, inorganic, or quasi-2D artificial freestanding or free-floating structure having a thickness less than 100 nm . In the The electrospun nanomembranes are being developed by incorporating different bioactive nanoparticles to improve tissue regeneration ability. Different nanoparticles such as bioactive glass nanoparticles , graphenTitanium dioxide nanoparticles have a wide range of applications such as reducing toxicity in dyes and prescription drugs; wastewater treatments; silkworm reproduction; space applications; food industry; etc. For industries, the atmosphere, and agriculture, the applications of nanoparticles synthesized with a biological approach would be favorable . In ordeThe tribological behavior of graphite nanoparticles were evaluated using vegetable based oil as lubricant with the help of a pin on disc tribometer. Investigation was performed on graphite nanopartilces grease instrument. The results indicated that the vegetable based oil reduced the friction and wear significantly .Azadirachta indica (Neem) extracts. In our real life, people suffer several kinds of bone diseases in which bone fracture very vital problem. So, in medical science, they try to reduce bone fracture by using metal. But metal is not safe for such a kind of operation. Metals contain toxic features and have bacterial effects. Therefore, this experiment will reduce those kinds of harmful effects of metals.In this study, an attempt has been made to reduce the toxicity and bacterial effect of the multi-nanoparticle's membranes for biomedical applications by infusing nanoparticles with the nanomembrane. The toxic and bacterial impacts of SiC–TiO2- Graphite membranes are purified by 5%, 10%, and 15% of 2-Graphene hybrid nanoparticles in PVA solution to increase the versatile multifunctional aspect of this bio-based nanomembrane to be applied as a biomaterial in different biomedical applications which cannot be obtained when a single nanoparticle is used. In nanofibrous membrane, a large surface area to volume ratio, flexibility in surface functions, and increased stiffness and tensile strength when compared to other materials since fiber diameters are lowered from micro to nanoscale. There is large surface porosity with small pore size in nanomembrane when the nanoparticles are absent there. This type of porous nanomembrane is suitable for water filtration, high energy generation, and some other areas. But when nanoparticles are considered as nanofillers coatings in PVA nanomembrane, the porosities are disclosed and reduced to enhance the stiffness, strength, wear resistance, fatigue resistance, reduction of thermal expansion, and dimensional stability. These nanoparticles also have their inherent properties; these have also a considerable effect on nanomembrane properties. The concept of this type of nanofibrous-nanofiller membrane can be used in dental implants to mitigate the complication of stress shielding and implant-related infections and inadequate osseointegration problem.The novelty of this study is to use SiC–TiOBesides this, GTR (guided tissue regeneration) and GBR (guided bone regeneration) are surgical methods used to regenerate the tooth-supporting tissues (GTR) and the alveolar bone (GBR) in edentulous areas, respectively (GBR). Recent techniques include the use of resorbable membranes/nanofiller. These materials have the advantage of disintegrating over time, eliminating the need for surgery to remove the scaffolds. Nanofibrous scaffolds have improved mechanical qualities, cell adhesion, biocompatibility, and antibacterial properties for GTR/GBR applications. This study can be used as a guideline for future research to resolve the problem using this type of nanofibrous-nanofiller membrane but before using this concept biodegradability should be ensured. Therefore, the choice of nanofiller in the nanofibrous membrane is important. The concept of the nanofibrous-nanofiller electrospun membrane in GTR/GBR dental application is illustrated in Titanium and its alloys are recognized one of the best biomaterials for dental implant applications for their excellent properties such as higher tensile strength, higher corrosion resistance, and effects resistance due to body fluid, flexible and ferromagnetic abilities. Unique mix strength, biocompatibility, and low reactivity made them suitable for biomedical applications , as wellTo ensure ion releasing, ion element identification, survival rate, non-toxicity, and cell growth confirmation, corrosion tests in artificial body fluids such as SBF, HBSS, in vitro test, cell viability test of degraded corroded solution using fluorescence microscopy, microwave plasma atomic emission spectroscopy, and cell attachment test using the chemical process are conducted. These tests are not observed in this study as there are many results available in the literature , 29, 30.22.12 nanopowder, and Graphene nanopowder. PVA is collected from sigma, having a molecular weight of 98,000g (99% hydrolyzed). SiCnano powder, TiO2 nanopowder, and Gr nanopowder are purchased from Shanghai Ruizheng Chemical Technology Co., Ltd of China. Their chemical and physical properties are available in the literature [The materials used in this experiment are PVA, SiC nanopowder, TiOterature , 33. Nee2.2Neem leaves were collected from the Dhaka University of Engineering and Technology garden during the winter season. The tree was around three meters tall. The collected leaves are washed three times with distilled water to remove the dirt and other impurities and then dried under the sun. Then the leaves are cut into small pieces. 20 gm of leaves are taken and kept in a glass jar with 200 ml ethanol and kept at 4 °C for 24 h. The next day, the solution turned to greenish color. The solution is then filtrated to remove any solid particles and kept in the refrigerator for further use .Figure 22.315 gm of PVA is put in a beaker and mixed with 300 ml distilled water at 70 °C temperature over a magnetic stirrer to form a uniform solution.2.42, and 0.2 gm Graphene nanoparticles are mixed together at room temperature by continuous stirring to obtain a combined mixture and the mixture was then dissolved with 20 ml PVA solution under a magnetic stirrer and kept in three different jars. The solution was stewed overnight at room temperature to remove bubbles. Then 0%, 5%, 10%, and 15% neem were added to the mixture in the jar. Later, the solution was injected into a syringe with having 0.9 mm inner diameter with a metal nozzle shown in 0.2 gm SiC, 0.2 gm TiOIn the electro-spun process, the syringe is filled with the requisite solution. The syringe is inserted into the pump and joined by a pipe to some needles. The outside of the drum collector is wrapped in aluminum foil. The positive and negative voltage terminals are linked to the needles and the collector, respectively. In relation to SiC–TiO2-Gr and NEEM extraction on the surfaces of aluminum foil, a magnetic field is produced between needles and the collector as a result of positive and negative voltage potentials, which electrifies the liquid droplet of the solution to produce a jet. The jet is then stretched and extended to produce a nanofiber network.The electrospinning had a 15 cm distance between the fiber collector and spinneret, 25 kV power supply, 0.5 mL/h spinning solution feed rate, and 25 °C temperature. Later, the membrane was dried at room temperature. 2.52.5.1−1 to 4000 cm−1.FTIR analysis has been performed by FTIR spectrometer made by PerkinElmer, the USA maintaining the frequency range from 500 cm2.5.2The surface roughness of the synthesized nanomembrane was estimated by a surface harshness analyzer . Three areas of the surface were estimated at any rate and the average value was considered.2.5.3The microstructure of the surface of the synthesized nanomembranes was examined by a scanning electron microscope made by Hitachi, Japan having model number SU1510.2.5.4The SEM images were analyzed then by SPIP software for better realization of the microstructure of the surface by 2D and 3D images.2.5.5The SEM images were further analyzed by SPIP software as well to analyze the particle distribution on the surfaces of the synthesized nanomembrane.2.5.6−1.The synthesized nanomembranes were analyzed by Bruker D8 advanced X-ray beam diffraction analyzer which was made in Germany. The X-ray beam force was estimated in the scope of 100 ≤ 2θ ≤ 600 with a sweep speed of 0.50 min2.5.7Staphylococcus aureus (ATTC 25923) as gram-positive bacteria were subjected to the tests in the disc having a size of 6 mm × 6 mm. The bacterial cell concentration was 1000 CFU/ml and the nanomembrane sample concentration was 200 mg/ml. The bacterial cells used in this research were collected from Dhaka University. The culture was spread on a nutrient agar plane after suspending in nutrient broth. The culture was incubated at 37 °C for 24 h. The culture was picked off from the agar plate with an inoculating loop and two single colonies were suspended in a nutrient broth that had a 10 mL volume. Later, the culture was incubated for 18 h at 100 rpm and 37 °C. 0.3 mM M and pH 7.2 sterile buffer solution were used to dilute and a final concentration of 1000 CFU/ml was obtained.The antibacterial test of the nanomembranes was accomplished by applying the Kirby-Bauer disk diffusion test method maintaining the ASTM E2149-01 standard. The bacterial cell 2.5.8The cytotoxicity tests of the nanomembrane were performed in a biological biosafety cabinet model NU400E, incubator, Trinocular microscope with camera, and Hemocytometer. All the equipment is made by Nuaire, USA. Vero cell line, a kidney epithelial cells extracted from an African green monkey, was maintained in DMEM (Dulbecco's Modified Eagles' medium) containing 1% penicillin-streptomycin (1:1) and 0.2% gentamycin and 10% fetal bovine serum (FBS). Cells (1.5 × 104/100 ul) were seeded onto a 48-well plate and incubated at 37 °C + 5 % CO2. The next day, a sample (Autoclaved) was added to each well. Cytotoxicity was examined under an inverted light microscope after 48h of incubation. Duplicate wells were used for each sample. Vero cell line, a kidney epithelial cells were supplied by the WAFFEN Laboratory, Dhaka, Bangladesh.33.1−1 because of less oxidation effect due to the absence of neem. Alkyl amine stretching vibration is shifted to 1090 cm−1 in the nanomembrane containing 5% neem extract due to oxidation. Further shifting of alkyl amine stretching vibration is observed attributed to 1088 cm−1. Medium stretching functional group O–H is attributed to 3320 cm−1 [−1 [−1 [320 cm−1 . Besidescm−1 [−1 . Medium  [−1 [−1 . The pre3.2Figures 3.3Figures 3.43.52, Graphene, and neem present on the surface of the nanofibrous membrane shown in SEM images of 2 decreased with 5% addition of neem but increased with the addition of 10% and 15% of neem. It is also an indication that the addition of neem increased the density of nanomembrane. However, the mean value increased with 5% addition of neem but decreased with 10% and 15% neem addition.Partilce analysis observed the presence of PVA, SiC, TiO3.6XRD analysis was performed to determine the crystallinity of the developed nanomembrane films shown in 3.7Staphylococcus aureus makes it resistant and the polycationic group present in the nanomembrane facilitates the disruption of gram-negative bacteria [Staphylococcus aureus virus indicating their combined effect. The leaching of antibacterial bioactive constituents from the nanomembrane may cause the incorporation of the neem extract. Besides, bacteria cannot penetrate the mesh structure of the developed nanomembrane due to the presence of micropores and thus the surrounding infections are nullified effectively [Staphylococcus aureus. The presence of tetratriterpenoids including azadirachtin in neem made this possible. The inhibition of the Staphylococcus aureus production polysaccharide intercellular adhesion is possible by these compounds due to the inhibition of the ica genes expression [The antibacterial activity of the synthesized nanomembrane can be seen in bacteria , 53. Howectively , 55. Thepression , 57.Figu3.8The cytotoxicity of the developed nanomembrane was observed on the Vero cell line and the results confirmed the survival of all cell lines. There are no significant changes in the cell line. The study revealed that the developed nanomembrane can safely be used against infections. Ion complexity by legends may be the reason for the zero or near zero toxicity of the developed nanomembrane.4Staphylococcus aureus with zero or near zero toxic effect. The morphological analysis confirmed the formation of smooth nanofiber with the development of micropores. FTIR spectra demonstrated the characteristic peaks and the peaks became sharper with the increased percentages of neem. Micropores present in the nanomembrane will inhibit the bacteria and keep the skin safe. The addition of neem increased the fiber diameter. Regardless of the percentage of neem, all the developed nanomembranes formed crystal structures confirmed by the XRD analysis. Besides, variation of surface roughness with the various constituent percentages of neem is confirmed by surface roughness analysis. The formation of inhibition zones by the developed nanomembranes was higher than amoxicillin. The overall findings of the present study suggest that the developed nanomembrane can successfully be sued as bio-based antibacterial wound dressing material. This nanofiller-nanofibrous membrane concept can be applied in dental applications with enhanced bacterial, structural, particle distribution, surface, and chemical bonding properties.A new way of developing nanomembrane with the help of electro-spinning techniques has been introduced in this work with antibacterial resistance against Dr. Mohammad Asaduzzaman Chowdhury: Conceived and designed the experiments.Mr. Nayem Hossain: Performed the experiments; Wrote the paper.Dr. Md. Abdus Shahid, Md. Jonaidul Alam: Analyzed and interpreted the data.Sheikh Monir Hossain, Md. Ilias Uddin, Md. Masud Rana: Contributed reagents, materials, analysis tools or data.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Data will be made available on request.The authors declare no conflict of interest.No additional information is available for this paper.