Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease. Multiple research has revealed that the extracellular matrix (ECM) may be associated with the development and prognosis of IPF, however, the underlying mechanisms remain incompletely understood.We included GSE70866 dataset from the GEO database and established an ECM-related prognostic model utilizing LASSO, Random forest and Support vector machines algorithms. To compare immune cell infiltration levels between the high and low risk groups, we employed the ssGSEA algorithm. Enrichment analysis was conducted to explore pathway differences between the high-risk and low-risk groups. Finally, the model genes were validated using an external validation set consisting of IPF cases, as well as single-cell data analysis.Based on machine learning algorithms, we constructed an ECM-related risk model. IPF patients in the high-risk group had a worse overall survival rate than those in the low-risk group. The model’s AUC predictive values were 0.786, 0.767, and 0.768 for the 1-, 2-, and 3-year survival rates, respectively. The validation cohort validated these findings, demonstrating our model’s effective prognostication. Chemokine-related pathways were enriched through enrichment analysis. Moreover, immune cell infiltration varied significantly between the two groups. Finally, the validation results indicate that the expression levels of all the model genes exhibited significant differential expression.Based on CST6, PPBP, CSPG4, SEMA3B, LAMB2, SERPINB4 and CTF1, our study developed and validated an ECM-related risk model that accurately predicts the outcome of IPF patients.The online version contains supplementary material available at 10.1186/s12890-023-02699-8. Idiopathic pulmonary fibrosis (IPF), an interstitial lung disease, is distinguished by rarity, persistent progression, and fibrosis, with an etiology and pathogenesis that remains incompletely understood . SmokingExtracellular matrix (ECM) is a critical component of tissues and organs, playing an essential role in the survival of multicellular organisms, and serving as a key regulator of cellular behavior. Comprising over 300 different proteins, including collagens (primarily types I and III), elastin, glycoproteins, and proteoglycans such as hyaluronic acid and fibronectin, these molecules influence cell adhesion, migration, and differentiation . InitialIn this study, we aimed at exploring the relationship between EAGs and prognosis in IPF. We established an ECM-related prognosis model through LASSO and Cox regression analysis, and verified its reliability using the GEO cohort. Furthermore, we studied the interaction between this model and immune cells. Our findings reveals the potential value of EAGs in treating IPF patients and help us comprehend the correlation between ECM and IPF prognosis even better.We retrieved 1,062 EAGs from the previously published literature . To furtFirstly, we carried out a multivariate Cox regression analysis on the selected model genes and constructed a risk score to assess the prognosis of IPF by calculating the formula: In this study, the ssGSEA algorithm was applied to dissect the gene expression profiles of high and low risk groups of patients, resulting in the identification of 28 immune cell subtypes and their scores in clinical samples . In ordeTo elucidate the molecular mechanisms and pathway differences between high and low-risk groups, we conducted gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) using the “clusterProfiler” package , 19. TheWe incorporated the external validation set GSE28042 to validate the expression levels of the seven model genes , 21. FurFigure P < 0.001, C-index, 0.707; 95% CI, 0.649–0.766), with the low-risk subgroup having a higher survival rate  , Type I Alveolar cells, Proliferating myeloid/Proliferating lymphocyte cells, and Dendritic cells Fig. A. NotablIPF is a disease with a currently incompletely elucidated etiology that poses a substantial global socioeconomic burden. Reducing morbidity and mortality from IPF requires early identification, prevention, and intervention of relevant risk factors. For predictive diagnosis, focused prevention, and individualized treatment of the disease, it is thought to be useful to screen for putative susceptibility genes and uncover their molecular mechanisms in IPF. In this work, we constructed an ECM-related risk model utilizing LASSO, Random forest and Support vector machines algorithms to forecast the prognosis of IPF patients. The results indicate this model, based on seven genes CST6, PPBP, CSPG4, SEMA3B, LAMB2, SERPINB4 and CTF1, is a reliable predictor of the survival rate of IPF patients. The model was validated in an independent dataset from the GEO cohort, GSE27957, which confirmed its good performance. In addition, the results of ROC analysis indicated that these seven genes could be used as biomarkers of IPF with high AUC values.The identification of these seven EAGs is consistent with earlier studies, which found that the ECM is closely associated with the development of IPF. With the exception of CST6, SEMA3B, LAMB2, SERPINB4 and CTF1, the remaining two genes have all been linked to the progression of IPF. PPBP (pro-platelet basic protein) is a gene that encodes a cytokine protein called CTAP-III, which stimulates the formation and maturation of white blood cells, red blood cells, and platelets . The resIn addition, enrichment analysis revealed that comparing the high-risk group to the low-risk group, chemokine-related pathways were considerably enriched in the high-risk group. It is well recognized that chemokines are crucial for the attraction and activation of immune cells, which may help to cause IPF. Moreover, GSEA analysis showed enrichment in the pathway for chemokine signaling, further supporting the importance of this pathway in IPF. KEGG pathway analysis showed that IL-17 pathway plays an essential part in the pathological mechanism of IPF. IL17 is secreted by CD4 + T cells and can induce the production and excretion of pro-inflammatory cytokines such as IL-6, IL-8, and G-CSF in epithelial cells, endothelial cells, and fibroblasts. This family includes six ligands  and five receptors  . CurrentOur findings also showed that the immune cell infiltration was different between the high and low risk subgroups. According to prior research revealing that immunological dysregulation is a crucial aspect of the disease, these findings imply that the immune response may be implicated in the etiology of IPF.One limitation of our study is that it is based on retrospective data from public database, therefore, prospective studies are required to validate our results. In addition, the precise relationship between EAGs and IPF remains to be evaluated and validated in experimental studies because we have no access to clinical specimens of IPF.In conclusion, our study offers fresh perspectives on the function of ECM in IPF and identifies a new ECM-related risk model for forecasting the survival rate of IPF patients. These findings aid to a deeper comprehension of the association between EAGs and the prognosis of IPF and may provide a basis for developing personalized therapies for IPF patients. Further studies are required to validate the clinical usefulness of our ECM-related risk model and to investigate the underlying mechanisms by which these genes contribute to the pathogenesis of IPF.Additional file 1.


Large spin-orbit coupling is often thought to be critical in realizing magnetic order-locked charge transport such as the anomalous Hall effect (AHE). Recently, artificial stacks of two-dimensional materials, e.g., magic-angle twisted bilayer graphene on hexagonal boron-nitride heterostructures and dual-gated rhombohedral trilayer graphene, have become platforms for realizing AHE without spin-orbit coupling. However, these stacking arrangements are not energetically favorable, impeding experiments and further device engineering. Here we report an anomalous Hall effect in Bernal-stacked tetralayer graphene devices (BTG), the most stable configuration of four-layer graphene. BTG AHE is switched on by a displacement field and is most pronounced at low carrier densities. The onset of AHE occurs in tandem with a full metal to a broken isospin transition indicating an orbital origin of the itinerant ferromagnetism. At lowest densities, BTG exhibits an unconventional hysteresis with step-like anomalous Hall plateaus. Persisting to several tens of kelvin, AHE in BTG demonstrates the ubiquity and robustness of magnetic order in readily available and stable multilayer Bernal graphene stacks—a new venue for intrinsic non-reciprocal responses. Intrinsic anomalous Hall effect has been observed in twisted graphene multilayers, but these structures are typically not energetically favorable. This study extends these observations to Bernal-stacked tetralayer graphene, which is the most stable configuration of four-layer graphene. Recently, intrinsic valley access in the absence of external magnetic field or circularly polarized light irradiation was demonstrated via orbital ferromagnetic ordering in moiré materials4, and rhombohedral trilayer graphene6, giving rise to an anomalous Hall effect (AHE). In these, valley polarization can be directly identified by the sign of the anomalous Hall response at zero magnetic field and switched hysterically7. However, consistently achieving these stacking configurations for device characterization is challenging8. In twisted bilayer graphene, AHE has only been found for twist angles that are simultaneously close to magic angle as well as aligned to a hexagonal boron nitride substrate8, while rhombohedral stacking arrangements are not the thermodynamic ground state in naturally occurring graphene trilayers11.Electrical access to the valley degree of freedom in two-dimensional Dirac materials requires the simultaneous breaking of inversion and time-reversal symmetries. For instance, by irradiating MoS10, as illustrated in Fig. 12, as well as topological phase transitions and helical states at high magnetic field13. In contrast to these previous works, we focus on the low-density and moderate displacement field region in BTG that displays pronounced valley-locked orbital magnetic moments and prominent density of states close to van Hove singularities . These features interact to produce an AHE.Here we uncover an AHE in Bernal-stacked tetralayer graphene (BTG), the most energetically stable allotrope of four-layer grapheneRxx and Hall resistance Rxy. The carrier density n = [Ct(Vt-Vt0)  +  Cb(Vb-Vb0)]/e and perpendicular displacement field D  =  [Ct(Vt-Vt0)  −  Cb(Vb-Vb0)]/2ϵ0 can be independently tuned by applying voltages on top and bottom gates  and a multi-peak like structure as a function of carrier density; these peaks can be controlled by D and have been attributed to gate-tunable Lifshitz transitions in BTG12. Similarly, Rxx at the CNP first increases with D and then saturates at larger D ( > 0.2 V/nm). These behaviors are signatures of Bernal-stacking order of BTG15.Figure Rxx  at a small out-of-plane magnetic field of B = 1 T in Fig. Rxx  are immediately apparent. First, and perhaps the most prominent, are vertical stripes that occur at uniform spacing of filling factor δν = nϕ0/B = 4, where ϕ0 is the magnetic flux quantum. The degeneracy of 4 isospin flavors  is often associated with a “full” metal (FM) phase, see also Supplementary Fig. Rxx  density plot with filling fraction labels. Additionally, superposed on top of the vertical stripes are diagonal fringes at moderate to high density that originate from the intersection between collections of Landau levels.To investigate the fermiology of BTG, we measure D , an unusual stitch-like ripple pattern emerges. These ripples arise from a complex pattern of Landau levels and their intersections that disperse with D12. Strikingly, this region is marked conspicuously by the absence of the clear vertical lines with δν = 4 degeneracy spacing. To further investigate the features in this region, the period of quantum oscillations is examined in Fig. B that can reveal the k-space area of the enclosed orbits. Extracting the oscillation frequency, fB, in the standard method16 via Fourier analysis of Rxx (1/B)  as f = fB / nϕ0; 1/f indicates the degeneracy of the Fermi surface.In contrast, at low density and moderate f ≈ 1/4 in agreement with the 4 fold-degenerate vertical stripes discussed above. In contrast, the “VM” region possesses f ≈ 1/2 indicating a lowering of the isospin flavor degeneracy with an orbit area that is approximately half the total Fermi surface size. As a result, this behavior is reminiscent of a gate-tunable transition from a full metal to a half-metallic-like regime, similar to other isospin-polarized phase transitions reported in rhombohedral trilayer graphene6 and bilayer graphene recently17. While the combination of an applied magnetic field  and D field (broken inversion) are sufficient to lead to externally broken valley symmetry18 and hence half-metallicity at low densities, as we now discuss, time-reversal remains spontaneously broken at B = 0. We denote this spontaneous time-reversal broken phase a valley metal state.For clarity, we examine the quantum oscillations for several illustrative points in the “VM” region (“FM” region) denoted by the empty (solid) polygons respectively. In the “FM” region, we find Rxy as a function of applied B field at D = 0.3 V nm-1 at two different densities 0.1 × 1012 cm-2 (deep in the “VM” region) and 1.0 × 1012 cm-2 (outside the “VM” region). Rxy for 1.0 × 1012 cm-2 exhibits a conventional linear in B dependence with near identical forward (red) and backward (blue) B-field sweeps. In contrast, Rxy for densities of 0.1 × 1012 cm-2 is hysteretic with a non-zero Hall resistance at B = 0 of several tens of Ohms  = ηB + RAH (M), where η = 1/ne is the Hall coefficient and M is the magnetization. Plotting RAH as a function of applied B in Fig. RAH increases rapidly before saturating at approximately 40–50 mT. RAH also highlights the hysteretic features of BTG AHE. In the small field window where hysteresis is most pronounced, the blue and red curves mirror each other to display the same stepped profile but displaced in the y-axis. This shift between forward and backward sweeps indicates that red (-) and blue (+) RAH curves track different ferromagnetic states  as they evolve with applied B.BTG AHE can be better visualized by delineating the total Hall resistance into a conventional component that scales as applied FK = Σp fK(p) ΩK(p), displays a rapid change as a function of chemical potential close to band edge; away from the band edge it saturates  is the electronic distribution function for valley K. When both valleys are equally populated , the net Berry flux Ftotal = FK + FK’ that tracks the strength of the AHE20 is zero. Crucially, close to the band edge in the VM region, BTG possess D tuned van Hove singularities with pronounced peaks in the density of states  ≠ fK’(p). Spontaneous valley polarization features a non-zero RAH and a non-zero Rxy at B = 0, as shown in Fig. AHE in BTG can be naturally understood by examining the Berry curvature distribution in the bands of gated BTG. In valley K, the Berry curvature distribution  ≠ fK’(p)] can be further tuned by B field  favouring one valley over the other, see also Fig. RAH with applied B. Importantly, because Berry curvature is concentrated at the band edge, dFK,K’/dμ is most pronounced at the band edge , RAH is flat with no appreciable hysteresis. As density is lowered, both the size of the hysteretic window and the saturated RAH increase, in agreement with the peaked dFK,K’/dμ close to the band edge. This density sensitivity allows BTG AHE to be switched “on” by tuning into the low electron density region where Berry flux changes rapidly. Similarly, since a finite D field turns on Berry curvature in the valleys of BTG, we find that AHE can also be switched “on” by D field  nature of the half-metallicity in rhombohedral trilayer graphene6. This arises due to the minimal spin-orbit coupling in graphene multilayers that yields a Berry curvature which has the same sign for opposite spins. We note that evidence for spin polarized half-metallic states have also been reported more generally in rhombohedral stacked few-layer graphene 27.It is interesting to compare BTG AHE discussed here with other AHE found in graphene stacks, e.g., in rhombohedral trilayer graphene4 where both spin- and valley- symmetry are broken. In sharp contrast, in BTG, we find that AHE occurs in the VM region where the frequency of quantum oscillations are consistent with a half metal while we found no signatures of a quarter metallic state. This indicates the valley polarized half metal origin of BTG AHE.Similarly, in twisted bilayer graphene heterostructures, AHE occurs near 3/4 filling of a moiré flat bandD field tuned van Hove singularities, which are naturally found in gated (even) layers of Bernal-stacked graphene. As a result, we anticipate that AHE and spontaneously valley-polarized states are likely ubiquitous in gapped graphene multilayers. Therefore our results imply that the energetically stable form of graphene multilayers can be used to probe and exploit graphene based AHE beyond the metastable configurations of moiré superlattices4 and rhombohedral trilayer graphene6. Perhaps the most exciting aspect is the ability to sustain valley polarization at zero magnetic field. Requiring both inversion symmetry breaking (controlled by D field) as well as spontaneous time-reversal symmetry breaking, such valley polarization mirrors the requirements for magneto-chiral responses28, which has implications for valley-mediated intrinsic non-reciprocity. Further, since BTG ferromagnetism is turned on by D field, they are ideal candidates to be readily combined with layered (out-of-plane) ferroelectrics30 to achieve stack- engineered multiferroic functionality.The key ingredients of BTG AHE are valley contrasting Berry curvature and the high density of states close to Tetralayer graphene, graphite and hexagonal boron nitride (hBN) flakes are exfoliated on silicon dioxide/silicon wafers. The whole heterostructure was assembled using dry-transfer technique using a PC (polycarbonate) film/PDMS stamp. We first pick up the top graphite and subsequently top hBN, BTG, bottom hBN and bottom graphite and put the whole stack on another wafer. The hBN layers are not intentionally aligned with BTG sample. Graphite gates are employed to reduce disorder and ensure high quality devices. Finally the device was etched into a Hall bar and contacted using edge-contact method with Cr (5 nm)/Au (70 nm).RAH are limited by the electrical noise in data acquisition.Transport measurements were conducted in a variable-temperature He-3 or He-4 Probe in Oxford Teslatron system. The lowest temperature reached is ~300 mK. Standard lock-in technique is used to measure the four-probe voltages using SR830. Low pass filters are used to suppress low frequency noises. Constant current source is applied with ranges from 10 to 100 nA. Gate voltages are applied using Keithley 2400 and 6430. Error bars in determining ΔSupplementary InformationPeer Review File


Thetopography of such graphene has two important properties. First, itssurface is comprised of folds that have different orientations, andsecond, it has several multilayer-graphene regions distributed overthe monolayer-graphene surface. On such folded graphene features,we vacuum evaporated pentacene and observed three-dimensional islandswith an average height of ∼15 nm. They are oriented eitherparallel or perpendicular to the folds, and they are also predominantlyoriented along the symmetry axes of graphene. Orientation of pentaceneislands on graphene evaporated at room temperature has a wide distribution.On the contrary, most of the pentacene islands evaporated at 60 °Care oriented at 30° with respect to the fold direction. We observedthat the folds act as a potential barrier for the surface transportof pentacene molecules. In addition, we interpret the 3D growth ofpentacene islands on graphene in terms of reduced polar componentsof the surface energy on graphene investigated with contact anglemeasurements.Using atomic force microscopy, we probed the growth ofpentacenemolecules on graphene that was fabricated by chemical vapor depositionand transferred onto 300 nm-thick SiO I2 Most often, the organicmolecules are attached to the graphene surface by the π–πinteraction, which leads to a non-negligible charge transfer at thegraphene/OS interface. The density and areal configuration of transferredcharges is crucially dependent on the molecular arrangement on thegraphene’s surface, i.e., order/disorder is one of the principalparameters characterizing the graphene/OS interface. Therefore, itis crucial to understand the adsorption of organic molecules on graphene.Pentaceneis one of the organic molecules that forms well-ordered polycrystallinelayers with a relatively high charge carrier mobility on a varietyof substrates. This stimulated a considerable interest in pentacene-basedorganic thin-film transistors.3 However, the growth of pentaceneis highly influenced by the topography of graphene and the substrateunderneath.6The principal drawbackof graphene, precluding it from being usedin a widespread manner as a staple material in electronic switchingdevices, is the absence of the electronic energy band gap and negligibledensity of states at the principal points of the Brillouin zone. Anincrease in the on/off ratio of graphene-based field-effect transistorscould in principle be achieved by integration of organic semiconductor(OS) molecules with graphene.7 Third, trapped molecules (hydrogen and hydrocarbon)between graphene and copper during the CVD growth process can be anotherreason for such unusual fold formations on graphene.8 The 6-fold symmetry of the graphene unit cell is brokenat folds, giving rise to the triangular pattern of atoms, while flatgraphene exhibits a honeycomb lattice.9 In a triangular pattern, out of six carbon atoms, only three areobserved in honeycomb rings. Nirmalraj et al.10 have highlighted the influence of folds on the growth of pentaceneand reported a large molecular energy gap for pentacene depositedon high folds (1–3 nm thick) as compared to flat graphene.They measured a large conductance gap of pentacene molecules depositedon such folds (3.0 ± 0.3 nm) in comparison to that on flat grapheneregions (1.3 ± 0.2 nm). This is due to the increased spatialdistance at folds that leads to significant screening from the underlyingCu substrate. When pentacene is deposited at such regions of surfaceexhibiting increased local curvatures (folds), it may acquire a differentgrowth configuration relative to the flat graphene. Moreover, thegrowth mode may depend on the orientation of folds itself with respectto the flat regions.While mechanically exfoliated graphene presentsitself as mostlydefect-free, the tens-of-micrometer-size flakes preclude its use ina large-scale production setting. Chemical vapor deposition (CVD)instead yields large-scale graphene foils, which are frequently plaguedby folds. There are several factors that contribute to the formationof folds. First, differences in thermal expansion coefficients ofcopper (or nickel) foil used as a template for graphene growth inducestrain in graphene layers. Second, folds may also emerge during thetransfer process of graphene to the desired substrate and may exhibitchemical reactivity superior to flat, defect-free graphene.2 and graphene/sapphireby performing contact-angle measurements.In light of the importance of the initialstages of growth of OSson graphene on the device performance, we elected to explore the growthdynamics of pentacene at low coverage on graphene. Graphene in ourstudies was composed of a high areal density of folds. It was mostlyof a single-layer type with a sizable fraction of a multiple-layertype. Our experimental results confirm that pentacene islands canbe aligned in the direction perpendicular to the fold regions andhighly depend on the characteristics of the folds. We show here thatfolds not only attract pentacene molecules but also determine thein-plane direction of the island nucleation and subsequential growth.We also observe that pentacene growth on folds is not hindered bythe poly(methyl methacrylate) (PMMA) residues on graphene in the submonolayerregime. Finally, we examined the cause of the Volmer–Webergrowth mode of pentacene layers by extracting the surface energy components(polar and nonpolar) of graphene/SiOII2 layers thermallygrown on Si(001) substrates using a wet chemical route.12 A 300 nm-thick layer of PMMA dissolved in chlorobenzene was dropcast onto graphene at 60 °C. After Cu foil was etched in ironchloride solution, the PMMA/graphene sample was left to dry and transferredonto the SiO2 substrate at 90 °C. Prior to transfer,the SiO2 substrates were immersed for 10 min in a sequenceof baths comprising acetone, isopropanol, and deionized water. Theywere further cleaned in piranha solution for 10 min. PMMA from graphenewas cleaned, as described in ref , Veeco CP-II operating in a non-contact mode using Si tips mountedon cantilevers with a resonance frequency of 325 kHz and a springconstant of 40 N/m. The relative humidity level was kept in the rangeof 25–30% during the AFM measurements. Gwyddion software wasused to perform the statistical analysis of the pentacene islands.Commerciallyavailable CVD graphene on Cu foil (from Graphenea)was transferred onto 300 nm-thick SiOSelected samples were examined by contact-angle measurements toassess the role of the substrate on the surface energy of graphene.It was determined by measuring the contact angle of deionized water,diiodomethane, and ethylene glycol. A μm-sized liquid drop wasreleased from a microsyringe onto the substrate surface. Images ofa liquid drop were obtained using a charge-coupled device camera.The liquid contact angle was determined by measuring the angle betweenthe tangent line and the droplet–substrate interface line.III2 recorded by AFM. The scan size is 2 × 2 μm2, and the bright tones represent the higher elevations; theheight scale (black-to-white) is 20 nm. Apart from atomically smoothregions, we see a network of bright lines of different widths andheights. These features are folds in graphene. The position of suchfolds is highly influenced by the topography of the underlying Cusubstrate that is preserved after transferring to the SiO2/Si substrate.13 Folds have variation in the heightand width. The mean height of the folds is in the range between 0.4and 2.5 nm, and their width was found to vary in the range between50 and 310 nm. So the fold height ranges from a simple wrinkle (0.4nm) to a multilayer graphene (MLG) (1.3 nm). Such a variance in morphologyis expected and is widely described in previous reports.15 Kim et al.15 performed electron diffraction upon scanning an electronbeam across the folds. Inside the folds, the intensity of the diffractionspot was twice as compared to the regions outside the fold. For example,in a trilayer fold, they observed a set of three hexagonal diffractionspots, and the total intensity of the diffraction spots was threetimes larger than that of a single spot diffracted off a flat monolayer.Another set of hexagonal spots appears from the middle layer of thefold. The intensity inside the folded region is twice that of monolayergraphene, and the spot shows splitting of two distinct diffractionspots with a small angle rotation of 0.4°. A simple folded geometryis the most frequent and results in moderately higher and wider featuresunder the AFM tip. If the amount of the folded graphene exceeds acritical value, such folds collapse and form bilayer or even trilayerfolds, which appear lower than simple folds but wider. Such a variancein the geometry of the folds is exemplified in 15 The interlayer interactions in folds and strain as a resultof the bending of the fold play crucial roles in the final shape ofthe fold. The height profiles 2 and 3 represent the fold along theline profiles 2 and 3, respectively. They are taller and narroweras compared to the fold on line profile 1. Such folds have a rangeof heights varying from 1 to 2.5 nm and widths from 60 to 90 nm. Suchhigher and narrower features are intermediate “standing collapsedwrinkle”.13 Their height is largeenough, so they collapse but remain standing below a certain heightof 8 nm based on energy calculations of interlayer interactions andelastic bending.2. Pentacene was depositedat a substrate temperature of 25 °C. Pentacene forms three-dimensional(3D) islands, which are represented in the image as white irregularlyshaped features and are located near or on the folds. From this, wespeculate that the onset of island nucleation occurs at or in closeproximity of the folds. Islands grow in close proximity to the folds.These islands are of various shapes from circular to elongated rod-likestructures. Some of them extend from the top of the fold away fromit, while some of them tend to align along the fold lines. Similarresults have been confirmed with other molecules also where foldsserve as a template for the growth of organic molecules.16 Khokhar et al.17 reportedthat folds play a critical role for the growth of 4,4′-bipheneyldicarboxylicacid (BDA) on graphene grown on Ir(111). Essentially, folds representpreferential sites for the nucleation of organic molecules. The localvariations in strain on or near the folds in graphene reduce surfacemobility of the molecules as compared to defect-free regions.18 Therefore, folds effectively immobilize theincoming molecules, thereby increasing the probability for islandnucleation and finally leading to a 3D island growth.Onto the graphene surface exemplified in 11–2, and the mean island area is 0.15 μm2. On the other hand, the island density on MLG is 22.5 μm–2, and the mean island area is 0.002 μm2. This indicates that indeed there is a strong drive for the pentacenemolecules to preferentially form islands. Such a 3D island formationcorresponds to the Volmer–Weber growth mode on graphene.In addition to the folds on graphene in 2 high-resolution AFM image of pentaceneislands on smaller folds, which are ubiquitous on graphene. The depositionconditions were kept the same as in 6 and was interpreted in terms ofthe reduced influence of the interface dipole field on the surfaceenergy of graphene, thereby lowering the molecular surface mobilityon MLG. This results in smaller islands on this region of graphene.We also compared the height distribution of pentacene islands onSLG and MLG regions, as shown in 2 AFM topography map of pentacene layer grown on graphene atsubstrate temperature Ts = 60 °C.The surface coverage is 19.5%. We see that the layer morphology ischaracterized by elongated islands (bright features) aligned in closeproximity to the fold lines. The island density is 22.8 ± 1.8μm–2, while the mean island area is 0.008μm2. The mean value of the length of the longer islandaxis is 84 ± 5 nm, and the average island height is 15 ±2 nm. The surface mobility of the molecules increases at a highersubstrate temperature during growth. On the other hand, a confiningeffect of the folds is still present and appears to be dominating,causing the molecules to attach to those already immobilized nearthe folds. This leads to the formation of elongated islands. The interactionof the molecules with graphene contained in folds is so strong thatit precludes the lateral growth of the islands, thereby favoring a3D island formation. Interestingly, this effect is much less evidenton the MLG regions .29 Such high values cannot be reconciled by dispersion forces. It isalmost three times higher than that at the interface of SiO2 and graphene (0.45 J/m2).30 We consider that our samples of graphene/sapphire have a slightlyhigher interfacial energy compared to that on graphene/SiO2.31 have examined the morphology of submonolayerthick pentacene films on polymeric substrates with a lower polar contributionto the surface energy, and pentacene islands appeared more compactand exhibited a strong tendency toward the 3D growth. On stronglypolar substrates such as SiO2, pentacene nucleates in alayer-by-layer mode, forming strongly dendritic islands.33So, far we have analyzed the role of folds on the pentacenemorphology.Folds dictate the arrangements of these 3D islands on graphene. Furthermore,we can relate the 3D formation of islands to the surface energy ofthe substrate. The shape and size of islands may vary depending onthe surface energy of different substrates. Rebernik Ribic et al.34 This approach considers thatthe free surface energy can be expressed as a sum of polar and dispersivesurface energy. The polar component of the surface energy resultsfrom the electrostatic intermolecular forces between permanent dipolesand multipoles of molecules and surfaces. In contrast, the dispersiveenergy is determined by the electrostatic interaction between fluctuatinginduced dipoles and multipoles. By measuring the contact angle ofdeionized water, diiodomethane, and ethylene glycol and using thevalues for the polar and the dispersive energy of the liquids fromrefs . We can conclude the following: (1)Graphene is nonpolar and the pentacene–graphene interactionis lower on a nonpolar surface in comparison to a polar surface. Graphenetransferred onto SiO2 and sapphire has a similar but verylow polar component of surface energy. The 3D island formation isa result of the weak molecule–substrate interaction. (2) Dueto the lower surface energy of graphene on sapphire (36.77 ±6.40 mJ/m2) as compared to graphene on SiO2 (42.38± 8.00 mJ/m2), we observe smaller islands on the formersurface; as a consequence of the reduced surface energy, the surfacediffusivity of pentacene molecules is lower on graphene on sapphirethan on graphene on SiO2.We have, therefore, examined the graphene surface from thestandpointof polar contribution to the surface energy and employed the Owens–Wendtapproach.romrefs , we have2) of surface energy could be a possible cause of the 3D growthof islands. The lower surface energy of graphene/sapphire (36.77 mJ/m2) results in smaller islands as compared to those on graphene/SiO2 (42.38 mJ/m2).Our systematic AFM study of the initialstages of the growth ofpentacene on CVD graphene reveals the 3D growth mode on the foldedregions of graphene and on the MLG patches. Pentacene islands cangrow either parallel or perpendicular to the folds. At room temperature,most of the islands have no preferred orientation. But evaporatingat 60 °C, the majority of them are aligned with 90 ± 5°,i.e., horizontally in the AFM image. In addition, we observe thatthe growth mode is also not influenced by the change of the substrateafter CVD graphene was transferred to sapphire. We obtained a similar3D growth on sapphire substrates confining most of the islands onfolds. We interpret the nucleation of pentacene on folds as a resultof the reduced mobility of pentacene due to the local strain presentat the folds. Also, with contact angle measurements on graphene, alow polar contribution (3.64 ± 1.70 and 2.83 ± 2.00 mJ/m


The synthesized system is exposed to a set of structural, morphological, and optical investigations. The apparent crystallite size is less than 30 nm, reflecting the nanoscale of the structure, and thus validating the preparation route as evident on XRD pattern. SEM images show a fibrous structure where polyaniline dominates and covers most of the structure’s surface. The evident bands of the FTIR spectrum are designated to the component used in synthesis confirming the chemical structure of the fabricated system. The humidity sensing study of the synthesized structure is carried out through a wide range of relative humidity (RH) levels range of 11–97%. The response and recovery times of the fabricated structure are found to be around 120 and 220s, respectively.Humidity sensing has been offering a noticeable contribution in different industrial, medical, and agricultural activities. Here, graphene quantum dots doped with polyaniline (PANI) and MnO Optimizing the sensor performance concerning stability, detection range, sensitivity, rapid response, fast recovery elapsed durations, and cost-effectiveness is the major challenge facing the employment of humidity sensors and expanding their scope in these directions15. From this aspect, the selection of the materials and synthesis protocol involved in humidity sensors design is essential19. Numerous materials such as ceramics, metal oxide semiconductors, polymeric matrices, and carbonic substances were investigated for the use in humidity sensing devices22. The operation of nanocomposites as a humidity sensor is favorable due to their elevated surface-to-volume ratio and their capability to provide enhanced physicochemical specifications. Graphene quantum dots (GQDs), a member of the carbon nanomaterials family comprising graphene sheet layers with reduced lateral dimension have been incorporated in many composite materials. GQDs possess few desirable physicochemical properties such as noticeable bandgap  and quantum confinement, thus, supporting potential applications in energy, medical, and sensing fields25.The manipulation and monitoring of the environments surrounding humidity are demanded in various applications in recent years. Inherently, humidity sensors emerged and evolved in versatile areas such as the agricultural sector, food storage, medical devices, and others2 is a promising metal oxide owing to its chemical stability, specific capacitance, ease of acquisition, tailorable morphology, and reduced cost27. It was reported that the combination of MnO2 and graphene derivates hinders the stacking of the graphene layers due to the contribution of van der Waals forces. Moreover, MnO2 gains a better dispersion due to the presence of the graphene layers28.MnO30. PANI was previously reported as a promising candidate in sensing applications32, anti-corrosion coatings33 Photocatalysis35, optoelectronics37, and energy storage appliances42.Owing to its superior conductivity, thermal stability, and sensitivity to gases, polyaniline (PANI) became an ideal polymeric candidate43. Characteristically, the humidity sensing devices using metal oxide constrain a few undesirable specifications such as increased lag, lowered response, and prolonged recovery and response times45.A few disadvantages are limiting the wide-spread employment of PANI and opposing its functionality and application such as insolubility in some solvents and its limited mechanical stability48. The implementation of PANI and metal oxide promotes the adsorption locations accompanied by raised thermal stability and conductivity. Furthermore, the incorporation of PANI into graphene or graphene derivatives can enhance the overall conductivity, mechanical stability, functional groups, and more.Therefore, the combination of GQDs with other metal oxide materials to acquire a hybrid binary or ternary system facilitates novel tailored characteristics and expands the scope for enhanced functionality. Researchers devoted a desirable set of efforts and experimentation towards overcoming the aforementioned limitations of PANI through the implementation of organic–inorganic hybrid composites for the humidity sensing devices2 metal oxide to work as a humidity sensor was investigated in this work. To the authors’ best knowledge, this work is the first to report on this structure as a humidity sensor.Motivated by the aforementioned points, the ternary system composed of PANI as a polymeric matrix and GQDs with MnO49. In another report, authors synthesized silver nanoparticles doped with GQDs through the hydrothermal process. The report signifies the 1:1 ratio of Ag: GQDs to deliver an optimum response of 98% within a 25–95% humidity range at ambient temperature50. The following report revealed a humidity sensor formed by graphene quantum dots doped with chitosan and chemically synthesized was reported. The outcomes revealed a fast response and recovery times of 3 and 36 s, respectively, with a limited hysteresis of around 1.6%. The humidity sensing studies for PANI-containing composites was also reported51.Khouloud Jlassi et al., fabricated functionalized GQDs films from graphene waste using the spin coating method for humidity sensing applications. The synthesized films provided a low hysteresis (~ 2%) at a 30%RH level. Furthermore, the response and recovery times were found to be 15 s and 55 s, respectively1. Besides, PANI-titania nanotube-rGO was also investigated for humidity sensing within a wide humidity range of 7–97% RH. The system sensitivity of 3.28 MΩ/RH% and a rapid response of 13 s was observed18. Polyaniline-doped WO3 was prepared by in situ deposition technique as a single-step polymerization procedure. The results elucidated a linear behavior throughout a humidity range of 10 and 95% RH52. MnO2-CaO was also studied for humidity sensing and reporting an average sensitivity of ∼2.225 μW/% RH with a nominal response and recovery times of 47 and 59 s, respectively53.B. Chethan et al., synthesized a polyaniline-yttrium oxide composite prepared via the mechanical mixing method and studied the composite for humidity sensing applications. The structure delivered an optimum sensing response of 99% with a nominal response and recovery times of 3 and 4 s, respectively 2 composite was not studied. In this report, GQDs-PANI- MnO2 nanocomposite via a feasible two-pot process is successfully synthesized. The structural, optical, and morphological implication of the desired structure is explored. The humidity sensing performance is introduced through a wide span of humidity levels (11–97% RH). The proposed composite is promising for humidity sensing and related applications.Despite these efforts directed towards the humidity sensing area, and to the best of our knowledge, the introduction of a humidity sensor based on GQDs-PANI-MnO4), Ammonium persulfate ((NH4)2S2O8) (APS), Aniline (C6H5NH2), hydrochloric acid , Citric acid (99.5%), and sodium hydroxide (≥ 97%) were obtained from Fisher chemical. Toluene  was purchased from Thermo Scientific Chemicals. The above-mentioned chemicals and reagents were used as delivered without any added purification or treatments. The deionized Milli-Q water was used during this experiment.Potassium permanganate (KMnO54. Citric acid was used as a carbon source to create graphene quantum dots (GQDs). In brief, 5 g of citric acid was melted at 180 °C for 12 h, 1.5 M NaOH solution was added dropwise to the melted thick citric acid solution at ambient temperature, and then hydrothermal treated at 180°C for an additional 12 h. The resulting dark brown powder was removed from the liquor using dialysis filtration, washed, and dried overnight in a vacuum oven at 70 °C.GQDs were prepared as per the previous report2, 0.6 g of KMnO4 were added to 45 ml of DI water, next, 1.5 ml of sulphuric acid  was added under sustained magnetic stirring for an hour. To obtain the final powder, the solution was then transferred into an autoclave and kept at 120 ºC for 4 h. Afterward, the powder was purified and cleaned using water, and ethanol till acquiring the final dried MnO2.To acquire the desired MnO54.The ternary nanocomposite was prepared through two successive steps comprising the hydrothermal route and in-situ polymerization as represented in Fig. 12/GQDs nanocomposite was obtained via hydrothermal technique. 0.6 g of KMnO4 was dissolved in 45 ml of deionized Milli-Q water under continuous starring, subsequently, 1.5 ml of Conc. H2SO4 was added carefully to the dark purple solution. The solution was kept under constant stirring for an additional hour. The obtained precipitates from the solution, it was washed thoroughly and dried overnight. The PANI/MnO2/GQDs nanocomposite was executed through an in-situ polymerization reaction. Two separate solutions were prepared individually, then mixed. In the first beaker, 150 mg of MnO2/GQDs was dispersed in 45 ml of 1 M HCl aqueous solution using an ultrasonic bath for 30 min, then 1 ml of aniline was added into the suspension, it was cooled down to 5 °C and kept under constant stirring for additional 30 min. The second solution composed of 2.6 g of APS and 45 ml of 1 M HCl aqueous solution was dropped slowly to the first solution for 2 h, while the reaction temperature was kept at 5 °C. The resultant dark green precipitates were recovered, washed many times, and dried overnight. The obtained material was investigated by different characterization techniques.The MnO−6 Torr then Ar (99.99%) gas for backfilling till reaching a pressure of 6 × 10−3 Torr as a base pressure where Ar gas flow was 20 standards cubic centimeters per minute (sccm). DC head was used to sputter a thin Cr layer of 8 nm thickness at 250° C as a wetting layer, after that, RF head was used to deposit 90 nm of Au layer at room temperature. Interdigitated electrode (IDE) patterns were fabricated using XT-Laser 50 W near-Infrared 1064 nm Q-switched Ytterbium pulsed fiber laser (Raycus RFl-P50QB) coupled with high-speed galvo scan head of 8000 mm/s maximum speed. 10 pairs of fingers of 8 mm length and 500 µm width were engraved using 20% power, 250 mm scan speed, 50 kHz frequency, and pulse duration of 13ns. The collected sensing material was mixed with Polyvinylpyrrolidone PVP 2%) and a suitable amount of de-ionized water till forming a homogeneous paste. The paste was applied on the fabricated IDE using spin coating machine at 2000 rpm. Silver paste and copper cladding were used to prepare the sensor for measurement. Figure The proposed microscopic soda–lime glass substrates were cleaned using an acidic piranha solution of 98% sulfuric acid with 30% hydrogen peroxide 3:1 to remove organic contaminations, after rinsing with DI water several times, base piranha of 3:1 mixture of Ammonium hydroxide with 30% hydrogen peroxide was applied to enhance surface quality. The slides were rinsed in type 1 DI water several times. An ultrasonic bath was used for 15 min of ethanol and acetone, respectively. Finally, the substrates were submitted to Ar plasma surface treatment to promote surface characteristics and adhesion. DC/RF sputtering VTC-600-2HD system was used for metallic layer deposition. The chamber was evacuated to a base pressure of 3 × 10The fabricated sensor was allowed to dry to eliminate mixing water at 60°C for 24 h. The sensor was subjected to two different levels of humidity (7% and 97%) while applying 1 VAC for 12 h to enhance its stability. The response of the sensor was evaluated using saturated salt solution in a humidity range 11%-97%. The measured parameters included sensitivity, repeatability, and response and recovery times. the sensing mechanism was explained based on complex impedance measurements up to 5 M Hz. The schematic representation of sensor fabrication is dipected in Fig. -1 scanning range and 4 cm-1 scanning resolution. Besides, Raman analysis was acquired by the device .The X-ray diffraction (XRD) planes and the pattern were investigated using the XRD Device  within a scanning scale from 5° to 75 º, UV–Vis absorption spectra of the prepared samples were acquired using Agilent Varian Cary 5000 double-beam UV–Vis-NIR spectrophotometer in the wavelength range of 200–800 nm to study the optical properties of the prepared samples. The Fourier Transform Infrared (FTIR) was analyzed using the KBr disks method by  within a 400–4000 cm2, GQDs and MnO2/GQDs/PANI were caried out from 2θ = 5 to 75º with a scanning rate of 2º/min as demonstrated in Fig. 2 were observed. The tetragonal system of the MnO2 with a space group of I4/m was verified. The diffraction planes were observed at the angles of 12.8°, 18.07°, 28.7°, 37.55°, 41.96°, 49.9°, 56.3°, 60.2°, 65.4°, and 69.5°, corresponding to the planes of (110), (200), (310). (211), (301), (411), (600), (521), (002), and (541), respectively, with the most intense peak residing at a degree around 37 º. The results are in acceptable agreement with prior reported works56. For the composite, the three components' card numbers and peaks are included as 00–012-0720, 00–048-1206, 00–065-0826 for MnO2, Carbon component, and the PANI, respectively.XRD is an important technique that is considered as a primary tool for investigating the structure of various composite materials. The phase identification and composition of MnO57.27The corresponding card no. is PDF 01–072-1982. While implementing other constitutes of the structure, the material's sharpness and the intensity of the peaks get a bit broader and become distorted. This might be attributed to the occurring rearrangement of the structure for the atoms, resulting in a deformation of the composite structure. The nominal crystallite size of the structure is calculated based on the Scherrer equation as previously reportedArea (c), Area (A) are the crystalline and amorphous areas respectively.The dislocation density existing is determined from the inverse of the crystallite size as–4, 44 × 10–4 for MnO2 and MnO2-GQDs-PANI composite respectively.The values of crystallite size (D), crystallinity degree (XC), and dislocation density (δ) are 20, 15 nm and 75%, 36%, and 25 × 10The HRTEM of the GQDs is illustrated in Fig. 2/GQDs/PANI were examined by scanning electron microscope (SEM) at two different magnifications as shown in Fig. The surface morphological features of the MnO2 particles, thereby no evidence for the presence of these particles (MnO2). More importantly, the dimensions of GQDs are too small to be seen by this technique. the XRD can penetrate through the sample to some specific depth, hence the presence of MnO2 particles was confirmed through XRD measurements. By combining the results of both techniques (XRD and SEM), it was concluded that the MnO2 nanoparticles and GQDs are present and covered with the PANI fibers. The elemental mapping of the ternary composite is shown in Fig. The low magnification SEM image demonstrates a fibrous-like structure of PANI. The entire sample just reveals the presence of PANI without any evidence of the presence of other materials. By looking at the higher magnified SEM image, the fibers are clearly demonstrated with some other irregular ferments. The PANI could have covered the entire MnO2/GQDs/PANI prove the presence of oxygen carbon and nitrogen as a proof fro the presence of PANI and GQDs. The elemental mapping analysis proves the presence manganese as a constuent elemnt of the ternary nano composite. the distribution of the individual elements seems to homogeniuos without any agglomeration.The elemental mapping of the ternary composite MnO−1 to 400 cm−1 with a spectral resolution 4 cm-1. The FTIR spectra of MnO2, GQDs and nanocomposite (MnO2/GQDs/PANI) are depicted in Fig. −1, 1381 cm−1, 1611 cm−1, and 1738 cm−1 are ascribed to the C–O stretching vibrations, C–H bindings, C = C, and C = O, respectively. The FTIR spectrum of MnO2 reveals an absorption band at 710 cm−1 that arises from the stretching vibrations of the Mn–O. The nanocomposite (MnO2/GQDs/PANI) demonstrates the characteristics band of MnO2, GQDs and PANI. The benzene ring presents a stretching vibration at 1487 cm-1 due to the presence of C = C60.The chemical bonding and interaction between elements were verified through the FTIR measurements from 4000 cm\. The disordered band (D band at 1339 cm−1) is related to the defects that originate from the carbon atom vibrations with dangling bonds. G band at 1564 cm−1, the vibration of carbon atom in the hexagonal lattice due to the E2g phonon mode of sp2 is resulted in the graphitization band. More additional wide band at 3022cm−1 is devoted to G\ or overtone. One of the most valuable parameters that are generally used to judge the quality and the intercalation of the prepared materials is ID/IG ratio. The intercalation between GQDs and PANI can be verified by measuring the ID/IG ratio. This ratio was estimated and found to 0.96, thus indicating significant defects, that promote the sensitivity of the materials to humidity measurement61.The Raman spectrum of the composite nanostructure is presented in Fig. 2/GQDs/PANI is recorded from 200 cm−1 to 800 cm−1 as displayed in Fig. * transition due to the C = C bonds. The absorption band at about 281 nm is due to the electronic transition of the pure MnO2 nanoparticles. The electronic transition of π → polaron for PANI demonstrates a characteristic peak at about 485 nm. The most surprising phenomenon is the formation of radical cations in PANI that improves the delocalization of charges in PANI thereby enhancing the electrical conductivity of the PANI. the characteristic peak of PANI is in the range 400 -430 nm, however, the absence of this band, blue shift in the UV spectra, and the presence of a broad peak, confirm the incorporation of MnO2 and PANI conducting matrix during the in-situ polymerization process62.MnO2 adsorption–desorption isotherm of the ternary MnO2/GQDs/PANI is illustrated in Fig. 2 adsorption–desorption isotherm is belong to type III. In this case (type III isotherm), the interaction between the adsorbent-adsorbate are relatively weak, and the adsorbed molecules are accumulated all over the active sites on the surface of the studied material. The BET surface area of the ternary MnO2/GQDs/PANI was found to be 54.9 m2/g. Different parameters have been measured and calculated from N2 adsorption–desorption isotherm and tabulated in Table The NThe humidity sensing experiments were carried out using a saturated salt solution to generate a fixed relative humidity level. The saturated salt solutions were stored in a closed vessel to generate the required humidity level. The investigated sensor was inserted above the saturated salt solution in the closed vessel for a while, then the impedance/capacitance change was recorded using LCR meter at different frequencies . The sensor was evaluated in a humidity range of 11–97%, at different frequencies from 50 Hz up to 100 kHz. All experiments were conducted at room temperature. In these measurements, the impedance and capacitance variations were recorded as a function of humidity level to evaluate the humidity sensing performance of the studied structure.18. Based on the measured values for impedance and capacitance, the optimal testing frequency was found to be 200 Hz. The impedance decreases when the capacitance increases as the humidity increases. The measured curves can be divided into two regions, low humidity up to 43% and high humidity from 75% up to 97%. In the first region the impedance/capacitance variation is less than that of the second region. The impedance variation seems to be linear at high humidity level (greater than 75%). The capacitance variation is mostly having an expositional increment trend. The sensitivity can be defined as the change of measured signal per change in relative humidity level. the sensitivity can be estimated using the following Eqs. 65:The impedance/capacitance variation as a function of relative humidity is displayed in Fig. 6 Ω/RH and 5.02 × 10−11 F/RH for impedance and capacitance measurements respectively.The calculated sensitivities are 1.18 × 10The repeatability of the sensor describes the ability of the sensor to offer the same value of change when subject to two different humidity levels many times. The repeatability could be a good way to describe the stability of the sensor over a short period of time. The repeatability of the sensor was tested for three successive cycles between 11 and 43%. The repeatability of the sensors is described in Fig. To get a closer vision, response and recovery time were studied as depicted in Fig. The humidity sensing mechanism can be explained based on complex impedance spectroscopy (CIS) measurements.3O). The synthesized emeraldine base polyaniline has two forms of oxidized (−N =) and reduced (—NH—) groups. The presence of un-bonded electron pair on the nitrogen atom resulted in the protonation of above stated forms (–N = and —NH—). This can be described as follows:The sensing mechanism can be divided into two regimes. The first regime comprises low humidity up to 43%, whereas the relationship between real and imaginary parts of impedance is linear as depicted in Fig. 2+) to the protonated oxidized form (NH+). Since (NH2+) is incapable to leave an electron without earlier leaving a proton, thus the electron hopping occurs conditionally before the transformation of a proton. This type of conduction occurs in the presence of water molecules66.The conduction mechanism of the polyaniline is governed by the electron hopping from protonated reduced form (NHThe proposed sensing mechanism is provided schematically in Fig. The results of the obtained humidty sensor was comapered with thous obtained from similar structures as shown in Table 2. The validity of the preparation strategy is explored via versatile examination apparatus such as XRD, SEM, FTIR, UV–Vis, and Raman spectra. The dimensions of the crystallite size and other related structural parameters are emerged from the XRD, and SEM which verifies the nanoscale and crystallinity of the structure. The feasible and resulted implantation of the components is promoted through the bands arising in FTIR, and Raman as well as UV–Vis spectra. The structure is investigated for the humidity sensing throughout a wide scale of humidity levels up to 97 RH% which verifies the decent response and recovery times in both capacitance and impedance modes. The delivered structure might pursue a desirable set of specifications directed towards humidity sensing for the upcoming years in various areas.In the studied work, ternary composite of polymeric matrix as polyaniline combined with graphene quantum dots and MnO


Large language models (LLMs) have demonstrated significant potential in diverse domains, including medicine. Nonetheless, there is a scarcity of studies examining their performance in medical examinations, especially those conducted in languages other than English, and in direct comparison with medical students. Analyzing the performance of LLMs in state medical examinations can provide insights into their capabilities and limitations and evaluate their potential role in medical education and examination preparation. This study aimed to assess and compare the performance of 3 LLMs, GPT-4, Bing, and GPT-3.5-Turbo, in the German Medical State Examinations of 2022 and to evaluate their performance relative to that of medical students. t tests for pairwise comparisons. The relative strength of the LLMs in comparison with that of the students was also evaluated. The LLMs were assessed on a total of 630 questions from the spring and fall German Medical State Examinations of 2022. The performance was evaluated with and without media-related questions. Statistical analyses included 1-way ANOVA and independent samples GPT-4 achieved the highest overall performance, correctly answering 88.1% of questions, closely followed by Bing (86.0%) and GPT-3.5-Turbo (65.7%). The students had an average correct answer rate of 74.6%. Both GPT-4 and Bing significantly outperformed the students in both examinations. When media questions were excluded, Bing achieved the highest performance of 90.7%, closely followed by GPT-4 (90.4%), while GPT-3.5-Turbo lagged (68.2%). There was a significant decline in the performance of GPT-4 and Bing in the fall 2022 examination, which was attributed to a higher proportion of media-related questions and a potential increase in question difficulty. LLMs, particularly GPT-4 and Bing, demonstrate potential as valuable tools in medical education and for pretesting examination questions. Their high performance, even relative to that of medical students, indicates promising avenues for further development and integration into the educational and clinical landscape.  The minimum duration of study for the medical degree in Europe is 6 years. In Germany, state examinations take place after the second, fifth, and sixth years. The first and second examinations are multiple-choice tests . In the Today, promising applications of large language models (LLMs), particularly ChatGPT, are already being observed in education and research, with the potential to trigger a paradigm shift in health care . New, hiChatGPT is a web-based platform using advanced AI-driven LLMs such as GPT-3.5-Turbo and GPT-4 developed by OpenAI . HoweverCompared to GPT-3.5-Turbo, its successor GPT-4 can understand more nuanced instructions and questions and is expected to provide false information less frequently. Moreover, it is more likely to refuse queries that could result in harmful responses . GPT-3.5Bing is an AI chatbot developed by Microsoft and has been unveiled in 2023. In the development process, technology from GPT-4 was used to enhance its accuracy and performance capabilities ,13. In cOverall, ChatGPT and Bing represent a significant advancement in the field of AI and natural language processing. The ability to generate human-like responses and adapt to new information make them valuable tools for a wide range of applications, including content creation, customer service, and research.This study critically examines the capabilities of ChatGPT and Bing in medical education, by addressing how Bing, GPT-4, and GPT-3.5-Turbo perform in answering multiple-choice questions on the German Medical State Examination of 2022. Specifically, we assessed the number of correct answers of these LLMs in German, which is not their main training language, and compared it to the performance of medical students to evaluate their usefulness in the medical field.precise settings [We conducted a retrospective analysis of the spring and fall 2022 German Medical State Examinations. A total of 630 out of 640 multiple-choice questions in German (including questions containing media) were analyzed. Questions that were excluded post hoc for factual incorrectness were excluded from further analysis . The questions were taken from the learning platform Amboss . We usedsettings . No datasettings .We asked examination questions based on a German translation of the scheme used in previous studies evaluating OpenAI’s models : “The fot tests were carried out with python statistical library scipy (version 1.7.3) to assess statistical differences between means.We used a MacBook M1 pro 14-inch 2021 device with macOS Ventura (version 13.4), with Python (version 3.8.11) installed, and the data analysis libraries numpy (version 1.21.6) and pandas (version 1.4.3). For visualization, we used matplotlib (version 3.5.2) and seaborn (version 0.11.2). One-way ANOVA and independent samples For analysis without media content , 38 out of 311 (12.2%) questions were excluded for the spring examination and 22 out of 319 (6.9%) questions for the fall examination. The models’ strength was calculated as a relative proportion of the number of correct answers provided by students; a value above 100% means that the model was outperforming the average student, whereas a value under 100% shows below average student performance.When writing this paper, the authors used Grammarly  and GPT-4 to improve the language of the manuscript and correct grammatical errors. After using these tools, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.We compared the performance of 3 LLMs, GPT-4, GPT-3.5-Turbo, and Bing, in the German Medical State Examinations of 2022, both in spring and fall, and with and without questions containing media. The performance of the models was also compared to that of the students who participated in the examinations.GPT-4 had the highest overall performance with 555 correct answers out of 630 (88.1%) questions. Bing followed closely with 542 out of 630 (86.0%) correct answers. GPT-3.5-Turbo lagged with 414 out of 630 (65.7%) correct answers. The students performed in between .F2=64.1, P<.001). Independent samples t tests were conducted to make pairwise comparisons between the models. There was no significant difference in performance between Bing and GPT-4 . However, both Bing  and GPT-4  performed significantly better than GPT-3.5-Turbo.One-way ANOVA revealed significant differences among the 3 models . However, both GPT-4  and Bing  significantly outperformed the students. In the fall 2022 examination, GPT-3.5-Turbo’s performance was significantly lower than that of the students . In contrast, both GPT-4  and Bing  again significantly outperformed the students . Further investigation into these differences was conducted using independent samples t tests for pairwise comparisons between the models. There was no significant difference in performance between Bing and GPT-4 . However, both Bing  and GPT-4  significantly outperformed GPT-3.5-Turbo. Since there are no officially published data on students' performance on individual questions, we were unable to compare the models’ performance on nonmedia questions with that of the students.Next, the 40 questions containing media content were excluded, leaving a total of 590 questions for analysis. Bing achieved the highest performance with 517 correct answers out of 590 (90.7%). GPT-4 was slightly behind, with 515 out of 590 (90.4%) correct answers. GPT-3.5-Turbo, on the other hand, answered 389 out of 590 (68.2%) questions correctly. One-way ANOVA revealed that there were significant differences among the 3 models  questions correctly, Bing answered 254 out of 311 (81.7%) questions correctly, and GPT-3.5-Turbo answered 195 out of 311 (62.7%) questions correctly. When questions containing media were excluded, the performance of all models increased, with Bing having answered 239 out of 273 (87.5%) questions correctly, GPT-4 also having answered 239 out of 273 (87.5%) questions correctly, and GPT-3.5-Turbo having answered 181 out of 273 (66.3%) questions correctly. In comparison, the students answered a mean of 230.7 (SD 22.6) questions correctly out of 311 (74.2%).When evaluating the performance of the models relative to that of the students for the spring 2022 examination, GPT-4 displayed a relative strength of 116.6% when including questions with media and 118.0% when including questions without media, whereas Bing demonstrated strengths of 115.4% and 118.7%, respectively. Slightly worse performance, but still exceeding that of the students, was observed for the fall 2022 examination. The relative performance of GPT-3.5-Turbo for both examinations, compared to that of the students, with and without questions containing media, ranged between 88.5% for the fall 2022 examination including questions with media to 95.1% for the spring 2022 examination with questions without media.P=.007, t628=2.71). Furthermore, there were significant differences in Bing’s performance . When questions with media were excluded, the differences remained significant, with GPT-4 showing +37 (+5.4%) correct answers  between the spring and fall 2022 examinations. Bing had +39 (+6.1%) correct answers  between the spring and fall 2022 examinations  or excluded . The difference in the number of correct answers for GPT-3.5-Turbo was +24 (+6.0%) with and +27 (+5.7%) without questions containing media; however, these differences were not significant.In contrast, for GPT-3.5-Turbo, there was no significant difference in performance between the spring and fall 2022 examinations, irrespective of whether questions with media were included  questions, and Bing correctly answered 288 out of 319 (90.3%) questions, whereas on average, students correctly answered 239 out of 319 (74.9%) questions. Thus, both models showed outstanding results. A comparison with the highest scoring students revealed that only 0.5% of participants achieved a score between 291 and 300 . Even thIn comparison, both Bing and GPT-4 showed a significant decline in performance in the fall 2022 examination. However, both LLMs were still able to significantly outperform the students and ChatGPT-3.5-Turbo. The performance of GPT-3.5-Turbo and that of the students did not differ significantly between the 2 examinations. Overall, there was a significant difference in performance between Bing and the students in both the spring and fall 2022 examinations, as well as between GPT-4 and the students in both examinations. While GPT-3.5-Turbo was not significantly worse than the students in the spring 2022 examination, a significant difference was observed in the fall 2022 examination. Therefore, not every model appears to be equally suitable for correctly answering medical examination questions. However, a noticeable improvement has been noted with the further developed models. To what extent this can be further improved in the future should be investigated through further comparisons of different models.An explanation for the poorer performance is the higher proportion of media-related questions in the fall 2022 examination, as there is no image recognition in the current version of the LLMs. This feature is already announced for GPT-4 and needs to be considered in future analyses. The consistent performance of the students and GPT-3.5-Turbo also suggests that there may have been more questions with a higher degree of difficulty in the fall 2022 examination; this also aligns with a current negative trend in the results of the state examinations, with a large variation among individual examinations [Another prominent point in the fall 2022 examination compared to the spring 2022 examination is the relatively high proportion of questions that were subsequently excluded. In the spring 2022 examination, only 1 question was excluded post hoc, whereas 9 were in the fall 2022 examination. Since only those questions that are factually incorrect or whose answer options are contentious were excluded, a high proportion of such questions can lead to uncertainty among students regarding their own decision-making process. Given the outstanding performance of GPT-4 and Bing, it might be worth considering using these LLMs to pretest questions for future examinations to reduce the number of contentious questions. This could be a relevant aspect for further examinations. Bing seems to be a qualified medium, as it can also provide sources for the given answer. The issue remains that incorrect answers from these programs are difficult to detect, especially as the examiner is presented with a seemingly correct solution with explanations. This should be tested in further investigations.In addition to the actual testing of examinations using the LLMs, the aspect of preparation for these is a crucial point. Besides traditional textbooks, LLMs appear to provide a valuable supplement to conventional learning by elucidating medical issues, offering students the opportunity to obtain rapid solutions for specific medical questions. This also plays a significant role in preparation for examinations. LLMs could be used to quickly inquire about specific medical queries, hence simplifying learning. For instance, extensive research could be made easier by Bing's citation of sources. Future research should investigate how image recognition functions in a medical context to provide, for example, support in radiographic diagnostics or dermatologic findings. ChatGPT is already being increasingly used in the field of radiology, where it can aid in education and assist in making clinical decisions . EspeciaCertainly, programs using LLMs such as OpenAI’s ChatGPT , MicrosoIn this study, the evaluation was limited to OpenAI's models and focused on single prompts and answers from just 1 year’s German Medical State Examinations. The inability of the models to process media content and the lack of diversity in examination content and languages confines the scope of insights. Additionally, the rapid evolution of LLMs means that the results may quickly become outdated. Moreover, there are implications regarding the accuracy of the LLMs’ outputs and the level of trust that should be placed in them, particularly in the context of medical education. The study did not investigate the potential for misinformation or inaccuracy in the responses generated by the LLMs, which is critical given that medical students might rely on these tools for preparation for examinations. Lastly, potential biases or errors intrinsic to the models were not explored. These constraints warrant measured interpretation of the results and indicate the need for more extensive and varied studies, as well as a critical analysis of the reliability of the LLMs in a medical education setting. Being the fastest growing web platform ever , LLMs wi


The reports on bone mineral loss or major osteoporosis fracture (MOF) in sarcoidosis are scarce and have conflicting outcomes. This study aimed to evaluate the prevalence and risk factors of MOF in sarcoidosis patients.In a single-center cohort of 382 patients with sarcoidosis (55.8 ± 11.6 years) we evaluated bone mineral density at lumbar spine, at femoral neck and at total hip and the presence of MOF. Lung function measurements including diffusion capacity for carbon monoxide (DLCO) were assessed. Chest X-rays were performed and radiological staging was done by Scadding score.T-scores were lower in sarcoidosis with MOF with respect to those without MOF, but the difference was statistically significant only for BMD at femoral neck (p < 0.05). Moreover, BMD values at total hip was positively correlated with DLCO (%) (p < 0.001). Prevalence of MOF was higher in patients with sarcoidosis with lung parenchymal involvement  than in patients with sarcoidosis in chest X-ray stages 0 and 1 . Moreover, multiple regression analyses showed that X-ray Scadding score was positively associated with MOF.Ninety patients (23.6%) with sarcoidosis have history of a MOF. BMD This study shows that MOF represent a common and important complication in patients with moderate/severe sarcoidosis. The chest X-ray evaluation and the pulmonary function test could allow to define the risk of MOF in sarcoidosis patients. Osteoporosis is defined as a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture .It is associated with an excess of mortality, a decrease in quality of life, and co morbidities; and has a high social and economic burden, representing a major public health problem. Osteoporosis affects mostly postmenopausal women and fewer men. It is mainly related to the normal aging process—primary osteoporosis. Nevertheless, it can also occur due to secondary causes such as bone-affecting treatments, clinical disorders, or lifestyle habits-secondary osteoporosis. Nine million osteoporotic fractures occur worldwide yearly . The lasSarcoidosis is a systemic inflammatory disorder characterized by the formation of granulomas, which are clusters of inflammatory cells, typically in the lungs, lymph nodes, and other organs .Several factors contribute to the increased risk of osteoporosis in sarcoidosis patients, including the chronic inflammation associated with the disease and the use of corticosteroids as a treatment. In fact, the chronic inflammation seen in sarcoidosis can disrupt the normal bone remodeling process. Inflammation leads to an imbalance between bone formation and resorption, resulting in increased bone loss and reduced bone quality –7. CytokAmong MOF in sarcoidosis patients vertebral fractures are the most serious. In fact, vertebral fractures can have various effects on lung diseases depending on the specific circumstances and the severity of the fracture. In particular, the vertebral fractures can cause pain and restrict the movement of the chest wall. This can lead to shallow breathing and decreased lung expansion, resulting in reduced lung function. Moreover, lung diseases such as chronic obstructive pulmonary disease (COPD) or pulmonary fibrosis such as sarcoidosis, can make individuals more susceptible to lung infections . FracturTo identify the prevalence of major osteoporotic fractures in patients with sarcoidosis.To assess risk factors for major osteoporotic fractures.The aim of this single-center cross-sectional study was twofold:We studied a cohort of 382 subjects (mean age 55.8 ± 11.6 years) affected by sarcoidosis, referred to the Regional Referral Center for Sarcoidosis and other Interstitial Lung Diseases at Siena University Hospital  from January 2018 to December 2022. Diagnosis of sarcoidosis was performed through a multidisciplinary evaluation according to the diagnostic criteria of the ATS/ERS/JRS/ALAT guidelines , 13. AllT value lower than − 2.5 was diagnosed as osteoporosis, and a T value between − 1.0 but higher than − 2.5 was diagnosed as low bone density. Sex-matched Italian reference data were used for the calculation of T-score [In all subjects we measured BMD at the lumbar spine [LS-BMD] and at femoral subregions  using a dual-energy X-ray absorptiometry device . Osteoporosis and osteopenia were diagnosed according to the World Health Organization (WHO) definition: a  T-score .The information on history of previous fracture was collected during osteoporosis visit. All surveys included details of fracture location, including spine, hip, wrist, clavicle, upper arm/shoulder, rib, pelvis, ankle, upper leg and lower leg. Besides, the occurrence of fractures was assessed by way chart review in the Carestream database, by reviewing progress notes, and by radiological evidence of vertebral fracture. Magnetic Resonance Imaging (MRI), CT with 18F-fluorodeoxyglucose (FDG PET-CT), chest high resolution computed tomography (HRCT) and Computed Tomography (CT) reports were also reviewed. In particular, all radiological images were examined for the presence of any vertebral fracture according to Genant’s method’s . Namely,In all subjects blood samples were collected in the morning under fasting conditions to evaluate serum levels of calcium (Ca), phosphate (P), creatinine (Cr), alkaline phosphatase (ALP), 25-hydroxyvitam D (25OHD), Serum1,25-hydroxyvitam2D  and intact parathyroid hormone (PTH). At the same time points 24-h urine samples were collected for the evaluation of calcium, phosphate and creatinine. Serum 25OHD was determined by a radioimmunometric method . In our Institution the intra- and inter-assay coefficients of variation for 25OHD were 6.8 and 9.2%, respectively. Serum1,25(OH)2D was assessed by chemiluminescence immunoassay . In our institution the intra- and inter-assay coefficients of variation were 4.1 and 5.3%, respectively. Serum PTH was assessed by an immunoradiometric assay . The results were expressed in picograms per millilitre, and the intra and inter-assay coefficients of variation were 3.6 and 4.9%, respectively. Urinary calcium, phosphate and creatinine were determined by a colorimetric method  in 24-h urine samples.All participants underwent pulmonary function tests performed according to the American Thoracic Society/European Respiratory Society (ATS/ERS) standards , 18, usiAll subjects with sarcoidosis performed a chest X-ray with radiological staging according to Scadding criteria . The radt-test. Categorical variables were compared by Chi-square test or Fisher’s exact test, as appropriate. The correlations between the groups were analyzed with the Pearson’s correlation test. Multiple linear regression models were used to assess the association of independent predictors such as sex, BMI, creatinine, vitamin D, FVC (%), FEV1 (%), DLCO (%), COS classification, Radiological stages, therapy for sarcoidosis, prednisone dosage to the presence of osteoporosis and MOF. All tests were two-sided, and p < 0.05 was considered statistically significant. All statistical tests were performed using SPSS 10.1 statistical software (SPSS 10.1).The Kolmogorov–Smirnov test was used to verify the normality of the distribution of the outcome variables. All the variables were normally distributed and were expressed as mean ± SD. The significance between the means was tested using Student’s p < 0.05). As expected patients with sarcoidosis and MOF were older than those without MOF.The clinical and biochemical characteristics of sarcoidosis patients with or without MOF are shown in Table N = 81), femur (N = 4), wrist (N = 7) and humerus (N = 5). Moreover, 52 patients had history of more than one MOF. The prevalence of vertebral fractures in patients with sarcoidosis, by spinal location, is shown in Fig. Ninety patients (23.6%) with sarcoidosis have presented a major osteoporosis fracture. In particular, the MOF reported involved vertebrae N = 8, femur  and BMD-TH (p < 0.01).Figure p < 0.001). Also BMD-LS and BMD-FN were significantly correlated with DLCO% (r = 0.20 p < 0.001 and r = 0.18 p < 0.001 respectively). Instead, no significant associations between BMD values with FVC (%) and FEV1 (%) were observed.Figure p < 0.05).Figure In multiple regression analysis, statistically significant associations were found for predictors of osteoporosis (model 1) and of MOF model 2). In particular, multiple regression analyses showed that BMI was positively associated with osteoporosis, while DLCO (%) was negatively associated  had a MOF. According to a classification based on the frequency with which they occur, MOF consider a grouping of the most common fractures which include hip, clinical vertebral, distal forearm and proximal humerus fractures , 24. MorAn important finding of this study is that approximately a quarter of sarcoidosis patients had a MOF and over a fifth of them had at least one vertebral fracture. Similarly, in the Maastricht study at baseline 20% of patients with sarcoidosis had vertebral fractures while non-vertebral fractures were rare . MoreoveThe severity of sarcoidosis has been suggested as a potential risk factor for the development of osteoporosis and major fragility fractures (MOF). In fact, we have found that patients with more severe sarcoidosis, as indicated by lung involvement, multiorgan involvement, or higher disease activity, may be at a higher risk of developing osteoporosis and experiencing MOF. However, it is important to note that the association between sarcoidosis severity and osteoporosis/MOF is not fully understood and requires further investigation. Other factors, such as age, gender, menopausal status (in women), vitamin D deficiency, and physical inactivity, can also contribute to the development of osteoporosis in sarcoidosis patients .Another important finding in this study is represented by the close correlation between the worsening of the respiratory decalages, expressed by the DLCO%, and the reduction of femoral BMD; moreover, the multiple linear regression model confirms that the DLCO% is able to predict the risk of osteoporosis. Multiple regression analysis also reported that BMI shows a protective effect against osteoporosis. This finding seems to be in agreement with a previous study by Nuti et al. witch reported that an association between the reduction in BMI and the risk of vertebral fracture in COPD patients . MoreoveMoreover, the extent of chest radiology involvement evaluated according by Scadding’s criteria is able to predict the risk of MOF in sarcoidosis patients independently of BMD. The better association of femoral BMD with disease severity and MOF with respect to lumbar BMD may be explained by the increased occurrence of spondyloarthritis in sarcoidosis patients .Our study has some limitations. Firstly, the cross-sectional nature of the study does not allow the establishing of any causality relationships between the parameters. Secondly, the lack of data about markers of inflammation. Nevertheless, this study presents several strengths: firstly, the large study sample of a rare condition such as sarcoidosis disease; secondly, it was a single-centre study and this may ensure its internal validity; thirdly, has confirmed that the severity of pulmonary involvement is related to the risk of osteoporosis and fragility vertebral fractures.Our data obtained from a large cohort of sarcoidosis patients confirm that MOF and particularly vertebral fracture represent a common and important complication in patients with moderate/severe sarcoidosis. In these patients, the radiological evaluation according to the Scadding’s criteria and the measurements of DLCO could allow to define the risk of MOF and to implement adequate therapeutics strategies.


Capacity distribution is a challenging issue for an airline under financial budget and resource consideration. It is a large-scale optimization problem covering both long-term planning and short-term operating arrangements. This study investigates on the airline capacity distribution problem with financial budget and resource consideration. It contains subproblems of financial budget arrangement, fleet introduction, and fleet assignment. Among them, financial budget is arranged in multiple decision periods, fleet introduction is decided under fixed time points, while fleet assignment is decided under all available time points. To tackle this problem, an integer programming model is formulated for descriptions. Then, an integrated algorithm of modified Variable Neighborhood Search (VNS) and Branch-and-bound (B&B) strategy is developed to find solutions. In detail, a greedy heuristic approach is utilized to generate an initial solution for fleet introduction, the modified B&B strategy is utilized to generate the optimal solution for fleet assignment and the modified VNS is applied to update current solution for a new one with better quality. In addition, budget limit checks are added for financial budget arrangements. Finally, the hybrid algorithm is tested on efficiency and stability. It is also compared to other algorithms which replace the modified VNS by basic VNS, differential evolution and genetic algorithm. Computational results show that performance of our approach is powerful in terms of objective value, convergence speed and stability. Airline capacity distribution problem (ACDP) is a combination of subproblems which contain financial budget distribution, fleet introduction, and fleet assignment. Airlines usually deal with this problem in batches  are applied. Rezgui et al.  apply VNAccording to studies above, VNS is an efficient framework to deal with multi-stage optimization problems which are similar to ACDP. In addition, to improve solution quality, exact method can be incorporated into VNS framework. As a result, in this study, ACDP under financial budget and resource consideration is presented and a hybrid algorithm of VNS and an exact method is proposed to maximize profits. The main contributions are as follows: (1) ACDP under financial budget and resource consideration is described mathematically as an integer programming model; (2) a greedy heuristic approach is designed to improve quality of initial solution; (3) VNS encoding process is utilized to integrate budget distribution and fleet introduction for dimension reduction; (4) a shaking strategy and special neighborhood structure are applied to generate efficient perturbation solutions; (5) an exact method is applied to improve solution quality; (6) the hybrid algorithm is tested on efficiency and stability and compared to other algorithms.The remaining chapters of this study are arranged below. Section In this study, ACDP under financial budget and resource consideration is studied. It is an integration problem involving three stages: budget distribution, fleet introduction, and fleet assignment. The notations used throughout this paper are given in Table During the first stage, a long planning term is divided as a set of Additionally, a proportion upper limitation mentclass2pt{minimFor example, an airline’s fleet prices, flying missions, flight demands and budget limits are defined in Tables 8. Total planning term is 10 years and divided into 5 periods with 2 years in the same period. Then, the budget can be distributed as 4 × 108, 2 × 108,0, 1 × 108 and 0 from H1–H5, not exceeding the proportion limit in each period of 50%, 30%, 20%, 20% and 20%. Then, the airline decides on fleet introduction. There are three decisions containing buying one 787 in H1, one 757 in H2 and leasing one 737 in H4. These decisions are shown in a square box, a circle and a triangle respectively in Table According to the example above, total financial budget is set as 8 × 10After fleet numbers are determined, an airline assigns available fleets to flying missions in all planning years. Results are shown in Table  How many budgets to be distributed in each planning period, Which time point is chosen to buy or lease an aircraft, Which type of aircraft is chosen, How many aircrafts is bought or leased, How to match flying missions with suitable available aircrafts.To summarize, for our ACDP study, a combination of subproblems containing budget distribution, fleet introduction, and fleet assignment, the following decisions should be made:Among the decisions, (1) belongs to budget distribution, (2–4) belong to fleet introduction and (5) belongs to fleet assignment. In the next sections, this problem is formulated as an integer programming model and then valuable solutions are searched by a designed hybrid algorithm.According to descriptions and assumptions above, an integer programming model for ACDP is constructed. As mentioned before, there are mainly three parts of ACDP containing financial budget distribution, fleet introduction, and fleet assignment. The fleet introduction part refers to formulation of Bazargan and Hartman . The fleEquation  illustraEquation  promisesThis section introduces the proposed hybrid algorithm of VNS and B&B in detail. Firstly, algorithm design motivations and framework are illustrated. Secondly, with regards to problem-specific characteristics, algorithm components, involving initial solution generation, encoding, decoding, modified B&B strategy and VND process, are presented respectively. Finally, the whole procedure of the hybrid algorithm for addressing ACDP is summarized.In ACDP, there exist mainly three subproblems containing budget distribution, fleet introduction, and fleet assignment. They are considered simultaneously. Since these subproblems are dependent, the whole problem has NP-hard property and its various variables cannot be dealt efficiently with exact methods in limited time. As a result, we adopt a VNS metaheuristic framework to solve this problem. In addition, as one of the subproblems, fleet assignment, can be handled with B&B  and (4);Buy-down-lease-up: combination of (2) and (3);Fleet-Change: change one fleet type to another randomly;Buy-Lease-Swap: swaps values of bought and leased fleets in one period;Yearly swap: choose two years randomly, swap all the fleet numbers.There are nine neighborhood structures to deal with encoding results involving: Add-Buy, Minus-Buy, Add-Lease, Minus-Lease, Add-Buy-Minus-Lease, Add-Lease-Minus-Buy, Fleet-Change, Buy-Lease-Swap, and Period-Swap. They are indexed in Fig. For these nine structures, (1–4) only change one value in the encoded results, (5–7) change two values, (8) changes six values, while (9) involves two periods and change 18 numbers totally. From a multi-layer view, (1–6) and (8) are concerned with buy-or-lease choices, (7) is related to fleet type decisions, while (9) represents time period choices.Once a neighborhood of fleet introduction is generated in Fig. Our budget limit checks are incorporated into neighborhood generation process in Sect. After comparison, if the fleet introduction cost of the new code exceeds relative period budgets or total budget, the code is invalid and should be deleted. The previous code is output for VND process.Algorithms of period and total budget checks are illustrated in Fig. This section intends to systematically evaluate our integer programming model and modified VNS-B&B hybrid algorithm. It is compared with three metaheuristics containing basic VNS, differential evolution (DE), and genetic algorithm (GA). They only replace the modified VNS part and incorporate with B&B. Three different sets of benchmarks are set and collected to compare their performance involving objective value, running speed and robustness. All experiments of the modified VNS-B&B hybrid algorithm and compared metaheuristics are coded in C++. They are run on a windows 10 system with a 1.10 GHz Intel Core i7-10710U CPU processor and a 16G RAM.To solve ACDP problem, we set four instances scaled with 3, 10, 20, and 50 routes, which covers airlines of small and medium sizes. For each instance, there are four sets of input data involving budget, fleet, route, and flying mission information shown in Fig. Data sources are open websites and reports. Because most of airlines keep data privacy, direct and complete company-specific data is difficult to obtain. As a result, open resources are referred for numerical settings. To be specific, total budget volume, period limit numbers, fleet capacity and fleet prices are estimated based on open reports on Statista Erick , b. OnlyTo test our modified VNS-B&B algorithm, the modified VNS part is tested and then replaced by other efficient metaheuristics involving DE, GA, and basic VNS. There are various parameters to be predefined before the experiments described in Table For all experiments, ACDP is planned in the future 10 years and they are divided into 5 periods with 2 years in each period. Assuming there are 365 days in each year, flying missions are repeated in each day, once a mission is assigned a fleet type, it would repeat in each day. Then, yearly operating profits can be calculated as daily profits multiplied by 365 days, which is a widely used simplification (Kenan et al. For our modified VNS, number of VND neighborhoods u et al. . To imprg et al. , populatm et al. , populatThis subsection intends to evaluate the performance of modified VNS-B&B algorithm and compare it with DE-B&B, GA-B&B and basic VNS-B&B algorithms. The first phases of the four hybrid algorithms deal with two subproblems of ACDP containing budget distribution and fleet introduction with metaheuristics. The second phases apply modified B&B strategy to solve fleet assignment, another subproblem of ACDP. The four algorithms are compared on running speeds, objective values and robustness. For robustness, an evaluator named Relative Percent Deviation (mentclasspt{minimaExperiments of the four instances in Sect. In terms of objective value, the modified VNS-B&B is dominant among all the four algorithms. Because the objective in Eq.  is to maIn terms of running speed, the modified VNS-B&B also has advantage over the other three algorithms. The running speed is measured as the running time in second, which starts from the beginning of our algorithms and ends after the final iteration is finished. A smaller value of seconds means a better running speed of an algorithm. In all cases, the modified VNS-B&B algorithm obtains the smallest values of running time with the numbers of 4.79 s, 18.60 s, 49.78 s and 265.82 s. To compare it with other algorithms, our modified VNS-B&B is approximately 1/3 faster than basic VNS-B&B and 1/2 faster than GA/DE-B&B. For example, in case 1, the modified VNS-B&B has a running time of 4.79 s while the basic VNS-B&B has a running time of 6.46 s, which means that the former is 25.81% faster than the latter. The proportion for case 2 is 33.62% and similar in other cases.Comprehensively, although there are adjacent objective values compared to those of modified VNS-B&B, their speeds are considerably slower than those of modified VNS-B&B. For example, the objective value of GA-B&B in case 1 with the number of 5.78E + 08 has the smallest deviation to that of the modified VNS-B&B, while the running time of the former with the number of 4.79 s is considerably slower than that of the latter with the number of 8.25 s. For the objective value of DE-B&B in case 4 with the number of 6.94E + 09, the situation is similar.In terms of robustness, as mentioned before, RPDs are utilized for evaluations. In Table In detail, in all cases and algorithms, iterative processes are tracked and recorded as convergence curves shown in Fig. Furtherly, to test out whether the robustness of modified VNS-B&B algorithm is sensitive to input disturbance, we make changes on input numbers of total budget and passenger demands, which are likely to fluctuate under volatile market conditions. To be specific, total budget and passenger demands are assumed to be 10% more or less than before while the other input numbers remain the same. Then, two more groups of experiments are conduced besides those shown in Table In Fig. To summarize, compared to DE-B&B, GA-B&B, and basic VNS-B&B hybrid algorithms, the modified VNS-B&B algorithm has advantages on objective value and running speed. In terms of robustness, the modified VNS-B&B algorithm performs good and only has slight deviations in limited conditions.This paper investigates on the ACDP problem with resource and budget consideration. It is an integration of subproblems containing budget distribution, fleet introduction, and fleet assignment. An integer programming model is formulated to describe the problem and a hybrid algorithm of modified VNS and B&B strategy is designed to find solutions. The hybrid algorithm consists of seven components containing initial solution generation, encoding, decoding, modified B&B, VND and shaking process processes. Moreover, budget limit checks are added once a new neighborhood is generated to avoid invalid solutions and improve search efficiency. The modified VNS-B&B algorithm is compared with the DE/GA/basic VNS-B&B algorithms on objective values, running speeds, and robustness. Experimental results show that the efficiency and stability of the modified VNS-B&B algorithm take advantage over the other compared algorithms.Our study contributes to reduce dimensions of the complex integrated ACDP problem and provide practical advice for airline planning decisions under budget and resource limits. In the future, relative research can focus on incorporate more limited considerations into ACDP problem and solve it with a more efficient method in larger cases.