Idiopathic pulmonary fibrosis (IPF) and sarcoidosis are two distinct clinical entities with different aetiology, epidemiology, risk factors, symptoms and chest imaging. A number of papers have reported an overlap of the two diseases and have suggested the existence of a distinct phenotype defined as combined sarcoidosis and idiopathic pulmonary fibrosis (CSIPF). We used the scoping review protocol to review the literature on CSIPF. We also enrolled a cohort of nine CSIPF patients and compared them with lone-IPF and fibrotic sarcoidosis patients. Our CSIPF cohort showed male prevalence and only ex-smokers. Functional assessment at baseline showed mild to moderate restrictive impairment of lung volumes in lone-IPF and CSIPF patients, associated with moderate-to-severe reduction in DLco percentages. Although all CSIPF patients were on antifibrotic treatments, functional impairment occurred in the two years of follow up. This suggests the importance of considering these patients at high risk of rapid deterioration and lung damage. Interstitial lung diseases are a heterogenous group of more than 200 lung disorders characterized by inflamed interstitium and fibrosis of the parenchyma leading to thickening of the alveolar walls. All are associated with substantial morbidity and mortality ,2,3,4. IIdiopathic pulmonary fibrosis (IPF) and sarcoidosis are two distinct clinical entities. The former is a chronic fibrosing lung disease that is diagnosed in the absence of any identifiable underlying pathological/clinical cause. It typically affects adult males with unexplained chronic dyspnoea but no other symptoms ,7,8. OldSarcoidosis is a systemic granulomatosis of unknown aetiology that mainly occurs in females in the 20–40 years old age range. It is reported to have a lower incidence among smokers, as well as seasonal and geographical variations ,16,17,18combined sarcoidosis and idiopathic pulmonary fibrosis (CSIPF).Idiopathic pulmonary fibrosis and sarcoidosis are very different diseases in terms of aetiology, epidemiology, risk factors, symptoms and chest imaging. It is not clear whether UIP is the common final pathway of advanced pulmonary fibrosis, or whether CSIPF patients have an overlap of the two conditions. This distinction may have important implications for prognosis and therapy. Recent papers reporting an overlap of sarcoidosis and IPF suggest the existence of a distinct disease which has been called The aim of the present study was to systematically review the literature on CSIPF patients. A secondary aim was to analyse and compare Italian cohorts of CSIPF, lone-IPF and fibrotic sarcoidosis patients in order to highlight possible differences and any relationship between these disorders.We used the scoping review protocol  and descThe inclusion criteria were peer-reviewed, empirical or prospective papers : (a) with pertinence to the study topic; (b) in English; (c) preferably in journals related to pneumology with full text or abstract; (d) in the form of review, case report, case series, original article or letter to the editor. Studies were excluded if they met any of the following criteria: (a) they were not pertinent to the topic of the study; (b) they were written in a language different from English; (c) the full text of abstracts was not (yet) available.A systematic search of the literature was conducted in the PubMed online database. The terms entered in our Boolean search syntax were: “combined sarcoidosis and idiopathic pulmonary fibrosis”, “CSIPF”, “sarcoidosis and pulmonary fibrosis”, and “sarcoidosis and usual interstitial pneumonia”. We did not search the grey literature .Three independent reviewers  screened the abstracts and titles, and ascertained availability of the full texts. If the papers met the eligibility criteria, they were considered. Any disagreement of the reviewers was resolved by consensus.One author (L.B.) extracted formal data items, including publication type, sources, geographies, objectives and main findings; a random of 5% of items were verified by another author (G.N.). Three independent reviewers  extracted text quotations on either of the following: (1) data collected for each cohort; (2) comparison cohort used to discriminate this possible new disease phenotype. These independent extractions were later paired for qualitative data synthesis, which was also supplemented with a brief summary of each paper, drafted independently by two reviewers. The content of these extractions and the reviewers’ combined summary of each paper were then merged. Nine CSIPF patients  seen at the Siena Regional Referral Centre for Sarcoidosis and other Interstitial Lung Diseases and at the Respiratory Diseases Unit, Florence University Hospital, Florence, Italy, in the period 2014–2021, were enrolled retrospectively. Control cases seen in the same period were randomly selected from the database and included 19 lone-IPF patients and 26 patients with fibrotic sarcoidosis. Diagnoses were performed according to international guidelines [Results are reported as mean ± standard deviation (M ± SD) or medians and interquartile range ) for continuous variables, as appropriate. The Shapiro–Wilk test was used to check normal distribution of the variables. Non-parametric one-way ANOVA  and Dunn test were used for multiple comparisons. The Chi-squared and Fisher tests were used for categorical variables. Two-dimensional ANOVA (Friedman test) was used to analyse 2 × 2 matrices of variables.The literature search yielded entire texts, of which we selected ten: two comparative studies, one case series, two case reports, three reviews and two comments/editorials. p < 0.0001). We did not observe any difference in mean age at diagnosis between patients with lone-IPF and CSIPF (p > 0.05). Patients with sarcoidosis were predominantly non-smoker females who were younger than patients in the other groups (p < 0.05). Among patients with CSIPF, sarcoidosis was ascertained by histopathology in 8/9 cases. Seven out of nine CSIPF patients (77%) had a family history of lung fibrosis compared to only two patients with IPF. Two out of nine CSIPF patients (23%) showed extrapulmonary manifestations of sarcoidosis  compared to nine (38%) of the fibrotic sarcoidosis group . The main comorbidities of lone-IPF and CSIPF patients were gastroesophageal reflux disease (GERD) and arterial hypertension, while those of the sarcoidosis group were infectious diseases and other lung disorders. All patients with lone-IPF and CSIPF underwent antifibrotic therapies , respectively; mean DLco% 89 ± 17 vs. 39 ± 12 (p = 0.02), respectively. Interestingly, at the two-year follow-up, a statistically significant decrease in FVC% emerged for CSIPF patients  and in DLco percentages for the fibrotic sarcoidosis cohort  , associated with moderate-to-severe reduction in DLco percentages ; however, no statistically significant differences emerged at baseline between these two groups. The fibrotic sarcoidosis group differed greatly from other two groups of patients; specifically, no restrictive impairment was found with respect to the CSIPF group: FVC% 103 ± 20 vs. 76 ± 29 (= 0.044) .In recent years, a disease phenotype consisting in an overlap between sarcoidosis and IPF, known as CSIPF, has been postulated. The present case series included nine patients with CSIPF. In line with the literature, these patients showed different features in terms of age, occupational exposure and granuloma localization ,27. TheyCollins BF et al. described a cohort of 25 patients who manifested combined clinical and radiological features of IPF and sarcoidosis (CSIPF). Clinical course and mortality rate were similar in groups of patients with lone-IPF and CSIPF. The authors concluded that sarcoidosis and IPF co-existed in these patients but that their onset occurred at different times . Two subIn 2019, Bianchi et al. described a case series of four patients with overlapping sarcoidosis and idiopathic pulmonary fibrosis. This study confirmed that this rare association is more frequent in males with a documented family history of ILD and that the sarcoidosis prevalently affected the lungs .Findings in our cohort confirmed the previous literature. In particular, our patients were prevalently male ex-smokers, with an age intermediate between those of the sarcoidosis and lone-IPF groups. Occupational exposure seemed to play a crucial role in this cohort. The main comorbidities were similar to those of the lone-IPF group and most cases did not show extrapulmonary localizations of granulomas. Interestingly, lung function deteriorated during the two years of follow-up, despite that fact that all CSIPF patients were on antifibrotic treatments. This result suggests that these patients are at high risk of rapid deterioration and lung damage.CSIPF showed different features in terms of age, occupational exposure and localization of granulomas. Faster functional deterioration than in lone-IPF and fibrotic sarcoidosis patients was observed during the two years of follow-up. These patients have a worse prognosis, making it opportune to investigate a large international cohort of CSIPF patients.


SBR was crosslinked in situ in the presence of OMMT nanoparticles by an efficient vulcanization technique using sulfur as a crosslinking agent and zinc diethyldithiocarbamate as a catalyst. The structure and morphology of the hybrid materials obtained were characterized by Fourier transform infrared spectroscopy, X-ray diffraction, and scanning electron microscope analysis. The thermal properties of these hybrid materials were studied by differential scanning calorimetry and thermogravimetric analysis/thermal differential analysis. The mechanical properties were studied by strength measurements. The results obtained occurred when the OMMT was incorporated in the CSBR matrix; a significant increase in the glass transition temperature of the SBR was observed which passed from −27 °C for virgin SBR to −21.5 °C for that containing 12 wt% of OMMT. The addition of OMMT nanoparticles to CSBR also improved the mechanical properties of this copolymer. When the OMMT content in the CSBR varied from 0 to 15% by weight, the tensile strength, the elongation at the nose and the modulus at 100% elongation increased from 3.45 to 6.25 MPa, from 162, 17 to 347.20% and 1.75 to 3.0 MPa, respectively. The results of pervaporation revealed that when the OMMT content varied between 3% and 12%, a significant increase in the total flux, the separation factor and the separation index by pervaporation increased from 260.67 to g m Styrene–butadiene rubber (SBR) is mainly synthesized by free radical copolymerization of styrene with butadiene ,2. This Bentonite, which belongs to the family of clays, is known as a better reinforcing agent used in rubber compounds. Its low cost, low weight, and environmentally friendly reinforcing filler is still a challenge in the rubber industry . It alsoConventional composites are obtained when the polymer is unable to intercalate (or penetrate) between the silicate sheets, and a phase-separated composite is obtained a; the inThe latter structure is the most interesting because of the greater clay–polymer interactions, which produce composites with much better mechanical, thermal and barrier properties . Nanocomposites involving SBR and clay are among the most interesting materials for researchers, especially during the last decade. Indeed, based on the character of separating clay into monolayers with a thickness of 1 nm, Zhang et al.  preparedThe prepared hybrid materials showed that the main structure of the clay dispersion in the SBR was a bundle of layers, the thickness of which was 4–10 nm and its aggregation formed by several or more bundles of layers. Gu et al.  preparedAlcohol-aromatic mixtures such as a toluene–methanol system are commonly used in the petrochemical and pharmaceutical industries. This system forms an azeotropic with 32 wt% toluene at a constant pressure of 101.3 kPa . Over thIn this work, a series of crosslinked poly(styrene-co-butadiene) rubber/Maghnia organoclay montmorillonite (CSBR/OMMT) membranes containing different OMMT contents were prepared by a solvent casting method. To achieve this goal, organomontmonrillonite or organophylic clay (OMMT) as a filler was synthesized according to the procedure described by Bhattacharya and Aadhar . The cro−1 was supplied by Sigma-Aldrich . Maghnia bentonite containing 98% montmorillonite [(Si8)IV (Al4−x Mgx)VI O20(OH)4], having a cation exchange capacity (CEC) of 89 meq·g−1 was provided by “Entreprise Nationale des substances utiles et des produits non-ferreux”(ENOF) . The chemical composition of this clay is presented in Poly(styrene-co-butadiene) rubber with 45 wt% of styrene content and a density of 0.965 g·mLOrganomontmonrillonite or organophylic clay (OMMT) was synthesized according to the procedure described by Bhattacharya and Aadhar . A totalSBR solution was prepared by dissolution of small SBR pieces in toluene during 24 h. A determined amount of organoclay filler was then added in small portions under continuous stirring for 24 h to prepare a suspension. Sulfur (crosslinker) and ZDC  were added to the previous mixture in a ratio of 1.5: 6 by weight, and the whole was left to stir for an additional 8 h. The contents were then carefully poured onto a horizontal glass plate and allowed to air dry for 12 h. Crosslinking of the SBR was then carried out in the presence of sulfur and ZDC by heating at 110 °C for 1 h. The cured thin film deposited on the glass plate was allowed to cool to room temperature then tromped into distilled water which was easily peeled off . Differe−1 under transmittance mode at 4 cm−1 increments. XRD patterns were obtained by a Siemens D-5000 X-ray diffractometer  with Co (wavelength λ = 1.788965 Å) irradiation at a scanning rate of 0.02°/min in the range of 2–30°. SEM images were performed on films coated with gold grid using JSM-6060LV (JEOL). The particle size of the OMMT filler was measured by dynamic light scattering (DLS)  at room temperature (25 °C) after dilution of the formulations with double-distilled water. The value of the particle size was taken from the arithmetic mean of three replicates. The surface morphologies of OMMT, CSBR particles and CSBR/OMMT hybrid materials were studied by transmission electron microscopy (TEM JEOL JEM-2100 F)  at an acceleration voltage of 200 kV. The DSC thermograms were carried out on a DSC Q1000  previously calibrated with indium. A total of 6–10 mg of film sample was packaged in a DSC aluminum pan before being placed in the DSC cell and heated under nitrogen gas from −80 to −10 °C at a heating rate of 20 °C min−1. TGA measurements of the samples were carried out under nitrogen gas on an SDT 600 device . A total of 4–10 mg of sample film was placed in an aluminum TGA pan and heated from 0 to 700 °C at a heating rate of 20 °C min−1. Mechanical strength measurements were performed on an Instron Zwick/Roell Z010  instrument at a heating rate of 0.3 mm·s−1. Tensile properties, such as tensile strength, 100% modulus and elongation at break were evaluated according to the norm DIN EN ISO 527-1. The crosslinking densities (v) for the pure CSBR and CSBR/AOMMT nanocomposites were estimated by a toluene swelling test. The film sample was immersed in an excess of toluene at room temperature (25 °C) until constant mass (equilibrium). The value of the crosslinking density was calculated using the Flory–Rehner Equation  is much lower compared to that of the SBR–Methanol system −3)0.5]. According to the Hildelbrand and Scott Equation (10) . This further promotes the transport of toluene across the membrane. The increased affinity of SBR/OMMT nanocomposite membranes for toluene is the driving force behind the remarkable difference in the separation of toluene from the methanol/toluene mixture . Some reDi values for the different CSBR/OMMT nanocomposite membranes are calculated from Equation (10) and the results obtained are collated in The mass transport of a binary liquid in a pervaporation membrane is usually explained by a solution diffusion mechanism, which takes place in three stages: sorption, diffusion, and evaporation. Thus, the rate of permeation and selectivity of the membrane are governed by the solubility and diffusivity of each component of the mixture. In the pervaporation technique, as long as the establishment of a distribution equilibrium is rapid between the feed and the upstream surface of a membrane, it is the diffusion step which controls the migration of the penetrant. Therefore, it is important to estimate the diffusion coefficient of penetrating molecules to understand the mechanism of the molecular transport. The diffusion coefficient is then an important kinetic parameter for estimating the diffusion of penetrants through a membrane. The Di values of toluene are almost three times higher than those of methanol for all membranes. These results once again confirm the observation of the greatest sorption of toluene compared to that of methanol. This fact clearly explains the greater selectivity of CSBR/OMMT membranes towards toluene. As shown by the data obtained, the Tg, max = 7.8 °C) and retains its flexibility regardless of the OMMT content incorporated in the hybrid material. In fact, the Tg value of the CSBR/OMMT nacomposite reached a maximum of −20 °C, when the OMMT/CSBR composition was 3 phr. Thermal analysis performed by TGA/ATD reveals that there is virtually no noticeable change in the thermal stability of the prepared materials regardless of the composition of the composite system. The mechanical properties of the CSBR/OMMT hybrid material in terms of tensile strength, elongation at break and modulus at 100% elongation showed a remarkable increase as the OMMT content in the membrane increased. Nanocomposite membranes involving organo-montmorillonite and SBR crosslinked in situ by an efficient vulcanization technique using sulfur as a crosslinking agent and zinc diethyldithiocarbamate as a catalyst have been successfully prepared. The uniform distribution of OMMT nanoparticles in the prepared CSBR/OMMT hybrid materials was confirmed by FTIR, XRD and SEM analysis. Thermal analysis of the resulting hybrid material performed by DSC revealed that the incorporation of OMMT nanoparticles into the CSBR matrix slightly improves the glass transition temperature of the pure SBR copolymer (ΔThe application of the hybrid materials prepared as membranes for the separation of the toluene/methanol azeotropic mixture by pervaporation revealed that the total flux improved much better compared to those in the literature using membranes based on SBR and carbon black. The trans-membranar total flux follows the same trend as the partial fluxes attributed to each component of the mixture. These fluxes and separation factors increase with the OMMT content to reach a maximum at 12 phr of the load in the membrane. The behaviors of the total and partial fluxes follow practically the same trend as the swelling degree of the membrane in the azeotropic mixture. The maximum amount of toluene/methanol mixture was reached with the CSBR/OMMT12 membrane. Finally, this study reveals that the membrane containing 12 phr of OMMT nanoparticles makes it possible to obtain the best performance, particularly in terms of total flux.


Graphene can be synthesized using top-down or bottom-up approaches and is used in the electronics and medical  fields as well as in photovoltaic systems. However, the mass production of graphene and the means of transferring monolayer graphene for commercial purposes are still under investigation. When graphene layers are stacked as flakes, they have substantial impacts on the properties of graphene-based materials, and the layering of graphene obtained using different approaches varies. The determination of number of graphene layers is very important since the properties exhibited by monolayer graphene decrease as the number of graphene layer per flake increases to 5 as few-layer graphene, 10 as multilayer graphene, and more than 10 layers, when it behaves like bulk graphite. Thus, this review summarizes graphene developments and production. In addition, the efficacies of determining the number of graphene layers using various characterization methods , atomic force microscopy (AFM), scanning electron microscopy (SEM), X-ray diffraction (XRD), Raman spectra and mapping, and spin hall effect-based methods) are compared. Among these methods, TEM and Raman spectra were found to be most promising to determine number of graphene layers and their stacking order.Graphene, a two-dimensional nanosheet, is composed of carbon species (sp The 6th element of the periodic table is truly fascinating . From thAround 70 years ago, scientists debated whether two-dimensional (2D) materials existed due to their thermodynamical instability . GraphenGraphite has a 3D structure with large particle sizes and a small surface area, whereas graphene is monoatomic in thickness and has small particle sizes and an extremely high surface area; these characteristics have remarkable effects on its properties ,5. AccorRegardless of the method used, the number of graphene layers per flake, importantly, determines the properties of the produced materials. Before we describe earlier work on graphene, one needs to understand what a 2D crystal is. Atoms arranged regularly in a single plane form a 2D crystal, and 100 layers of such planes would be considered a thin 3D film, which prompts the question of “how many layers are needed to make a 3D structure?” It has been shown that electronic structures rapidly evolve with the number of layers and graphite approaches the 3D limit at 10 layers . In thisMonolayer graphene can be produced in various ways, but is usually produced by micromechanical cleavage or liquid phase exfoliation (top-down methods), or using chemical vapor deposition (bottom-up method):(a) Micromechanical cleavage (MMC): MMC has been used for decades to synthesize graphene . Geim et(b) Liquid-phase exfoliation (LPE): High-quality graphene can be obtained by exfoliating bulk graphite by mechanical shear in a liquid . LPE of (c) Chemical vapor deposition (CVD): CVD involves the deposition of thin films from solid, liquid, or gaseous precursors. Upon heating, carbon-based compounds decompose and the carbon diffuses through and saturates metal lattices. Upon cooling, the carbon is expressed from the metal surface and forms graphene. This process can be used to manufacture large quantities of graphene . Several(d) Molecular beam epitaxy (MBE): MBE is used widely for growing graphene layers of high carbon purity  on many (e) Chemical synthesis of graphene: Chemical methods have been widely used to synthesize graphene in high yields and throughputs . ExfoliaMonolayer graphene has exceptional mechanical, electrical, and thermal properties  and it hMechanical properties are well known to be strongly influenced by graphene layer numbers. For example, Zhang et al. studied the effects of the number of layers on mechanical properties . The resCoefficients of thermal expansion (CTE) were found to depend on temperature and layer number. Optical transmittance was found to depend on layer number . A graphRestacking involves the auto assembly of graphene layers and reduces the properties of graphene in various industrial applications, such as barrier properties in batteries. One approach involves increasing the interlayer spacing using intercalants and then removing them by rapid evaporation. For example, when graphite is soaked in a strong acid , the acid penetrates the layers and increases the interlayer spacing. Promptly increasing the temperature can cause intercalant removal, and leave behind exfoliated sheets of graphene . FurtherTEM involves the transmission of a beam of electrons through an ultra-thin (<100 nm thick) specimen. TEM can provide high-resolution images down to the atomic level, and is frequently used to determine the number of graphene layers per flake. Nakamura et al. used TEM to study evenly stacked graphite-like thin sheets (GLSs) prepared by the laser ablation of graphite in argon . The proAl-Hagri used CVD to synthesize monolayer graphene, which was then studied using TEM . The proScanning electron microscopes provide images by scanning sample surfaces with a focused beam of electrons. These electrons interact with atoms and produce signals that contain information about surface morphologies. SEM is frequently used to determine the number of layers in graphene flakes. Ying et al. synthesized bi-layer and multi-layer graphenes on Cu foil in one step . Figure 4Ti5O12 anode in the context of achieving a high Li ion storage capacity [AFM is a high-resolution type of microscopy with a resolution at the atomic level. AFM can be used to produce images of 3D topological features and thickness histograms of specimens. AFM is also used to determine the number of graphene layers in nano graphitic flakes; however, due to the surface roughness of graphene and variations introduced by AFM, graphene thicknesses are difficult to determine accurately. Kim et al. examined an SLG wrapped Licapacity . Figure capacity . The gracapacity .2 and thickness 4–5 nm) of mostly hydrocarbon species on graphene flake surfaces. Singh et al. also studied the thicknesses of multilayer graphene using AFM [Prakash et al. studied epitaxial FLG grown at different temperatures using CVD with AFM microscopy . AFM revsing AFM  and founsing AFM .Yao et al. demonstrated the use of histograms and found that the flake thicknesses of 1, 2, and 4-layer graphene were 1.5, 1.9, and 2.73 nm, respectively, by AFM . MargaryOptical microscopy which is also known as light microscopy is often used to study morphologies at low magnifications. This type of microscope uses visible light and a system of lenses to generate magnified images of small samples, but it can also be used to estimate layer numbers per flake. A plasmon is a quantum of plasma oscillation and can be considered as a quasiparticle arising from the quantization of plasma oscillations. XRD is based on the differential diffractions of an incident X-ray beam caused by sample crystal structures. Using the intensities and angles of diffractions, 3D images of electron densities within crystals are generated. XRD was recently used to determine layer numbers in graphene flakes . Mauro e−1 for 3-layer graphene to 2720 cm−1 for 10-layer graphene and 2725 cm−1 for a graphite of thickness 40 nm [D and I2D/G band depend on layer number. For example, the intensity of the D-band increases with layer number, and the intensity ratio of the 2D/G–band decreases with layer number [Raman spectroscopy provides information on the vibrational modes of molecules, and is frequently used to obtain the structural fingerprints of molecules, but it has also been used to determine graphene layer numbers in flakes. The most prominent spectral feature of carbon-based materials is the appearance of a D-band, G-band and a 2D band  Figure a. Furthess 40 nm . Figure r number  and optir number . The RamNiavol et al. studied a single layer graphene-based sensor for monitoring toxic gases  and confD/IG ratio, which allowed the determination of strain type and layer number [Haehnlein et al. investigated epitaxial graphene of different types, such as defective/single-layer graphene to multilayer graphene with small defect densities by Raman spectroscopy and determined graphene layer numbers using strain types . A novelr number . Kolomiyr number . Yitzhakr number . The aver number .Silva et al. used Raman spectra to analyze graphene layer numbers of mass-produced flakes using 2D- and G-bands. The 2D-band provided information about exfoliation in graphene flake whereas the intensity of the G band provided information about layer numbers . Li et a2D/IG) and peak widths of the 2D-band to determine layer numbers. The 2D peak of single-layer graphene (SLG) consisted of a single Lorentzian peak with a width of 24 cm−1, but this was split into four, six, and five components for 2-, 3-, and 5-layer graphenes, respectively [Papanai et al. examined graphene layer numbers of mechanically exfoliated graphene flakes using Raman spectroscopy . The autectively . Niiliskectively . Raman sectively . Maruyamectively . Resultsectively .Raman mapping is a laser-based microscopic technique used to obtain performing Raman spectra. Recently, Raman mapping was used to determine graphene layer numbers and homogeneities of graphene flakes. Barbosa et al. prepared single-layer graphene using oxygen-free N-octane as a precursor . Graphen2 area [D/IG and the I2D/IG ratio to study the homogeneity of the synthesized bilayer graphene and demonstrated the high uniformity of bilayer graphene over deposited areas [Raman mapping was used to determine the thicknesses and graphene layer numbers of synthesized flakes. Raman maps of CuNi alloy after annealing at 900 °C for 30 min showed clear G and 2D modes. Coverage was approximately 63% of 10-10 µ2 area . Bayram 2 area . Shen et2 area  and repo2 area . The Ramed areas . Wang eted areas . Raman med areas . The stued areas . Woo et al. prepared graphene using CVD and used Raman maps on channels of graphene to study the uniformities in the graphene layers. Raman maps of individual graphene flakes showed that, in addition to the FWHM of 2D peak, the G peak position showed a lighter color at the edges compared with inside the graphene channels . Liu et The spin Hall Effect is a transport phenomenon, which manifests as spin accumulation on the lateral surfaces of electric-current-carrying specimens and opposing surface boundaries with spins of opposite signs. STM provides high-resolution images of surfaces at the atomic level. The technique involves sensing the surface of a specimen with a sharp conducting tip. Resulting images can capture features smaller than 0.1 nm and have a depth resolution of 0.01 nm. SCEM is used to measure local electrochemical behavior at different interfaces. Interpretation of SCEM technique is based on the concept of a diffusion-limited current. Wain et al. presented topological features, as observed by AFM-SECM . In grapGraphene flakes have remarkable effects on the physicochemical properties of graphene-based nanocomposites. Graphene layer numbers can be characterized microscopically, spectroscopically, or using plasmon exciton coupling spectroscopy, or the spin hall effect. Graphene can be synthesized using several bottom-up or top-down approaches, and because of its superior properties it has many applications, especially in the flexible electronics field. The stacking of monolayer graphene to form few-layer or multilayer graphenes significantly influences the properties of graphene. This review summarizes various techniques used to determine the layer numbers of graphene flakes. Greater layer numbers from monolayer graphene to few-layer graphene and multilayer graphene were found to significantly diminish the properties of graphene. This review summarizes the use of graphene with different layer numbers for various applications  and provides guidance regarding the effects of graphene flake thickness on the physicochemical properties of graphene.


The differential diagnosis of interstitial lung diseases (ILDs), particularly idiopathic pulmonary fibrosis (IPF) versus other non-IPF ILDs, is important for selecting the appropriate treatment. This retrospective study aimed to explore the utility of gremlin-1 for the differential diagnosis.Serum gremlin-1 concentrations were measured using an ELISA in 50 patients with IPF, 42 patients with non-IPF ILD, and 30 healthy controls. The baseline clinical data, including pulmonary functions, prognosis, and three serum biomarkers , were obtained through a medical record review for analyzing their associations with serum gremlin-1 concentrations. To evaluate the origin of gremlin-1, we performed immunostaining on lung sections.Serum gremlin-1 concentrations were significantly higher in patients with IPF , followed by those with non-IPF ILD (8.8 ng/mL) and healthy controls (1.6 ng/mL). The area under the curve for IPF versus non-IPF ILDs was 0.759 , which was superior to that of KL6/SP-D/LDH. The sensitivity and specificity for gremlin-1  was 72 and 69%, respectively. By contrast, serum gremlin-1 concentrations were not associated with the pulmonary functions nor the prognosis in all patients with ILDs. In immunostaining, the gremlin-1 was broadly upregulated in IPF lungs, particularly at myofibroblasts, bronchiolar/alveolar epithelium, and CD163-positive M2-like macrophages.Gremlin-1 may be a useful biomarker to improve the diagnostic accuracy for IPF compared to non-IPF ILDs, suggesting a role of this molecule in the pathogenesis of IPF. Interstitial lung disease (ILD) is a heterogeneous disease in terms of its variety, particularly in regard to its clinical course and treatment . Among IThe diagnosis of IPF requires evidence of a typical “usual interstitial pneumonia (UIP)” pattern either through radiology or pathology, as well as exclusion of the possibility of other non-IPF ILDs . HoweverWe recently performed a microarray analysis using bleomycin-induced myofibroblasts and steady-state fibroblasts directly isolated from mouse lungs . In the +Sca-1−lineage (CD45/TER119/CD31/CD324/Lyve-1/CD146)− population from bleomycin-treated mouse fibrotic lungs and in a PDGFRα+lineage− population from normal untreated mouse lungs, respectively. We evaluated the transcriptome data of these cells by microarray analysis . Gene expression profiling with RNA amplification was performed by Takara Bio, Japan.The detailed methods for the experiments using mouse samples have been described in one of our previous articles . BrieflyA database review between 2000 and 2017 at Hamamatsu University Hospital identified 50 randomly-selected patients with IPF and 42 age-matched patients with non-IPF ILD, including idiopathic interstitial pneumonias other than IPF , collagen vascular disease-associated ILD , and chronic hypersensitivity pneumonitis (3 patients)  on room air, the disease extent on chest high-resolution computed tomography (HRCT), the radiological UIP compatibility  on chest HRCT [The patients’ medical records were reviewed to obtain the following clinical data at the time of serum sampling: sex, age, percent predicted forced vital capacity (%FVC), percent predicted diffusion capacity for carbon monoxide (%DLCO), partial pressure of arterial oxygen PaO on room We obtained serum samples from the healthy volunteers during the health check-ups and from the patients with ILD at the time of the ILD diagnosis. Each participant agreed to undergo the procedure by providing their written informed consent. Serum gremlin-1 concentrations were quantified using sandwich enzyme-linked immunosorbent assay based on the manufacturer’s protocol .We evaluated formalin-fixed paraffin-embedded lung specimens from 3 patients with IPF, 5 patients with non-IPF ILD , and 3 healthy controls. Deparaffinized Sections (4-μm-thick) were preheated at 105 °C for 15 min (pH 9).2O2 for 10 min, the sections were incubated with a blocking/permeabilizing solution  for 60 min and then incubated with anti-gremlin-1 antibody  at 4 °C overnight. The next day, the lung sections were incubated with peroxidase-labeling goat anti-rabbit IgG antibody  for 60 min. The immunoreaction was visualized using 3,3-diaminobenzidine  and then counterstained with hematoxylin.After inactivating endogenous peroxidase with 0.3% HAfter incubating with a blocking/permeabilizing solution for 60 min and then incubated with primary antibodies at 4 °C overnight . The sections were then incubated with Hoechst 33342  with Alexa Fluor conjugated secondary antibodies  at room temperature for 60 min.p < 0.05 was considered statistically significant.We performed statistical analyses using EZR software (ver. 1.41). Coefficient values between each parameter were evaluated using Spearman rank correlation coefficient method. Group comparisons were performed using Fisher’s exact test, Mann–Whitney U test, or Kruskal–Wallis test with post-hoc Bonferroni test as appropriate. Multivariate analyses were done using a logistic regression model. In the analyses of receiver operating characteristic curves, area under the curve (AUC) values were compared using DeLong test. Cox proportional hazards model was used for a prognostic analysis. A Grem1) was significantly upregulated, while BMP4 mRNA was downregulated in bleomycin-induced lung myofibroblasts compared with steady-state fibroblasts  mRNA, a canonical marker of myofibroblasts. In contrast, the mRNA expression of other BMP antagonists, including noggin and chordin, was not significantly different between the cells.According to our published mouse microarray dataset GSE111043), gremlin-1 mRNA , suggesting that this molecule would be not useful as a prognostic biomarker.As shown in Table p < 0.001 vs. gremlin-1), SP-D , and LDH . Although we explored the possibility of these 3 biomarkers in combination with gremlin-1, none of them could not significantly increase the AUC value, compared to that of gremlin-1 alone. When the gremlin-1 cutoff was set at 10.4 ng/mL, the sensitivity and specificity for discriminating IPF from non-IPF ILD was 72 and 69%, respectively.To evaluate the potential of gremlin-1 as a biomarker for the differential diagnosis of ILDs, we constructed receiver operating characteristic curves between IPF and non-IPF ILDs Fig. d. The AUIn this study, we demonstrated that the protein of gremlin-1 was upregulated in fibrotic lungs, particularly at myofibroblasts, CD163-positive M2-like macrophages, and bronchiolar/alveolar epithelium in IPF lungs. The serum gremlin-1 concentration was significantly higher in patients with IPF than those with non-IPF ILD, suggesting a pathophysiological role of this molecule in IPF.Gremlin-1 is an endogenous BMP4 antagonist, and the antagonizing regulation employs extracellular and intracellular pathways . This moGREM1 mRNA expression in fibrotic lungs is significantly upregulated, even in alveolar type 2 epithelial cells (approximately 14.7-fold) and alveolar macrophages (approximately as much as 116.2-fold) compared with normal control cells [Based on our immunostaining results, the origin of lung gremlin-1 appears to be not only myofibroblasts but also the bronchiolar/alveolar epithelium and CD163-positive M2-like macrophages. Consistent with these results, a recent bulk RNA-seq data analysis of human cells also showed that ol cells . Anotherol cells . In contol cells . GremlinThe increase in serum gremlin-1 concentration was significantly prominent in IPF compared with non-IPF ILDs. Although the exact mechanism underlying this gap is unclear, it could be mostly due to the difference in the quantity of myofibroblasts or fibroblastic foci, which are significantly more numerous in IPF than in non-IPF ILD lungs or in UIP pattern than in non-UIP pattern , 20, 21.A previous pathology study evaluated the utility of gremlin-1 for the differential diagnosis of ILD and reported that the mRNA and protein expression of gremlin-1 in lung samples was more increased in IPF than in NSIP , which iThere have been numerous potential IPF biomarkers identified for evaluating disease severity and prognosis, including KL6, SP-A/D, matrix metalloproteinases, and osteopontin , 9, 24. In summary, gremlin-1 was upregulated in fibrotic lungs, particularly in IPF, and serum concentration measurements may be useful for improving the diagnostic certainty of IPF versus non-IPF ILDs. Our results again highlight the importance of gremlin-1 in the pathogenesis of IPF.


Biosensors hold great potential for revolutionizing personalized medicine and environmental monitoring. Their construction is the key factor which depends on either manufacturing techniques or robust sensing materials to improve efficacy of the device. Functional graphene is an attractive choice for transducing material due to its various advantages in interfacing with biorecognition elements. Graphene and its derivatives such as graphene oxide (GO) are thus being used extensively for biosensors for monitoring of diseases. In addition, graphene can be patterned to a variety of structures and is incorporated into biosensor devices such as microfluidic devices and electrochemical and plasmonic sensors. Among biosensing materials, GO is gaining much attention due to its easy synthesis process and patternable features, high functionality, and high electron transfer properties with a large surface area leading to sensitive point-of-use applications. Considering demand and recent challenges, this perspective review is an attempt to describe state-of-the-art biosensors based on functional graphene. Special emphasis is given to elucidating the mechanism of sensing while discussing different applications. Further, we describe the future prospects of functional GO-based biosensors for health care and environmental monitoring with a focus on additive manufacturing such as 3D printing. Detecaccuracy ,6.2/g for single layer graphene), and the fact that it is easy to functionalize with biomolecules as functional graphene has large number of groups and high electron transfer properties. Owing to direct electron transfer and high electrocatalytic properties, functional graphene is in high demand for developing sensitive biosensors.Graphene has shown great promise in the development of biosensing devices and is of increasing interest for the development of ultra-sensitive biosensors due to its single-atom layer thickness, extremely high carrier mobility, unique electrical conductivity, and inherently low electrical noise . Since tAmong the graphene derivatives, GO-based transducers exhibit excellent unique characteristics that enable the development of ultrasensitive devices for detection of minute amount of target analytes  due to their high surface area to volume ratio, high electron mobility, good water dispersibility, biocompatibility, and size controllability ,11. In aLately, microfluidic systems have paved a generic way for miniaturization, integration, automation, and parallelization of (bio-)chemical processes such as isolation/detection of analytes from biological fluids on a single chip . CurrentAnother problem in clinical diagnosis is disease heterogeneity i.e., single marker detection is not enough in clinical settings. For example, there are reports about various mutations existing in the environment of the COVID-19 viruses requiring more robust diagnostic tool to capture all the variants of single virus type. Similarly in cancer treatment, instead of detecting individual analytes, it might be possible to characterize the molecular composition of a tumor indirectly, by sampling the blood and searching for alterations in the serum .Thus, de−5–10−1 M), and shorter detection time (≤15 s). This indicates that the application of functional graphene can be potentially used for agriculture monitoring for precision and sustainable farming.In addition to biomedicine, GO-based sensors have been applied in agriculture monitoring such as measurement of soil moisture and nutrient sensing, and as natural bio-imaging agents with photoluminescent potassium-doped GO from agricultural waste used for developing stable fluorescence probes for bio-imaging applications. Functionalized GO/iron (GO-Fe) composite was also utilized as a fertilizer to supply phosphate ions to soil for better agricultural product development ,20,21. IIn this article, we critically review the application of graphene and functionalized graphene such as GO towards health monitoring. We have emphasized different sensing modalities based on functionalized graphene and its derivatives for the detection of biomarkers and chemicals. Specially, we cover examples of enzymatic biosensors, immunosensors, DNA sensors, and pathogenic biomarkers. Further, we critically discuss and summarize the future of biosensor technology based on graphene using the industry 4.0 manufacturing outlook that focuses on additive manufacturing . 3D printing enables the potential impact of constructing prototyping and developing production/commercial quality platforms in multidisciplinary areas such as electronics, biotechnology, aerospace and defense, and chemical engineering . Figure In enzymatic biosensors, enzymes have been incorporated as bioreaction elements which allow catalytic biochemical reactions with specific target biomolecules. However, the main concerns are the enzyme functionalization and stability on a given transducing layer. GO has been repeatedly used to develop enzymatic biosensors and some of the novel reports are described in this section with a focus on the mechanism of detection. Zhou et al.  reported3). The rGO-BiFeO3 acted as a photoactive material possessing accelerated charge transfer with improved visible light absorption. The biosensor involved an anchor DNA-conjugated magnetic bead (MB-aDNA), PSA aptamer/trigger DNA (Apt-tDNA), and two glucose-oxidase-labeled hairpins (H1-GOx and H2-GOx). When the target PSA reacted with the aptamer, initially the trigger DNA was released, which partially hybridized with the anchor DNA on the MB. Consequently, the unpaired trigger DNA on the MB allowed the opening of the hairpin DNA structures in sequence and this propagated a chain reaction of hybridization events between two alternating hairpins resulting in the formation of a long-necked double-helix with numerous GOx enzymes on it. Following this, hydrogen peroxide (H2O2) was generated as an enzymatic product and consumed the photo-excited electrons from rGO-BiFeO3 under visible light irradiation thereby enhancing the photocurrent. This intelligent combination of target-triggered hybridization chain reaction and enzyme-catalyzed photoelectric reaction enabled the biosensor to achieve an LOD of 0.31 pg/mL within the linear range of 0.001–100 ng/mL, in phosphate buffer saline (PBS) and showed remarkable performance in human serum. Huang et al. [2O2. This reduced the silver (Ag) ions in the cholesterol-containing silver nitrate (AgNO3) solution to metallic Ag. Anodic stripping voltammetry was used as the electrochemical technique and the reported LOD was 0.001 μg/mL. Covalent modification of GO is achieved by a standard carboxylic activation/amidation approach in the presence of available amino groups in chitosan. The composite GO−Ch was deposited on standard screen-printed electrodes (SPCE) by a drop-casting approach. Comparison between a chitosan—GO blend and pristine GO demonstrated the superior reliability and efficiency of the electrochemical response for glucose as a consequence of the high number of enzyme binding sites and of the partial reduction of GO during the carboxylic activation synthetic step. Zhou et al.  have repg et al.  have demImmunosensors ,35 are aGO presents unique mechanical, optical, electrical, and chemical performance for construction of DNA-based biosensors. Among many sensing modalities, it serves as an effective acceptor of fluorescence resonance energy transfer (FRET) to quench the fluorescence of labeled DNA samples upon adsorption ,51,52. S+ on GO, which switches on the fluorescence, as shown in + from GO due to the π–π stacking effect, thus quenching the fluorescence. Stine et al. employed nanometer-thick layers of reduced GO (rGO) to covalently attach with single-stranded DNA (ssDNA), and formed a field-effect transistor (FET) device to implement sensitive, real-time, and label-free detection of DNA hybridization [Various types of GO biosensors for DNA detection have been developed over the past few years. Zhou et al. applied a chemically reduced GO modified glassy carbon (CR-GO/GC) electrode for the DNA sensing , and shodization . Large-adization . Noncovadization , as showdization , as showIt is important to note that the oxygen concentration in GO may vary considerably based on different synthesis protocols or procedures, which could potentially influence the DNA-sensing effect. Quite a few investigations have been conducted to quantify this GO compositional factor. Hong et al. noticed that the oxidation level of GO has a strong impact on the binding interaction to ssDNA and the fluorescence-quenching ability . They di2) and tungsten disulfide (WS2) for DNA sensing [2 and WS2 absorb DNA primarily by using of van der Waals force. Liu et al. conjugated GO with the nucleobases of DNA, while screening metal oxide nanoparticles to interact with the phosphate backbone of DNA, so as to reach highly sensitive DNA detection in serum and avoid the non-specific DNA displacement induced by proteins [Considerable efforts have also made to explain and clarify the mechanism that govern the GO biosensing for DNA detection, force generation, and nonspecific interactions. Liu et al. reported a mechanism of DNA sensing on graphene oxide . They di sensing , and expproteins . They reThe exceptional properties of GO biosensors make them an ideal choice for DNA sensing with high precision, selectivity, and sensitivity. Many types of GO biosensors in combination with glassy carbon, organic dyes, aptamer, and quantum dots have been reported to achieve the above goals with progressive improvement. Meanwhile, the oxidization level of GO serves as an important factor in affecting DNA-sensing efficiency. More specifically, the more reduced GO presents stronger binding with DNA strands and greater fluorescence quenching efficiency. Finally, the displacement of probe DNA, hydrogen bonding and π–π stacking, and nonspecific interaction induced by protein, are unveiled as generic mechanisms that govern GO biosensing for DNA detection.The presence of various oxygen-containing functional groups on the GO surface provides good dispersibility and favorable binding sites for functionalization in designing high-quality biosensors for pathogen detection . AnalyteKlebsiella pneumoniae (Kp), Escherichia coli (E. coli) and P. aeruginosa (Pa) for in vivo and in vitro studies. They showed that GO inhibited the growth and killing of Kp in macrophage and mouse models after GO solution were introduced with harvested bacterial suspension for 2 h at 37 °C and results were recorded. Researchers also explored the electrochemical properties of GO for sensing various biomolecules. Tiwari et al. developed a nucleic acid sensor using GO-modified iron oxide–chitosan hybrid nanocomposite (GIOCh) film for detection of Escherichia coli O157:H7 (E. coli) [−14 M. Researchers also fabricated GO-based devices to clean the environment using pathogen-like hyphae fungus to fabricate a mechanically stable thin film sensor. Zhang et al. developed highly flexible porous film for dye removal by graphene oxide–fungus interaction. They designed a flow-through adsorption device using GO and fungus hyphae which absorbed the target dye pollutant to clean the environment [Among graphene derivatives, GO has been found to have the most active antibacterial activity . Liu et E. coli) . The pDNironment .Virus infection is a global phenomenon, and the COVID-19 pandemic has caused havoc by infecting and killing almost 1.7 million people worldwide between late 2019 and mid-2021. Therefore, we require more robust and sensitive early detection systems to control the global pandemic caused by deadly viruses such as the corona virus. One of the earliest works for pathogen detection using GO was led by Lu et al., who demonstrated water-soluble GO as a new platform for the sensitive and selective detection of DNA and proteins . They exOne interesting work was reported by Song et al. who developed a novel GO-based label-free method to capture and disinfect environmental viruses (enteric EV71 and H9N2) . They deGO-based microfluidic immunosensors are becoming attractive alternatives to traditional pathogen-detection techniques such as ELISA, cell culture, and rt-PCR for better clinical tests due to rapid diagnosis, cost effectiveness, easy application, and high reproducibility. In the current scenario, we require highly sensitive, rapid, and early detection tools for quick diagnosis of highly infectious disease such as COVID-19 and the Zika and Ebola viruses. Graphene-based nanomaterials as transducing biosensing materials have shown great promise due to their large surface area, compatibility with biomolecules, electron transfer rate, and ability to immobilize a variety of different biomolecules. Although graphene-based materials are excellent electrode materials for biosensing applications, their application in industrial products is limited. In addition, manufacturing of compact biosensors at low cost is an important factor in biosensor technology since it involves development of reproducible, robust, and reliable sensors with high sensitivity, specificity, and low limit-of-detection. Traditional manufacturing processes such as screen printing of graphene electrodes provided two-dimensional surfaces which limited the performance of devices. So far, many techniques have been explored to produce graphene and graphene-derived nanomaterials such as thermal decomposition , mechaniRecently, Ghanam et al., introduced a simple, scalable technique called laser-scribed graphene (LSG) which employed a laser beam in order to convert carbon or polymer precursor films into three-dimensional graphene electrodes without using any lithographic mask . This maOwing to the excellent electrical and electrochemical properties of graphene and their integration into flexible devices, graphene-based materials can provide versatile neural recording probes which help to solve several problems in neural interface design . GraphenThe fourth industry revolution  is an innovative approach and shows the massive capability of manufacturing biomedical devices including biosensors . In rece2O2) in human serum and had a stable response after 7 days of incubation. This sensor did not require any mediator to detect H2O2, and thus realized a third-generation biosensor [4 showed increased current density for the redox probe ferrocene-methanol in contrast to PLA surface treated only by DMF. This is attributed to a better reorganization of the rGO surface with reduction in defects and enhanced exposure of electroactive sites validated using characterization through SEM, AFM, and Raman spectroscopy. The sensor produced encouraging results in determining catechol in natural water samples.Three-dimensional platforms of graphene electrodes have enhanced biosensing performance compared to two-dimensional electrodes in terms of sensitivity, limit-of-detection, and selectivity indicating their importance in next-generation biosensor development. Jakus et al., created a 3D printed scaffold structure consisting of graphene sheets and a biocompatible material of polylactide-co-glycolide with minimum features of 100 μm and improved electrical conductivity . Polylaciosensor . Howeveriosensor . This seiosensor  have alsA review paper was recently published on additive-manufactured-based electrochemical sensors . That reIn summary, functional graphene and its derivatives are exciting materials in the world of nanotechnology. The excellent properties of graphene have revolutionized the development of electrochemical, fluorescence, and optical biosensors for point-of-care health diagnostics. Continuous and growing applications of graphene are replacing the current technologies and opening a new direction of biomedical sensors with high performance. In this review, we covered the application of graphene and its derivatives for the detection of a range of biomarkers including toxins, antibodies, pathogens, and DNA. As the functionality of the graphene can be tuned for a specific application of biomarker sensing, we covered a set of examples of biosensing with different modalities. We also critically reviewed the fundamentals of functional GO and rGO towards DNA sensing, enzymatic biosensors, monitoring of pathogens, and immunosensing. It is noted that GO is more amenable to mass-scale production from the viewpoint of cost optimization and manufacturability when compared to graphene. GO can be easily used in composites with other polymers to produce electrochemically active, mechanically robust, functional transducers for biosensors. GO-based platforms have been successfully used in electrochemical-, FET- and surface-plasmon-resonance-based biosensors. Moreover, rGO offers the advantage of various functionalization schemes for the recognition of elements of biosensors and thus provides multidimensional advantages compared to other biosensing transducer materials. In addition, we described the possibilities of the next-generation graphene-based biosensors using advanced manufacturing such as 3D printing. It is noted that the production of high-quality defect-free graphene sheets and their use in industrial manufacturing of biosensors are still limited. This will need to be overcome for scaling up the production of graphene. We are expecting that future research would address these challenges for the commercial production of graphene at a low cost. In the coming years, we believe that the additive manufacturing of graphene combined with IoT, and wearable technology can potentially transform the market of traditional biosensor technology for health monitoring.


Pseudogymnoascus destructans (Pd), has caused severe population declines of several bat species in North America. We conducted the first experimental infection trial on the tri-colored bat, Perimyotis subflavus, to test the effect of temperature and humidity on disease severity. We also tested the effects of temperature and humidity on fungal growth and persistence on substrates. Unexpectedly, only 37% (35/95) of bats experimentally inoculated with Pd at the start of the experiment showed any infection response or disease symptoms after 83 days of captive hibernation. There was no evidence that temperature or humidity influenced infection response. Temperature had a strong effect on fungal growth on media plates, but the influence of humidity was more variable and uncertain. Designing laboratory studies to maximize research outcomes would be beneficial given the high costs of such efforts and potential for unexpected outcomes. Understanding the influence of microclimates on host–pathogen interactions remains an important consideration for managing wildlife diseases, particularly in variable environments.Disease results from interactions among the host, pathogen, and environment. Inoculation trials can quantify interactions among these players and explain aspects of disease ecology to inform management in variable and dynamic natural environments. White-nose Syndrome, a disease caused by the fungal pathogen,  Research to determine whether management actions are effective at reducing disease effects can be challenging to conduct, especially in dynamic and complex ecological systems4. Controlled experiments are often used to determine disease susceptibility of hosts or quantify aspects of host–pathogen-environment interactions5. Experimental inoculation trials in laboratory settings provide the advantage of controlling and randomly assigning specific conditions of interest to enable causal inference and determine disease susceptibility. Ideally, these inferences can be used to explain key aspects of disease ecology and inform management actions taking place in variable and dynamic natural environments4.Infectious disease can threaten wildlife populations by causing rapid population declines, particularly when pathogens first emerge or are introduced into novel areas with naïve hostsPseudogymnoascus destructans; Pd)9 that was first detected in eastern North America in 200610 and has caused rapid and extreme population declines of several species of hibernating bats14. Over the past 15 years, extensive research has revealed much about the disease ecology of WNS, including the interactions between the pathogen, environments where bats hibernate, and the physiology of hibernating bats (see12 for recent comprehensive review). Research efforts have also focused on testing potential management actions aimed to reduce transmission or spread, lower disease severity, or improve survival18. One of the challenging aspects of managing WNS is that the pathogen persists in the environment where bats hibernate, exposing bats to infection at the start of each hibernation season20. Understanding the specific conditions that affect disease severity and population declines where the pathogen invades and establishes22 remains important for informing management response11.White-nose Syndrome (WNS) is an infectious disease of hibernating bats caused by a fungal pathogen 33 and there is a recognized need to study the interactions among host, pathogen, and environmental conditions in other bat species whose populations are declining from WNS12. In natural habitats, P. subflavus hibernate across a range of environmental conditions, including at temperatures associated with high Pd growth rates and high disease mortality34.P. subflavus to test the effect of temperature and humidity conditions on infection and disease severity of WNS. We asked three questions: (1) What is the effect of temperature and humidity on WNS disease severity of P. subflavus? (2) Does pre-hibernation condition of bats influence disease outcome? and (3) What is the effect of temperature and humidity on growth of Pd on environmental substrates? We predicted that disease severity would be higher at warmer and more humid microclimate conditions based on evidence from field studies that indicate these conditions are associated with higher fungal loads and more severe declines in winter colony size after Pd arrival (reviewed in12). We designed the experiment to determine behavioral selection of specific temperature and humidity conditions. Most natural hibernacula have a range of temperature and humidity29 and behavioral selection by bats of beneficial microclimates would be necessary if management strategies of providing thermal refugia are to succeed35. We hypothesized that pre-hibernation body condition, specifically fat mass and stress indicator (measured by fur cortisol) would be related to disease outcomes. We also addressed how microclimate conditions may affect the environmental reservoir of Pd given its important role in transmission dynamics19. Using the same environmental chambers and microclimate conditions, we conducted a separate experiment testing the effects of temperature and humidity gradients on fungal growth and persistence on artificial and natural substrates.We conducted the first experimental infection trial on https://arriveguidelines.org/).All methods in this study were approved by the Institutional Animal Care and Use Committee at Texas Tech University (protocol 18032-12). All procedures were performed in accordance with relevant guidelines in the manuscript and the ARRIVE  guidelines (36. A second group of bats (n = 14) was free to move among three chambers with a common VPD condition  but different temperatures   in a blocked experimental design, controlling temperature and humidity in each chamber  evenly across both wings, following established protocols37; treatments were conducted blind without knowledge of which bat was being assigned to what group and bats were inoculated in no particular order to reduce the confounding influence on the order of treatment. We used a Pd strain collected by Karen J. Vanderwolf at Trent University from naturally infected Myotis lucifugus. We cultured Pd on Sabouraud Dextrose Agar with chloramphenicol and gentamicin (SabDex)  and incubated subcultured plates at 10 °C for 60 days to allow the formation of conidia. We then harvested conidia by flooding plates with phosphate buffered saline solution containing 0.5% Tween20 (PBST). Conidia were resuspended in PBST, enumerated, and diluted to the inoculum concentration8.We inoculated each bat by spreading 20 µL of P. subflavus in natural hibernacula29. We set humidity in environmental chambers to achieve specific levels of water vapor pressure deficit (VPD) between the surface of the bat and the environment because relative humidity varies by temperature36. Higher VPD corresponds to drier air resulting in higher potential evaporative water loss (pEWL). We used three levels of VPD: 0.05, 0.10, or 0.15 kPa corresponding to low pEWL (high humidity), medium pEWL (medium humidity), and high pEWL (low humidity) levels  and used quantitative magnetic resonance  to determine pre-hibernation fat at the start of the experiment40. As an indicator of pre-hibernation stress, we collected a fur sample from the dorsal intrascapular region to quantify fur cortisol concentration with a commercial ELISA kit, following the manufacturer’s protocol  . Fur is moulted once per year in the late summer period41 and therefore fur cortisol reflects the level of circulating cortisol during the period of fur growth prior to hibernation. We attached a uniquely marked, modified datalogger42  to the back of each bat using ostomy cement to record skin temperature39. Prior to inoculation, we swabbed bats with a sterile polyester swab (Fisherbrand synthetic tipped applicators 23-400-116) five times on forearm and five times on muzzle to determine if any bats were naturally infected with Pd at time of collection. Swabs were stored in RNAlater at  − 20 °C until testing using quantitative polymerase chain reaction (qPCR) at Northern Arizona University43.We used 98  tricolored bats collected on 10 December 2018 from culverts in Mississippi and transported directly to Texas Tech UniversityDuring the experiment, we provided ad libitum drinking water in each cage but did not provide food. We secured a motion-activated infrared camera  above each cage to monitor bats throughout the experiment. Because one camera failed at the start of the experiment, we combined bats in that treatment chamber into a single cage Fig.  and repl39. We took respirometry measurements on a subset of animals38, and swabbed for Pd as described above. We photographed the left ventral wing using ultraviolet (UV) transillumination (368-nm wavelength and 2-s exposure) to detect and measure florescence associated with Pd infection44. For histology, we removed the wing section from the fifth digit and the body and rolled wing tissue around dental wax dowels and 10% neutral buffered formalin. We collected a 90–110 µL blood sample in lithium-heparin-treated capillary tubes for immediate analysis of blood chemistry with a handheld analyzer . Using an EC8+ cartridge, we measured sodium, potassium, chloride, anion gap, glucose, BUN (urea nitrogen), hematocrit, hemoglobin, pH, pCO2, TCO2, HCO3, and base excess  of 40 and quantified using a quantification curve from serial dilutions /3.34789), r2 = 0.986)47. Load values were averaged across multiple runs and then converted to attograms by multiplying loads in nanograms by 109.We used several metrics to determine pathogen and disease presence and severityPd prevalence, Pd loads, and WNS prevalence by histology) to determine whether infection status varied by microclimate treatment conditions. Low sample sizes of positive infection status by UV detection (n = 4) precluded use in statistical analyses  varied by sex and cortisol separately from an a priori candidate model set  (Table We used three different response variables (es Table . We usedet Table  to cope se Table . Bats beS) Table ; Fig. 2.P. subflavus, we investigated the effects of temperature and humidity on the hibernation physiology of the species39 and how physiological markers  may be associated with disease. To determine if physiological indicators were related to infection status at the end of the experiment, we compared total number of torpor arousal bouts during the experiment and 13 different blood chemistry metrics from blood samples taken at the end of the experiment and used t-test comparisons (at α = 0.05) for each metric between Pd/WNS positive and negative bats. We designated bats as Pd/WNS positive if a bat tested positive for either Pd or WNS by qPCR, UV, or histology. We used Program R version 3.6.2 to conduct all analyses.Because this was the first captive hibernation experiment with We used five environmental chambers  to test for the effects of temperature and humidity on fungal growth on natural and artificial substrates Fig. . Our expWe constructed modified plate lids to prevent contamination while allowing humidity to equilibrate across the plate lid. We drilled 14 equidistant holes (5.5 mm diameter) into each plate lid and hot glued a piece of circular filter paper to the top of the lid. Lids were then disinfected thoroughly with a hydrogen peroxide wipe before being placed in a disinfected, sealed storage container.Pd inoculum as described above for the infection trial on bats. We inoculated 30 SabDex plates with 100 µL of inoculum at a concentration of 20 conidia µL−1 by serial dilution with a starting concentration of 2.0 × 104 conidia µL−1 diluted four times by a factor of 10. We used sterile, individually wrapped 1-µL plastic inoculation loops to spread the inoculum evenly across the surface of the plates, added the modified plate lids, and immediately transferred plates into environmental chambers. We included six replicate plates in each of the five microclimate conditions.We prepared 2).We took weekly digital photographs  of each plate for the 5-week duration of the experiment Fig. A. Our caPd on each plate as a sigmoidal curve  across each sampling square and immediately transferred the rock to an environmental chamber.To evaluate fungal growth and persistence on a natural substrate, we inoculated pieces of sandstone flagstone. We etched a 4 × 6 sampling grid, composed of 5 × 5 cm squares, onto the surface of each sandstone rock , where each square served as a sampling unit Fig. . Each ro43. We quantified fungal loads for each swab sample from qPCR using the quantification curve provided above and normalized fungal loads to the value at day zero for each rock respectively. We then used linear models to test for effects of temperature and humidity on changes in fungal load (log transformed) over time.We measured fungal growth at days 0, 14, 28, and 56. We used a sterile cotton swab to collect fungal DNA from each sampling square. Swabs were moistened with RNAlater and rolled horizontally, vertically, and diagonally across the surface of the sampling square to ensure contact with the total surface area. One researcher collected all swabs to maximize consistency among swabs collected throughout the experiment. Swabs were placed in RNAlater and stored at − 20 °C until shipped to Northern Arizona University for qPCR analysisPd, we swabbed the entire inoculated surface of each rock at the end of the experiment and vortexed the swabs in RNAlater for one minute to release fungal DNA from the swab. We then applied 100 µL of RNAlater fungal solution from each rock to a respective SabDex media plate, using a sterile inoculation loop. After 2 weeks of incubation at 11 °C and 92% RH, we visually assessed plates for presence of fungal growth to determine viability of Pd collected from rocks at the end of the growth experiment.To evaluate viability of Pd by qPCR, 11% (10/95) scored as positive by histology, and only four bats showed signs of Pd infection by UV illumination (Table Only 37% (35/95) of the bats showed any infection response by the end of the experiment with 33% 31/95) testing positive for /95 testi38.Of the 14 bats that could behaviorally select temperature condition, seven bats spent > 89% of their captive days in the 11 °C chamber (4/7 spent 100% of captive days), one bat spent 100% of captive days in the 5 °C chamber, and three bats moved between chambers spending < 60% of captive days in any one temperature condition. Of the 14 bats that behaviorally selected humidity conditions, four bats spent 100% of captive days at the low pEWL/high humidity condition (0.05 kPA), three bats spent > 89% (2/3 spent 100%) of captive days at medium pEWL/humidity (0.10 kPA), and seven bats moved among chambers and were inconstant in selecting their humidity conditions. Detailed results on behavioral selection of microclimate conditions by bats in this study are provided inPd infection response between males and females (likelihood ratio test (LRT): Pd prevalence: χ2 = 1.44, df = 1, P = 0.23; Pd load: χ2 = 0.01, df = 1, P = 0.92) nor any evidence of a relationship with pre-hibernation cortisol . There was some support that WNS disease by histology differed between males and females  as 16% (n = 9/55) of males scored positive by histology compared to 2% (n = 1/44) of females. Because only one female scored positive by histology, bats of both sexes were pooled in subsequent analyses of WNS prevalence. We found marginal support for a weak positive relationship between pre-hibernation cortisol and prevalence of WNS disease by histology .We found no evidence for a difference in P. subflavus during this captive experiment  but there was no relationship between initial fat mass and Pd loads  and WNS prevalence .Model selection results indicate no support for an effect of temperature or humidity on infection response in nt Table ; Fig. 2.Pd/WNS positive or negative at the end of the experiment  and humidity , but the effect of temperature was much more pronounced . Similarly, humidity did not affect latency , but latency decreased with increasing temperature .The results for individual colony growth were similar to the patterns observed for total growth. There was no effect of humidity on maximum growth rate , but all three temperatures were distinct with growth rate increasing with temperature (LRT: χ10) ag; range: 5.69 – 9.33 (log10) ag). Neither temperature  nor humidity  affected fungal growth on the natural rock substrate . Fungal load increased in the first two weeks  and then remained consistent for the remainder of the experiment . After the 8-week experiment concluded, we successfully cultured Pd from swabs of the four extreme treatment rocks, confirming the continued viability despite the absence of fungal growth beyond day 14 of the experiment.Although there was no visible fungal growth on the rocks at any timepoint Fig. , fungal P. subflavus in this captive hibernation experiment. Why so few bats got infected from experimental inoculation with the fungus is unclear. Nearly two-thirds of the bats showed no signs of Pd infection at the end of 83—87 days of captive hibernation  more commonly chosen by bats for hibernation. When converted to a physiologically relevant measure of air moisture (VPD), our humidity gradient (VPD: 0.05–0.16) roughly overlaps with the two most humid conditions tested by Marroquin et al28 . Changes in humidity across this range had little effect on growth of Pd. The highest VPD in our experiment was well below the highest VPD (0.446 kPA) tested by Marroquin et al28, so we did not detect a decrease in growth in dry air as reported by that study.The results of our experiment concurrently testing the effects of temperature and humidity on growth of Pd can persist for long periods in the absence of bat hosts62. Although we observed slight fungal growth over the first sampling period (14 d), there was no change in fungal load over the remainder of the experiment. The growth of Pd on natural substrates depends on the interaction of the availability of nutrients and competition with other microbes. For example, Pd thrives when grown on sterile guano but growth is prevented by competition from other microbes on fresh guano63. We autoclaved the rocks in our experiment to limit competition from other microbes, but a lack of nutrients may have prevented additional growth. Substrates like cave soils or sediments likely provide more nutrients. Indeed, Pd has been isolated from cave soils60, although growth and persistence in soils can be greatly inhibited by competition from other soil fungi or other microbes63.The results from our natural substrate experiment are consistent with a growing body of literature that indicates that P. subflavus surveyed in culverts in Mississippi have not yet shown typical patterns of the progression of increased Pd prevalence and loads and the associated declines in winter colony counts, as has been documented extensively across the range of P. subflavus where WNS now occurs21. The mechanisms of why bats in Mississippi have not yet succumbed to Pd invasion and WNS mortality remain unexplored. Given what we know about how Pd infection disrupts hibernation physiology and energy balance65, there is a working assumption that regions with shorter, milder winters could serve as regional refugia from severe impacts66. However, the idea that hibernating bats may experience less severe impacts in areas with milder winter conditions assumes a behaviorally mediated response from bats being able to spend fewer days in torpor, sustain sufficient euthermic activity to mount an effective immune response, or potentially replenish fat stores with intermittent feeding activity during periodic warm spells67. The majority of bats (70/98) in our captive hibernation experiment were placed in controlled microclimate conditions for 83–87 days, which did not permit a behaviorally mediated response. Yet, two-thirds of the P. subflavus in this experiment failed to even have detectable levels of Pd on their skin tissues at the end of the experiment. Further research would be beneficial to understand what mechanisms could explain why P. subflavus in some habitats, such as culverts in Mississippi, have not yet experienced typical WNS progression.We explore a few post hoc hypotheses for why infection response was so unexpectedly low. Winter colonies of Pd infection and WNS disease to manifest sufficiently. We ended the experiment at 83–87 days based on our best estimate of the duration of bat hibernation in Mississippi, where we collected the animals. We terminated the experiment prior to bats becoming moribund to ensure we were able to collect sufficient data on physiological and disease condition and to comply with IACUC protocols. In captive M. lucifugus, death occurs 88–114 days after experimental infection9. To compare our results to other similar infection trials with M. lucifugus, we plotted Pd loads sampled on M. lucifugus by days since inoculation from two experimental infection trials conducted with the same inoculation protocols37 compared to the results of this study . We used a strain of tes Fig.  and rockPd in captive hibernation settings have also experienced challenges in producing results that are consistent with patterns observed in wild populations32. For example, Johnson et al32 used four levels of Pd inoculum and measured survival and Pd loads on M. lucifugus after hibernating at two different temperatures (4 °C and 10 °C) for 148 days. The results from this experiment were difficult to explain mechanistically given that mortality in the study did not correspond with patterns of Pd detection on bats across treatment groups at the end of the experiment.Other studies that experimentally inoculated bats with 39 highlight the similar relative influence of temperature and humidity on both host and pathogen when evaluated separately. Temperature has a greater influence than humidity for both host and pathogen and decreasing temperature reduces growth of the fungus and decreases energy expenditure in bats55. The effects of humidity are less pronounced, but decreasing VPD  reduces energy expenditure in bats39 while having less effect on the fungus. Behavioral selection of microclimates and the influence of variability in microclimate conditions in natural hibernacula are important to consider in applying these results to inform potential management actions to improve over-winter survival of bats in areas where Pd has established and to assess its role in infection as Pd spreads into new areas with different environmental conditions.WNS remains a severe and continuing threat to hibernating bats in North America. Concerted effort has been made over the past 15 years to understand the disease ecology and population impacts and much research attention continues to focus on potential treatments or actions that could improve survival of bats susceptible to the disease. Here, we aimed to augment field studies by providing experimental evidence of the effects of microclimate conditions on the host, pathogen, and environment. Our experiments here and our companion studies of bats hibernating in the same conditionsSupplementary Information.


An equivalent circuit model (ECM) using a MATLAB code to analyze a tunable two-layered graphene-based chiral dual-function metamirror, is proposed in this work. The investigated metastructure is composed of complementary U-shaped graphene resonator arrays in the terahertz (THz) region. The ECM analysis could be used for any two-layered chiral metastructure for any frequencies, containing resonators with a thickness less than λ/50. The characteristics of the proposed tunable metamirror were analyzed numerically using the finite element method (FEM) in CST Software to verify the ECM analysis. The proposed metamirror can be used in polarization-sensitive devices in the THz region with simpler biasing without a need for ion gels or similar. It works as a broadband TE and multiband (four bands) TM mirror in the 0.3–4.5 THz bandwidth with a strong linear dichroism (LD) response (up to 96%). The designed mirror is a dynamically tunable, dual-functional structure, requiring only 90° rotation of the incident electromagnetic fields to switch between broadband and multiband spectral behavior making it a promising candidate for future THz intelligent systems. The proposed ECM is in agreement with the FEM results. The ECM analysis provides a simple, fast, and effective way to understand the metamirror’s behavior and guides for the design and analysis of graphene-based chiral metastructures in the THz region. Graphene-based chiral metastructures have been designed and developed recently to achieve tunable CD and/or LD chirality responses6. Graphene, 2D layer of graphite, with a thickness of 0.335 nm7, has become a promising material in electromagnetics and optoelectronics metastructures as it is capable of dynamically tuning electromagnetic waves9. These proposed graphene-based chiral metastructures have circular conversion dichroism (CCD) and LD up to 20% and 94%, respectively. It is highly desirable to design and introduce dynamically controllable metamirrors with dual functionality, which can show both broadband and multiband reflection spectra by altering the type of the incident wave.Chiral structures do not coincide with their mirror images. Chiral metastructures are applicable platforms due to their chirality responses such as circular dichroism (CD)15, but none of them are dynamically controllable, dual-functional, or function in 0.3–4.5 THz frequency region, or use the proposed circuit modeling process. Dual-functional metamirrors are urgently needed in THz intelligent systems to greatly reduce the size of the THz system without needing two different metastructures to produce broadband and multiband reflections. For example, to be used in imaging objects under the broadband reflection or distinguishing them in case of multiband reflection16. In addition, these metasurfaces could also be favorable for other potential applications such as biosensors20, spatial modulators, attenuators16, and absorbers21.Some chiral metamirrors have been introduced and designed, recently22 or stacking resonators as a multilayer structure with resonators with different geometrical parameters and separate with dielectric spacers23.Metastructures, containing a resonator per unit cell with single-layered geometry, have a limited number of reflection bands and broadband reflection properties. Multi-band or broadband metamirrors are typically designed by combining multiple resonators with different sizes into a super unit cell27 without the need for ion gels28 or thin graphene strips29, thus reducing material usage, costs, and time in the fabrication process.A few graphene-based metastructures composed of complementary patterns have been proposed to ease the biasing procedure and fabrication of graphene based metastructures32, investigating the different aspects and applications of this configuration. However, the detailed investigation of this configuration with 2D materials like graphene is lacking. By the design, analysis and optimization of this metastructure with graphene-based complementary resonator arrays in THz region, their performance in tunable metamirror applications was investigated in this work.Chiral metastructures, composing of two-layered gold U-shaped rotated patterns, were previously proposed in following papers28, we developed a chiral metamirror consisting of two-layered complementary U-shaped, 90° rotated, patterns. The metastructure in28 is a multiband absorber but the proposed structure could act as broadband and multiband metamirror just by switching the type of the incident electromagnetic waves. Such dual function surfaces are expected to be a key element of intelligent THz systems in the future since they could act as a compact broadband and a multiband mirror. Our earlier paper28 presented a procedure to obtain an EMC model of the metastructure but in this work we used equivalent conductivity relations and reflections to obtain the equivalent conductivities of the graphene patterns. Then, the transfer matrix elements were determined for the structure to obtain the reflection characteristics of the metasurface containing graphene/dielectric/graphene/dielectric/gold layers. In28, we used transfer matrix elements and reflections to obtain the impedances of the graphene layer, which were then used to achieve the transfer matrix of the whole structure containing ion gel/graphene/dielectric/gold layers to obtain the metastructure absorption behavior.In contrast to the chiral metastructure composed of common single-layered graphene-based U-shaped patterns28, the graphene patterned layers are a complementary shape in our design. Thus, there is no need to use ion gel layers for biasing the graphene layers making its tunability much easier to implement and making the fabrication procedure easier. The dielectric substrates are made of quartz with a refractive index of 1.9633. The metamirror metastructure is backed with a gold layer with a thickness of 0.5 μm to ensure that electromagnetic waves cannot pass through it (the transmission is zero for both TE and TM waves). The conductivity of the gold layer is 4.56 × 107 S/m34. Simulations were performed using the CST Microwave Studio Software4. The device works as a broadband and multiband metamirrors when it is illuminated by TE and TM waves respectively. The structural parameters and their optimized values for the proposed dual-functional metamirror are given in Table 36. In the optimization technique in CST, we took into account that the unit cell dimensions, Px = Py = 18 μm, have to be smaller than λmin = 66.67 μm if fmax = 4.5 THz (the maximum frequency in the simulated region) to avoid excitation of high order Floquet modes39.3D schematic views of the periodic and unit cell of the proposed dynamically controllable graphene-based dual-functional chiral metamirror composed of two-layered, complementary 90° rotated U-shaped resonator arrays are respectively shown in Fig. μc = 1 eV and for the bottom graphene, μc = 0.95 eV are considered. The relative permittivity of graphene, by assumption of the incident electromagnetic wave as ejωt, is4:σ, ω, ε0, and Δ are respectively the graphene surface conductivity, angular frequency, vacuum permittivity, and graphene thickness. Δ is considered to be 0.335 nm7. σ is the sum of the inter- and intra-band electron transition contribution terms based on the Kubo formula reported in4.For the upper graphene, 4:β, k0, and η0 are respectively the propagation constant of the electromagnetic wave in a graphene-vacuum setup, the wave vector of the incident light wave, and the vacuum impedance.The propagation constant for the electromagnetic wave propagation in a graphene-vacuum setup can be obtained according torTE/TM were defined by CST simulations considering the front and the back patterned graphene layers respectively on one half-space  approach to the designed dual-functional metamirror structure is based on the modeling of each patterned graphene layer as the equivalent conductivity mentclass2pt{minimmentclass2pt{minimrTE/TM for the setups considered in Fig. 41:The TE and TM reflection coefficients The ECM of the introduced dual-functional chiral metamirror is presented in Fig. The transfer matrices of the patterned graphene layers and the dielectric layers are:The matrix components are calculated and given as follows:The input impedance of the chiral metamirror is calculated by:The scattering parameter Additionally, So,So,So,So,So,So,RTE/TM of the dual-functional chiral metamirror is calculated by:Finally, the reflection coefficients 42. The boundary conditions and the mesh type are considered the same as in Ref.4. The metamirror was excited by TE and TM electromagnetic waves in the z-direction.Numerical simulations were done using the finite element method (FEM) in the frequency domain solver of CST 2018μc, the resonance frequencies of the real and the imaginary parts of β in Eq.  vs μc spectrum for the metamirror is given in Fig. 45. Chiral metastructures exhibit strong interaction with light and they can improve the chirality response of biomolecules by orders of magnitude46. Using of graphene-based chiral metastructures, it is possible to dynamically tune the chirality response which can greatly improve the detection sensitivity3. By the increasing μc, the maximum LD value increases. The maximum obtained LD is 96% occurred for μc = 1 eV for the upper graphene and 0.95 eV for the bottom graphene. Additionally, the maximum LD vs τ and vs θin are respectively given in Fig. τ or θin, the maximum LD could be tuned without a need to refabricate the metamirror. By increasing τ, the maximum LD value increases. By increasing θin, the maximum LD value decreases. As a conclusion from Fig. μc = 1 eV for upper graphene and 0.95 eV for the bottom graphene, τ = 1 ps, and θin = 0°.The maximum linear dichroism 16 or they have to be refabricated with different dimensions49. This has potential applications in THz intelligent systems by greatly reducing the system size and could act as a broadband and multiband metamirror by just alternating the incident electromagnetic wave from TE to TM. Our proposed metastructure could be used for imaging objects under the broad-band reflection or distinguishing them in case of multi-band reflection16.The proposed chiral metamirror was compared with previously published chiral metamirrors in Table 51 at least for the complementary pattern design, which does not require ion gel to properly bias the graphene pattern. The fabrication steps of the proposed chiral metamirror could be done as follows: (1) the bottom quartz dielectric is deposited on a gold metal sheet through thermal evaporation, (2) the bottom graphene layer is coated on the quartz by chemical vapor deposition (CVD), (3) the bottom graphene pattern is written by electron beam etching, (4) the upper quartz layer is transferred on the combination by thermal evaporation, (5) the upper graphene layer is coated on the upper quartz by CVD, and (6) the upper graphene pattern is written by electron beam etching53.The fabrication of a structure containing two graphene layers is more difficult than the fabrication of a device containing a single graphene layer. However, the fabrication of multilayer graphene structures has been shown to be possibleThe proposed metamaterial model assumes that all the layers are fabricated without any defects. The fabrication of the proposed device and its influence on metamaterial performance needs further investigation.2 or to refabricate the structure with new dimensions for switching between broadband and multiband reflection spectra. The reported structure acts as a broad-band TE mirror and multi-band TM mirror but with a 90° rotation of the structure, it could act as a broad-band TM mirror and a multi-band TE mirror. Additionally, the proposed metastructure could find some other potential applications in tunable polarization sensitive structures in the future.In this paper, an equivalent circuit modeling (ECM) approach to a tunable graphene-based dual-functional chiral metamirror composed of complementary 90° rotated U-shaped resonator arrays by using simple and fast MATLAB code was proposed, designed, and analyzed in the terahertz (THz) region. The simulation results were done using the finite element method (FEM) in CST Microwave Studio Software and were in good agreement with the ECM ones. The proposed ECM approach could be used for modeling other two-layered chiral metastructures for any frequencies, containing resonators with a thickness in order of less than λ/50. Our metamirror device is tunable, and it has strong linear dichroism (LD) response of 96%. It is a dynamically tunable, dual-functional mirror in the 0.3–4.5 THz range which makes it very promising for future THz intelligent systems. Using one device to achieve two different functions can greatly reduce the size of terahertz system thus saving material, costs, and time as there is no need to use a phase change material such as VO