With the continuous advancement of wireless communication technology, the use of electromagnetic radiation has led to issues such as electromagnetic interference and pollution. To address the problem of electromagnetic radiation, there is a growing need for high-performance electromagnetic shielding materials. Graphene, a unique carbon nanomaterial with a two-dimensional structure and exceptional electrical and mechanical properties, offers advantages such as flexibility, light weight, good chemical stability, and high electromagnetic shielding efficiency. Consequently, it has emerged as an ideal filler in electromagnetic shielding composites, garnering significant attention. In order to meet the requirements of high efficiency and low weight for electromagnetic shielding materials, researchers have explored the use of graphene–polymer nanocomposite foams with a cellular structure. This mini-review provides an overview of the common methods used to prepare graphene–polymer nanocomposite foams and highlights the electromagnetic shielding effectiveness of some representative nanocomposite foams. Additionally, the future prospects for the development of graphene–polymer nanocomposite foams as electromagnetic shielding materials are discussed. With the increasing convenience and rapid connectivity provided by wireless telecommunication networks, the use of numerous telecommunication devices has resulted in an unprecedented level of electromagnetic interference (EMI). Electromagnetic radiation in the transmission process interferes with other electronic devices via electromagnetic induction, causing the disturbance or malfunction of appliances. Moreover, the growing electromagnetic pollution poses potential risks to human health ,2. To ta−1, makes it an ideal nanofiller in polymer nanocomposites for electromagnetic shielding applications [Polymer nanocomposites attract great attention since they combine the advantages of both polymers and nanoparticles ,4. Varioications ,13. Therications ,16,17,18When electromagnetic waves contact the surface of the electromagnetic shielding material, electromagnetic radiation will be attenuated in three ways: reflection, the absorption of electromagnetic waves by the shield material, and multiple reflection attenuation inside the shield. Good EMI shielding materials require high electrical conductivity in order to effectively reflect electromagnetic radiation. This is because the shielding material reflects radiation through direct interaction with electromagnetic fields, facilitated by charge carriers. On the other hand, absorption occurs when the radiation interacts with electric or magnetic dipoles in the shielding material. Additionally, scattering at the interface between materials plays a crucial role in the multiple reflection mechanism ,19. TherA range of publications have examined the EMI shielding properties of carbon-based composites ,16,17,18There are three primary pathways for preparing nanocomposite foams consisting of graphene and a polymer . The intThe widely used dip-coating method can be facilely implemented to prepare graphene–polymer nanocomposite foams by using polymeric foams as a template ,26,27,28The second route to graphene–polymer nanocomposite foams is covering the graphene foams with a polymer layer. Graphene foams are typically made by the chemical vapor deposition (CVD) method using a nickel foam (Ni foam) as a template . Since o−1. Coating the graphene foam with a polymeric layer is a straightforward process that involves immersing it in a low-concentration polymer solution [For the CVD graphene foam, the Ni foam scaffold is typically treated with a HCl solution after applying a thin polymer layer onto the graphene film . The ressolution ,31,32,33solution ,35,36. B2) for foam preparation [2 at a certain temperature. When the pressure is released, CO2 gas bubbles appear and grow, leading to foam formation with the necessary equipment  contributes less than microwave absorption (SEA) to the total shielding efficiency . Specifically, as 1.8 vol% graphene is incorporated, microwave absorption is the dominant mechanism of microwave shielding. This is presumably due to the conductive dissipation of the incident microwave in foam cells with graphene in the foam frame. Multiple reflections can explain the result as a reasonable shielding mechanism. When the incident microwave is frequently reflected and/or scattered by the microcell–frame interfaces, it is difficult for the trapped microwave to escape, and it is eventually absorbed. The specific shielding efficiency (SSE), which is calculated from EMI SE divided by the density, of the composite foam with 1.8 vol% graphene is 17–25 dB cm3/g. Meanwhile, a large number of microcells in the foam greatly enhanced the toughness in comparison with its solid counterpart. This work proved that graphene–PMMA microcellular foams would be a light and tough candidate suitable for EMI shielding applications.Zhang and coworkers dispersed graphene sheets into a PMMA matrix to fabricate their nanocomposite foams. Microcellular cells with a size dispersity from 1 to 10 μm were made by using subcritical COng agent . The add3 loading. They are labeled GPS045 and GPS027, representing their densities of 0.45 g cm−3 and 0.27 g cm−3. Correspondingly, their porosities are 60% and 76%. Since the same content (30 wt%) of graphene was loaded, the conductivity of GPS045 (1.25 S m−1) was higher than that of GPS027 (0.22 S m−1). As shown in total of GPS045 was higher than that of GPS027 in the whole frequency range. The average SEtotal of GPS045 was approximately 29.3 dB, while that of GPS027 was 17.3 dB for both 2.5 mm thick samples. It indicates that superior conductivity contributes more to the shielding performance than higher porosity. On the other hand, a much greater contribution of SEA to SEtotal indicates that more microwaves are absorbed through dissipation as heat rather than being reflected. As for this conductive composite foam, electromagnetic absorption is the primary EMI shielding mechanism in the X-band. The SSE of the porous graphene–PSt composite with a thickness of 2.5 mm is surprisingly 64.4 dB cm3/g.As mentioned before, Yan et al. proposed and implemented a novel method combining compression molding under high pressure and salt leaching to fabricate porous graphene–PSt (GPS) composites . Two pro2SO4 and HNO3 mixture and then mixed functionalized graphene (f-G) with PVDF and the foaming agent  in dimethyl formamide (DMF). The composite film was casted in a Petri dish and dried in the oven. The f-G/PVDF foam with cell sizes of 0.5–2 μm was prepared by hot pressing due to the decomposition of the foaming agent [−16 S m−1 for neat PVDF to conducting 10.16 S m−1 for the PVDF composite foam with 2 wt% f-G. The f-G fillers form conductive paths throughout the PVDF matrix above the percolation threshold (pc = 0.5 wt%)  a. The hi0.5 wt%) b. In conT and SEA both increased, but SER changed little. The incident electromagnetic waves were attenuated by multiple internal reflections, absorption, and scattering due to the heterogeneous structure of hybrid fillers and the porous structure of the composites.Recently, a binary polymer matrix of PVDF and epoxy resin (EP) was used to disperse and fix graphene nanosheets (GNS) functionalized with nickel–cobalt (Ni-Co) alloy particles to form high-performance EMI shielding composites . The gra3. Transitioning from the solid nanocomposite structure to the foam structure led to a decrease in the percolation threshold from 0.21 vol% to 0.18 vol%. This could be attributed to the growth of cells that facilitated the flow of graphene sheets, resulting in the orientation and accumulation of graphene along the cell walls. At the same loading of graphene (10 wt%), the electrical conductivity was 4.8 × 10−6 S/cm for graphene–PEI nanocomposite solids, while for the foam counterpart, it was 2.2 × 10−5 S/cm. Additionally, foaming significantly increased the EMI SSE from 17 to 44 dB/(g/cm3). These graphene–PEI nanocomposite foams also demonstrated a high Young’s modulus and extremely low thermal conductivity.Zhai et al. employed the WVIPS method to facilely prepare graphene–PEI nanocomposite foams . The hom3O4 (G@Fe3O4)/PEI composite foam by introducing Fe3O4 nanoparticles into the graphene–PEI system [3). The incorporated Fe3O4 nanoparticles improved impedance matching and attenuated the incident electromagnetic wave. The microwave was thus eventually absorbed via the combination of multiple reflections. The as-prepared G@Fe3O4/PEI foam with 10 wt% content of G@Fe3O4 had an EMI SSE as high as ∼41.5 dB/(g/cm3) in the X-band region. Meanwhile, the introduction of Fe3O4 nanoparticles endowed the resulting foams with superparamagnetic behavior, which facilitated its use in EMI shielding and magnetic actuation applications.Zhai and coworkers further developed a high-performance graphene@FeI system . The fleThe heatproof aromatic polyimide (PI) was also used to prepare a graphene composite foam in three steps . The pol−1 and an impressive electromagnetic interference specific shielding effectiveness (EMI SSE) of 693 dB cm−2 g−1. Similar to the previous study, the incorporation of PI into the composite film provided exceptional thermal stability and mechanical properties.In contrast to the previous approach of directly using reduced graphene oxide (rGO), Yang et al. implemented a different method to create lightweight rGO/PI composite foams . They in3O4 nanoparticles were both adsorbed onto the PI foam by dip coating. The impregnated PI foam with graphene–Fe3O4 fillers exhibited an EMI SE of 60.6 dB at a thickness of 6.0 mm with low reflection. When the loading of Fe3O4 nanoparticles increased, the conductivity and EMI SE of composites rose as well due to the synergistic effect of graphene and magnetic Fe3O4 nanoparticles. Additionally, dilute graphene could reduce reflections on the external surface, make more microwaves enter the interior, and realize the absorption domination of microwaves.Li and coworkers prepared a PI open-cell foam with pyromellitic diester and catalysts as component A and polyphenylene isocyanate as component B by a free-foaming method . Apart f−1. The relationship curves of the EMI shielding efficiency versus frequency for composite foams with the incorporation of different rUL-GO are shown in −1 cm−3) at 8–12 GHz were achieved due to the method of foam preparation and a uniform dispersion, together with a high aspect ratio for rUL-GO. The introduced rUL-GO improved the mechanical properties of the PU matrix without a decline in flexibility.Graphene–PU foams have been made by different groups using different methods due to the unique properties of the PU matrix. Gudarzi and coworkers fabricated multifunctional graphene–PU composite foams via in situ polymerization with reduced ultralarge graphene oxide (rUL-GO) . The add3. More importantly, they showed high-performance EMI shielding efficiency, which was possibly contributed to by an absorption-dominant mechanism, conductive dissipation, multiple reflections, and the scattering of EM waves by the conductive graphene network  of methane at 1000 °C . To enha3 at a low graphene content of <0.8 wt%, and its EMI SE reached 30 dB with a specific EMI SE of up to 500 dB · cm3/g  and conductive nanoscale carbon nanotubes (CNTs) . These m3O4/rGO as fillers, and PDMS as a binder to anchor the fillers onto the framework by dip coating [3O4/rGO on the skeleton synergistically gained excellent impedance matching and electromagnetic absorption performance, while the conductive Ag formed on the bottom helped the asymmetric composite achieve a superior EMI SE of 70 dB and a high absorption coefficient of 0.86 in the X-band.Wang et al. designed and prepared porous composites with polyaniline-modified melamine foam as a framework, Fe coating . With Ag−3 g/cm3 and a high porosity of 98.8%. The electrical conductivity was enhanced from 11.8 to 43.2 S/cm after the incorporation of the conductive PEDOT:PSS. The composites had an incredible EMI SE of 91.9 dB and a high SSE of 3124 dB·cm3/g because of their high electrical conductivity, porous structure, and effective charge delocalization :poly(styrenesulfonate) (PEDOT:PSS) foam composites were prepared by drop coating PEDOT:PSS on freestanding graphene foams (GFs) . GFs werlization . The exc3O4 particles [4 · H2O via nonsolvent-induced phase separation (NIPS). The Fe3O4 particles were grown in situ in NH4 · H2O by the co-precipitation method. The incorporated graphene–carbon nanotubes enhanced the conductive loss of incident microwaves, while the magnetic particles contributed to the dielectric loss and magnetic loss. When the content of Fe3O4 was 3.55 wt%, the composite foam had the highest EMI SE of 38 dB and the highest absorption ratio of 94%. The PEN matrix also rendered good thermostability for the composite foam, which paved the way for its practical application in EMI shielding.Zhang and coworkers recently reported a porous absorption-dominated EMI shielding material composed of poly(arylene ether nitrile) (PEN), graphene–carbon nanotubes, and Fearticles . The com(1)Enhancing the EMI shielding effectiveness of polymers: Although polymers have advantages such as light weight, corrosion resistance, and easy processing, most polymers have weak intrinsic shielding properties against electromagnetic waves. To enhance their EMI shielding effectiveness, conductive polymers such as polyaniline and polypyrrole can be selected as EMI shielding scaffolds.(2)Incorporating other functional materials: Adding metallic powders, semiconductors, inorganic/organic compounds, and magnetic nanoparticles can improve impedance matching and increase the dissipation capacity of electromagnetic waves. Additionally, new two-dimensional materials such as oxides, nitrides, and black phosphorus can be added as impedance amplitude modulators and media to improve the impedance matching of materials and enhance polarization and multiple reflections at the interface.(3)Investigating the role of porosity and cell size: Although some studies have mentioned the influence of porosity and cell size on the EMI shielding effectiveness of composite foams, a systematic investigation is still needed to understand the general rules governing how and why the cell density and size affect the reflection or absorption of incident electromagnetic waves.(4)Considering thickness-specific efficiency: Currently, the weight-specific shielding efficiency of foams is often emphasized, while the thickness-specific efficiency is usually ignored. It would be more reasonable to evaluate the shielding efficiency by dividing it by the thickness dimension of samples.This mini-review does not aim to provide a comprehensive summary of all the recent progress made in this field, as there has been an increasing amount of research and continuous presentation of smarter designs with better results. Several good reviews have already provided different perspectives on related topics, such as the foaming of polymeric composites and the structural properties of effective EMI shielding materials ,63,64,65With rational design and effective implementation, graphene-based foam materials are expected to play a significant role in electromagnetic shielding due to their ultralight weight, high strength, and exceptional efficiency.


Staphylococcus aureus (MRSA) biofilms can be resilient to the human immune system and antibiotics, a challenge augmented by acquired antimicrobial resistance. Phage therapy is being evaluated for biofilm-associated infections.Methicillin-resistant Here, model phages SaMD07ø1 and SaRB105030ø5 were evaluated against their hosts SaMD07 and SaRB105030, respectively, grown as biofilms in 96-well plates, as biofilms on glass beads and planktonically. Trypticase soy broth (TSB) and phosphate buffered saline (PBS) were evaluated as testing media . Biofilms were incubated with phages in the presence of media containing tetrazolium dye in 96-well plates and assessed for hold times  over 48 hours (h) using a Biolog Omnilog® system and by assessing colony forming units (CFUs). Subsequently, using the beads biofilm assay in TSB medium performed on the Omnilog® (BBTO) , test phage SaMD22ø1 was evaluated in quintuplicate against biofilms formed by MRSA isolates IDRL-6130, IDRL-6927, IDRL-6987, IDRL-7381, IDRL-7542 and IDRL-10947, all recovered from periprosthetic joint infection. Test phage K was evaluated against GFP-labelled MRSA AH1263 using BBTO method and simultaneously by fluorescence microscopy.A comparison of result of testing of biofilms in wells and on beads and against planktonic bacteria is shown in Figure 3. Phage SaMD07ø1 showed no growth at 4 h; whereas phage SaRB105030ø5 showed ∼1-log decrease in CFUs at 4 h and ∼3-log decrease in CFUs at 8 h . BBTO hold-times of 37, 28 and 15 h were observed for phages SaMD07ø1, SaRB105030ø5 and phage K respectively. Phage SaMD22ø1 had an average hold time of 9 h and showed hold times >6 hours for all isolates tested . Microscopic inspection showed eradication of GFP-labelled MRSA biofilm by phage K .The solid blue line indicates a hold time of 8 hours.The authors are thankful to Dr. Alexander Horswill at University of Colorado for providing the GFP-labelled MRSA strain.In conclusion, BBTO is a promising method for testing biofilm phage susceptibility although clinical correlation and testing of larger numbers of phage and bacterial isolates are needed.Joseph Fackler, n/a, Adaptive Phage Therapeutics, Inc: Employee|Adaptive Phage Therapeutics, Inc: Stocks/Bonds John M. Sowers, n/a, Adaptive Phage Therapeutics: Employee|Adaptive Phage Therapeutics: Stocks/Bonds Robin Patel, MD, Abbott Laboratories: Advisor/Consultant|Adaptive Phage Therapeutics: Grant/Research Support|Adaptive Phage Therapeutics: Mayo Clinic has a royalty-bearing know-how agreement and equity in Adaptive Phage Therapeutics.|BIOFIRE: Grant/Research Support|CARB-X: Advisor/Consultant|ContraFect: Grant/Research Support|Day Zero Diagnostics: Advisor/Consultant|HealthTrackRx: Advisor/Consultant|Mammoth Biosciences: Advisor/Consultant|Netflix: Advisor/Consultant|Oxford Nanopore Technologies: Advisor/Consultant|PhAST: Advisor/Consultant|See details: Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic|See details: continued, patent on an anti-biofilm substance issued|TenNor Therapeutics Limited: Grant/Research Support|Torus Biosystems: Advisor/Consultant|Trellis Bioscience, Inc.: Advisor/Consultant


Our new therapeutic antibody and caninized PD-L1 mouse model will be essential translational research tools in raising the success rate of immunotherapy in both dogs and humans.Immune checkpoint blockade therapy, one of the most promising cancer immunotherapies, has shown remarkable clinical impact in multiple cancer types. Despite the recent success of immune checkpoint blockade therapy, however, the response rates in patients with cancer are limited (∼20%–40%). To improve the success of immune checkpoint blockade therapy, relevant preclinical animal models are essential for the development and testing of multiple combination approaches and strategies. Companion dogs naturally develop several types of cancer that in many respects resemble clinical cancer in human patients. Therefore, the canine studies of immuno-oncology drugs can generate knowledge that informs and prioritizes new immuno-oncology therapy in humans. The challenge has been, however, that immunotherapeutic antibodies targeting canine immune checkpoint molecules such as canine PD-L1 (cPD-L1) have not been commercially available. Here, we developed a new cPD-L1 antibody as an immuno-oncology drug and characterized its functional and biological properties in multiple assays. We also evaluated the therapeutic efficacy of cPD-L1 antibodies in our unique caninized PD-L1 mice. Together, these Our cPD-L1 antibody and unique caninized mouse model will be critical research tools to improve the efficacy of immune checkpoint blockade therapy in both dogs and humans. Furthermore, these tools will open new perspectives for immunotherapy applications in cancer as well as other autoimmune diseases that could benefit a diverse and broader patient population. Immune checkpoint blockade therapy, one of the most promising forms of cancer immunotherapy, has been successful in multiple cancer types, including invasive urinary bladder cancer, the focus of this study . In partStrategies to improve PD-1/PD-L1 blockade therapies in bladder cancer and other cancers include: (i) identifying host factors including genetics, immune state, and molecular subtype that drive a relevant response, (ii) assessing biomarkers and combinations of biomarkers to predict response and to personalize therapy, (iii) developing better tools to monitor immune effects, and (iv) selecting combination drug approaches/regimens to address multiple “defects” in the immune response in addition to PD-1/PD-L1 blockade. Relevant preclinical animal models are essential to developing these strategies and testing multiple combination approaches. Factors that are likely to affect the PD-1/PD-L1 axis and thus must be represented in animal models, include aggressive and metastatic cancer behavior, tumor heterogeneity, mutational landscape, genetic and epigenetic cross-talk, cancer molecular subtypes, immune cell responsiveness, and innate and acquired mechanisms of drug resistance. Experimental rodent models, including carcinogen-induced, engraftment, and genetically engineered models, are instrumental in research of different types of cancer including bladder cancer . HoweverWhile these collective features are lacking in rodent models, we have demonstrated that pet dogs with naturally occurring invasive urothelial carcinoma  can provide this crucially needed relevant model in an immunocompetent host. The canine model can complement other models to drive preclinical research to understand and optimize drug activity in humans. Canine InvUC mimics human InvUC in presentation, pathology, local invasion, distant metastases (lung and other organs in >50% of cases), and chemotherapy response . Canine Canine PD-1/PD-L1 blockade antibodies are not commercially available for dogs with InvUC. The development of canine PD-L1 (cPD-L1) antibodies has been described by several academic groups . Tumor rin vitro and in vivo. Furthermore, we validated the therapeutic efficacy of cPD-L1 antibodies using our newly established caninized PD-L1 mice, which express cPD-L1 on the cell surface. Our cPD-L1 antibody, a new immuno-oncology drug, and caninized PD-L1 mice will be essential translational research tools in raising the success rate of immunotherapy in dogs and humans.In this study, we developed a new immunotherapeutic cPD-L1 antibody and characterized its functional/biological properties ex vivo canine peripheral blood mononuclear cell (PBMC)-mediated killing assay, and (vii) initial safety and pharmacology study in dogs, and is summarized in The work included: (i) generation of cPD-L1 antibodies in mice, (ii) selection of the superior clone to further develop, (iii) evaluation of the cPD-L1 antibody in mice expressing cPD-L1 on the cell surface and implanted with tumor cells expressing canine PD-1, (iv) creation of a chimera antibody of the top clone, (v) characterization of the chimera antibody through cell-based and cell-free cPD-L1 antibody binding and cPD-L1/cPD-1 blockade assays, and other assays of essential characteristics, (vi) assessment of the antibody activity in an Mycoplasma using the Mycoplasma PCR Detection kit (ABM). The cells were grown for no more than 15 passages and discarded. Cells were grown in DMEM or DMEM/F12 medium supplemented with 10% FBS. For stable expression of cPD-L1, the cDNA of cPD-L1  was inserted into the pGIPZ vector (Horizen Discovery) as described previously (cPDL1), we infected mPD-L1 knockout MB49 cells with lentivirus carrying pGIPZ-Flag-cPD-L1 followed by selection with puromycin.The BT549 human breast cancer and MB49 mouse bladder cancer cell lines were obtained from ATCC and Millipore Sigma, respectively. The canine bladder cancer cell line, K9TCC was obtained from the Dr. Deborah W. Knapp Lab . The humeviously . Using aeviously , we estamAbs were generated via conventional hybridoma procedures using A/J mice immunized with the extracellular domain of cPD-L1 (attached to a human Fc tag) at the Vanderbilt University Antibody and Protein Resource Core Facility. Splenocytes were isolated from the immunized mice and then fused with SP2/0 myeloma cells see . SupernaCD274 knock-in mouse) was generated by Easi-CRISPR (Efficient additions with ssDNA insert-CRISPR) strategy using a long single-strand DNA (ssDNA) donor and CRISPR ribonucleoproteins  was injected with preassembled guide RNA  and Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. The ssDNA and single-guide RNA were synthesized at Integrated DNA Technologies. C57BL/6N female mice at 4 weeks of age (Envigo) were superovulated and then mouse zygotes were obtained by mating C57BL/6N males with the superovulated females. Pronuclei of one-cell stage fertilized mouse embryos were injected with 20 ng/μL Cas9 protein, 10 ng/μL sgRNA, and 5 ng/μL ssDNA. Microinjections and mouse transgenesis were performed as described previously (CD274.The caninized PD-L1 mouse (canine proteins . Brieflyeviously . Mouse gcPD-L1 [2 × 105 cells in 25 μL of medium mixed with 25 μL of Matrigel Basement Membrane Matrix (BD Biosciences)] were injected into the flank of the caninized PD-L1 mice . Mice were divided according to the mean tumor volume in each group. For treatment with antibodies, 100 μg of cPD-L1 antibody (12C10E4 or 3C8D3 clone) or control mouse IgG (Bio X Cell) was injected intraperitoneally on days 4, 6, 8, 10, and 12 after tumor cell inoculation when tumor size was approximately 30 to 40 mm3. Tumors were measured every other day with a caliper, and tumor volume was calculated using the following formula: π/6 × length × width2.MB49 Tumor masses were frozen in optimal cutting temperature blocks immediately after excision. Cryostat sections of 5-μm thickness were attached to saline-coated slides. Cryostat sections were fixed with 4% paraformaldehyde for 30 minutes at room temperature and blocked with blocking solution  at room temperature for 30 minutes. Samples were stained with primary antibodies against CD8 and granzyme B overnight at 4°C, followed by secondary antibodies at room temperature for 1 hour. Nuclear staining was performed with Hoechst 33342 (Thermo Fisher Scientific). The stained sections were visualized by automated microscopy . Granzyme B–positive area and the number of CD8-positive CTL were assessed per high power field (200X). Fourteen randomly chosen microscope fields from four serial sections in each tissue block were examined for the number of CD8-positive CTL and granzyme B–positive areas for each tissue.2. Cells were collected 10 days posttransfection by centrifugation at 4,000 × g and 4°C for 20 minutes. The antibody supernatant was passed through a 0.22-μm filter and neutralized with 10XPBS buffer  and preincubated with protein A agarose for 2 hours. The agarose A-conjugated antibodies were applied to the column . The column was washed with low-endotoxin PBS . Bound antibody was eluted with elution buffer  into Neutralization Buffer . Purified antibody was concentrated and buffer exchanged with PBS, pH7.0. The antibody concentration was determined by UV absorbance at 280 nm.The codon optimized for CHO variable light (VL) and heavy (VH) chains were cloned into pTRIOZ-hIgG1 vector (InvivoGen), and then the constant light and heavy chains were replaced with canine kappa light constant chain and canine IgG2 heavy constant chain: pTRIOZ-cIgG2-12C10E4. Plasmids encoding 12C10E4 chimeric antibody, pTRIOZ cIgG2 12C10E4, were transfected into ExpiCHO-S cells following the transfection kit instructions . ExpiCHO-S cells were cultured with ExpiCHO Expression Medium (Thermo Fisher Scientific) in a shaker incubator set at 120 rpm, 37°C and 8.0% CO450 value with a Synergy LX multi-mode reader. For the cPD-L1/cPD-1 blockade assays, Pierce Ni-NTA–coated 96-well plates (Thermo Fisher Scientific) were coated with cPD-L1-His protein and cPD-1-hFc protein , and anti-human IgG Fc-specific HRP-conjugated secondary antibodies (Thermo Fisher Scientific) were added. Then the cPD-L1 12C10E4 antibody was added. The bound PD-1-Fc protein was quantified by measuring OD450 value with a Synergy LX multi-mode reader.ELISA-based assays were performed to compare the receptor-ligand and receptor-antibody binding. The 6X His-tagged extracellular domain of cPD-L1 proteins was expressed in the ExpiCHO cell system (Thermo Fisher Scientific) and purified by the Ni-NTA agarose (Thermo Fisher Scientific) according to the manufacturer's protocol. For the cPD-L1/cPD-L1 antibody binding assay, Pierce Ni-NTA–coated 96-well plates (Thermo Fisher Scientific) were coated with cPD-L1-His protein and 12C10E4 antibody, and anti-canine IgG-specific horseradish peroxidase (HRP)-conjugated secondary antibodies (SouthernBiotech) were added. The bound PD-L1 antibody was quantified by measuring OD4 BT549cPD-L1 cells per well in 96-well plates and then incubated the plates with cIgG control , or 12C10E4 antibody, and anti-canine Alexa Fluor 488 dye conjugate (SouthernBiotech). Every hour, green fluorescent signal was measured and quantified by IncuCyte S3 (Sartorius). To measure PD-1 protein on the cells, we seeded 1 × 104 BT549cPD-L1 cells per well in 96-well plates, and then incubated the plates with cIgG control , or 12C10E4 antibody, cPD-1-hFc protein , and/or anti-human Alexa Fluor 488 dye conjugate (Thermo Fisher Scientific). Every 3 hours, green fluorescent signal was measured and quantified by IncuCyte S3 (Sartorius). The Image analysis was performed according to the manufacturer's protocol.The antibody binding and blockade assays were performed as described previously . BrieflycPD-L1, K9TCC, or K9TCCnRFP cells were washed twice with ice-cold cell staining buffer (BioLegend) and stained with cIgG control or 12C10E4 cIgG for 1 hour at 4°C. After three washes with staining buffer, cell samples were stained with Alexa Fluor 488-conjugated anti-canine IgG-specific secondary antibody for 30 minutes at 4°C. Cell samples were loaded on BD LSRFortessa (BD) for analysis. Data analysis was performed on FlowJo v9 software (BD).MB49, MB49D) of cPD-L1 protein and cPD-L1 antibody (12C10E4) was determined by Octet Biolayer interferometry using the Octet RED384 system (Sartorius). Briefly, His-tagged cPD-L1 protein was loaded on the Octet NTA biosensor at a concentration of 200 nmol/L. The association step was performed by submerging the sensors in three concentrations of the 12C10E4 antibody  in the kinetic buffer. Dissociation was performed and monitored in fresh kinetic buffer. Data were analyzed with Octet Analysis HT software (Sartorius).The binding affinity (KThe purity and isoelectric point (pI) of the purified antibodies were determined by SDS-PAGE and isoelectric focusing (IEF), respectively. SDS-PAGE or IEF gels were purchased from Bio-Rad Laboratories or Thermo Fisher Scientific. The SDS-PAGE, IEF, and Coomassie blue staining were performed according to the manufacturer's protocol. Image acquisition and quantitation of band intensity were performed using Odyssey CLx infrared imaging system (LI-COR Biosciences).Size exclusion chromatography (SEC) analysis was performed to detect antibody aggregates and monomers. The AKTA Pure 150 M (Cytiva) and Superdex 200 Increase 10/300 GL column (Cytiva) were used to analyze antibodies at a flow rate 0.3 mL/minute for 135 minutes. Elution was monitored using UV absorption at 280 nm, and data were processed by Unicorn 7 software (Cytiva). The SEC analysis was performed in the Molecular Evolution, Protein Engineering, and Production core facility at Purdue University .The peptide mapping comparison for each 12C10E4 batch was performed. Briefly, the antibody was enzymatically digested with trypsin on S-trap micro columns from Protifi after reduction and alkylation . PeptideN-glycomic analysis of 12C10E4 antibody was performed by methods described previously  using the AB SCIEX TOF/TOF 5800 mass spectrometer . The structural assignments of the N-glycans were based on molecular weight and followed the principles of the N-glycan biosynthesis pathway. The carbohydrate analysis was performed at the Complex Carbohydrate Research Center, the University of Georgia .eviously . BrieflyPrimary canine PBMCs (cPBMC) were isolated from dog blood using SepMate PBMC isolation tubes (Stemcell Technologies) and Histopaque-1077 (Millipore Sigma) per the manufacturer's protocol. The activation of canine T cells by anti-canine CD3 and CD28 antibodies has been well established in the previous studies . BrieflyP < 0.05) were considered significant. Data were visualized using heatmap, volcano plot, and histogram for specific genes.RNA was isolated from activated cPBMCs as described previously  and submitted to the Stark Neurosciences Research Institute Biomarker Core, Indiana University School of Medicine, Indiana University, Indianapolis, IN, for detection of modulation of genes upon activation using the nCounter Canine IO Panel (NanoString Technologies). Data were analyzed using Rosalind . Groupwise comparison was conducted using control cPBMCs and compared with activated cells from three dogs. Differentially expressed genes (Fold change (FC) ≥ 1.5; The tumor cell killing assay was performed according to the previous description . To analMycoplasma free and contained <0.5 EU endotoxin/mg antibody (consistent with the endotoxin limit for human PD-L1 antibody solutions). After being acclimated to the facility, the dogs were treated with the 12C cPD-L1 chimera antibody diluted in sterile PBS for intravenous administration  and administered through an intravenous catheter over 1 hour. Six dogs were treated and received 2 or 5 mg/kg antibody. Blood was collected for pharmacokinetic analyses prior to treatment and at 1, 6, 24, 48, and 72 hours after the start of the antibody administration, and then once weekly for 4 weeks. Monitoring for adverse events included physical exam before and during treatment, then twice daily for 7 days, and then weekly for 4 weeks; daily observation for 4 weeks; and a complete blood count (CBC), serum biochemistry panel, and urinalysis before treatment and weekly for 4 weeks. Additional tests specific to any adverse events observed could be added. Adverse events were categorized using Veterinary Cooperative Oncology Group (VCOG) criteria [Veterinary Cooperative Oncology Group – common terminology criteria for adverse events (VCOG-CTCAE) following chemotherapy or biological antineoplastic therapy in dogs and cats v1.1] . The antibody solution was ts v1.1] .450 value with a Synergy LX multi-mode reader.The concentration of cPD-L1 antibody in dog serum was determined in pharmacokinetic analysis. Pierce Ni-NTA–coated 96-well plates (Thermo Fisher Scientific) were coated with cPD-L1-His protein and serially diluted dog serum , and anti-canine IgG-specific HRP-conjugated secondary antibodies (SouthernBiotech) were added. The standard curve was obtained from the standard samples of 12C10E4 antibodies prepared in standard sample dilution buffer: 0, 7.81, 15.63, 31.25, 62.5, 125, 250 ng/mL. The bound PD-L1 antibody was quantified by measuring ODt test. P < 0.05 was considered statistically significant.All quantitative results were displayed as the mean ± SD, with at least three biological replicates. The intergroup statistical significance was calculated by two-tail Student The data generated in this study are available upon request from the corresponding author.Anti-cPD-L1 mAbs were successfully generated using conventional hybridoma procedures . Please in vivo model. To do so, we established caninized PD-L1 (C57BL/6 background) mice. Briefly, we generated mice that expressed cPD-L1 on the cell surface by replacing the mouse Cd274 with canine CD274 using a CRISPR knock-in mouse strategy (cPD-L1) by knocking out mPD-L1 and reexpressing cPD-L1  constant domains. Briefly, we sequenced full-length VH and VL RNA transcripts obtained from hybridoma clones by 5′/3′ RACE and cloned these into the pTRIOZ-cIgG2-ck vector, which is designed for high-yield production of whole mAbs from a single plasmid . The chiD) of the chimeric antibody as determined by Octet was 8.6 nmol/L  are cocultured with activated canine PBMCs was established. The canine immune-oncology panel analysis (nRFP cells expressing nuclear-restricted RFP (nRFP) and confirmed the expression of endogenous cPD-L1 protein and mRNA in both K9TCC parental and K9TCCnRFP cells upon IFNγ treatment  was not matched between the cPBMCs and K9TCC cells, the 12C chimeric antibody enhanced tumor cell killing activity and IFNγ secretion  were transient and resolved without intervention  and measured the concentration of 12C chimeric antibody in dog serum . In an irvention .Companion dogs naturally develop several types of cancer that in many respects resemble clinical cancer in human patients . AlthougWe successfully developed a new cPD-L1 antibody as an immuno-oncology drug and characterized its functional and biological properties using multiple assays including a cPBMC-mediated canine tumor cell killing assay. It is recognized that in this assay  and N, tin vivo concentrations of cPD-L1 antibody that had good in vitro activity in PD-L1 binding and blockade assays and cPBMC-mediated killing of tumor cells. The shorter half-life of the cPD-L1 antibody compared with human checkpoint inhibitors  with the human PD-L1 gene (CD274) using CRISPR/CAS9 methods. The humanized PD-L1 mouse can be used to evaluate the therapeutic efficacy of anti-human PD-L1 antibodies in vivo. However, no caninized PD-L1 mouse model has been previously reported. The lack of a suitable mouse model is a major obstacle for developing canine ICIs such as PD-1/PD-L1 blockade antibodies, in which the ideal approach would be to move successful treatment approaches in mice to studies in dogs, and to further translate those with the highest success in dogs into human trials. To overcome this obstacle, we established a caninized PD-L1 mouse model as a preclinical tool, and validated the therapeutic efficacy of immunotherapeutic cPD-L1 antibodies in vivo (cPD-L1, are unique and powerful tools for preclinical canine immuno-oncology research.In the development of immuno-oncology drugs, particularly immunotherapeutic antibodies, translation of discoveries in mouse models to clinical trials has been hindered by multiple biological differences between mice and humans, such as the lack of cross-reactivity between species. For example, if an anti-human or cPD-L1 antibody does not recognize mPD-L1 protein, the therapeutic efficacy of that antibody cannot be evaluated in a syngeneic mouse model. Mice with an engrafted human immune system have been developed for translational research to help overcome this constraint. Indeed, pembrolizumab, an anti-human PD-1 antibody, showed tumor growth inhibition and CD8nografts . Despitenografts . As an a in vivo . Our canIn conclusion, our cPD-L1 antibody and unique caninized mouse model will be critical research tools to improve the efficacy of immune checkpoint blockade therapy in both dogs and humans. Furthermore, these tools will open new perspectives for immunotherapy applications in cancer as well as other autoimmune diseases that could benefit a diverse and broader patient population.Table S1The list of genes that were altered in the activated canine PBMCs from the nCounter canine immune-oncology panel analysisClick here for additional data file.


R (radius of the hexagon), Tp (thickness of the hexagon) and µc  to obtain optimum characteristics suitable for quantum plasmonic sensing applications. The study demonstrates that the proposed hexagonal-shaped nano-antenna exhibits gain of 4.9 dBi, 2.46 dBi, 14.99 dBi, 8.25 dBi, 5.15 dBi, 10.87 dBi and 2.4 dBi at 29.87 THz, 30 THz, 35 THz, 113.5 THz, 132.5 THz, 85 THz and 24 THz, respectively. The field enhancement factors observed at these frequencies are 794, 779, 584, 255, 234, 654 and 217, respectively.In this manuscript, a hexagonal-shaped graphene quantum plasmonic nanopatch antenna sensor is designed and investigated on silicon dioxide, zinc oxide and silicon substrates for quantum plasmonic biosensing applications. The optical properties of graphene are demonstrated using Kubo modeling to analyze the plasmon resonance characteristics of the nanopatch antenna. Nano-circuit modeling of the hexagonal-shaped graphene nano-antenna is proposed and validated using CST simulations. The parametric analysis of the hexagonal-shaped nanopatch antenna is performed using design parameters such as  Quantum plasmonic nano-antennas have prominent applications in biosensing since they can enhance optical energy extensively at the nanoscale, near the size of biomolecules6. These devices are based on the optical properties of the metals and their plasmon resonance frequencies are dependent on the refractive index of the surrounding media8. Conventionally, quantum plasmonic nanostructures are created using noble metals9. The mid-infrared frequency range has shown much importance in biosensing applications since most biological materials possess vibrational frequencies in this region11. Noble metals show plasmon resonance in the near visible region and have constraints9 such as low mobility, high losses, low sensitivity and tuning difficulty in the mid-infrared region13. Graphene, a two-dimensional material that produces surface plasmons within the infrared and terahertz regions, possesses extensive optical characteristics suitable to create plasmonic nano-antennas14 for biosensing applications. Among various nano-antenna structures, nanopatch is often used as a nanosensor since it can be easily designed and used15. In this regard, hexagonal-shaped graphene quantum plasmonic nano-antenna sensor is designed and studied on silicon dioxide, zinc oxide and silicon substrates.Plasmonics is a subfield of nanophotonics involving the study of surface plasmon polaritons (SPP)16. Graphene has dynamically tunable plasmon resonance characteristics that can be achieved through electrostatic biasing or doping16. It shows low dissipation and high plasmon confinement with extended life times compared to conventional metals14 used in the plasmonic antenna design. Moreover, graphene exhibits high quantum efficiency for quantum optical wireless communication17. Additionally, graphene is optically transparent, possesses high mobility, stability and it is abundantly available20. These properties of the graphene make it attractive for quantum plasmonic sensing applications15.Graphene plasmonics is an active research area that emerged recently by exploiting the unique properties of the graphene22. Rodrigo et al. demonstrated that the tunable plasmon resonance characteristics of graphene can be exploited to implement a graphene-based plasmonic biosensor11. Verma et al.23 showed that a layer of graphene in the sensor can enhance the performance of the biosensor. The enhanced sensitivity of the sensor by including a layer of graphene in a gold-based biosensor is reported by Zeng et al.24. A glucose sensor using graphene transistors is developed by Zhang et al. to increase the sensitivity of the sensor25. Zhou et al.26 studied and implemented a single-layer graphene sensor for the detection of cancer molecules. A DNA sensor using six graphene transistors is demonstrated by Xu et al.27 to increase the sensitivity of the biosensors. These all-reported works use graphene sheet, layer or transistor on the existing biosensing devices to increase the sensitivity. The inherent property of nano-antenna, which is the high field enhancement and the extensive properties of graphene can be exploited to build a nano-antenna using graphene to increase the performance of the biosensor.The invention of graphene in 2004 by Novoselov, stimulated the recent publication of research papers on graphene quantum plasmonic nano-antennas for sensing applicationsIn Graphene nanomaterial carbon atoms are connected in a hexagonal lattice structure so it is compatible to design a hexagonal-shaped nano-antenna using graphene. Moreover, hexagonal shape has six vertices therefore field concentration will be more near the edges. This will enhance the gain of the nano-antenna. In this regard, hexagonal shape for the graphene nanopatch antenna is chosen. Since graphene supports hexagonal shape, sensitivity of the graphene nano-antenna sensor will improve and also leads to easy fabrication of graphene patch antenna.30. But dispersive properties of the graphene are modelled using Kubo model. Kubo conductivity formula describes30 the surface conductivity of graphene, but the Drude permittivity model have to use volume conductivity of material. Since the thickness of graphene is very thin, the surface conductivity and volume conductivities supposed to be approximately same. Moreover, for the terahertz frequencies, the interband part of conductivity is negligibly small comparing with the intraband conductivity of graphene. Therefore, the surface conductivity from the Kubo model and volume conductivity from Drude model will be approximately same. The surface conductivity and plasma frequency (Drude Model) method of graphene modeling produce nearly the same simulation result for the same graphene antenna structure if they are compared. Hence dispersive properties of the graphene material is studies and analyzed using Kubo conductivity model.Traditionally, the Drude and Lorentz models are used to describe the dispersive properties of plasmonic metals and dielectrics respectivelyIn this manuscript, a hexagonal-shaped plasmonic graphene nanopatch antenna is designed and studied on different substrates for quantum biosensing biosensing applications. The nanocircuit modeling of the nanopatch antenna is proposed and validated using the finite integration technique. The next section of the paper portrays the design of a nanopatch graphene plasmonic antenna.R and Tp are the radius and thickness of the hexagonal-shaped patch, respectively. The proposed nanostructure is fed by a waveguide port matched to 50 Ω using a nanostrip line of width W. The entire nanostructure is designed on a square-shaped substrate with length Ls = 700 nm and thickness Ts = 36 nm. Graphene is modeled using parameters such as temperature Tg = 300° K, relaxation time tr = 0.1 psec, thickness t = 10 nm and chemical potential µc = 0.1 eV. A designed nanopatch antenna is studied in the CST Microwave Studio using silicon dioxide, zinc oxide and silicon dielectric substrates. The electromagnetic simulator used to analyze the hexagonal-shaped nanoantenna is the finite integration technique (FIT) in CST. The parametric analysis is performed on the hexagonal-shaped graphene nano-antenna to optimize the characteristics for quantum plasmonic sensing applications.Figure 19. Even though most of these methods are decade old, because of the popularity of the graphene these methods are used to produce graphene nanostructures recently19.Graphene nanoptach can be produced using several methods. Micromechanical cleavage can produce millimeter sized single layer graphene sheets; Liquid-phase exfoliation method can produce graphene nanoribbons with widths less than 10 nm; using graphene oxide large dispersion-processed graphene flakes can be produced; Chemical vapor deposition can produce single and few layer grapheneσg′(ω) and σg″(ω) in Eq.  voltage or with chemical doping. The variation of the bias voltage results in change in the conductivity of graphene. In this manuscript, graphene patch antenna is designed on a substrate whose gate voltage (bias voltage) which is connected in between the substrate and graphene sheet can modify the chemical potential and thus changing conductivityNano-circuit modeling is required to theoretically understand the behavior of nano-antennas. The concept is essential for interconnecting plasmonics with existing electronics. This will model the graphene nano-antenna using nano-inductor, nano-resistor and nano-capacitor. In this regard, nano-circuit modeling of a hexagonal-shaped graphene nanopatch antenna is designed and validated through CST simulations. Based on the fundamental concepts of nano-circuit theory, elements that exhibit negative real permittivity are modeled as nano-inductors and those that exhibit positive permittivity are modeled as nano-capacitors. The imaginary part of permittivity of the material is modeled as a nano-resistor; this signifies the ohmic losses contributed by the material. In this paper, a hexagonal graphene nanopatch is designed on silicon dioxide, zinc oxide and silicon substrates. The substrate materials exhibit positive real permittivity at terahertz frequencies and graphene exhibits negative real permittivity at the plasmon resonance. Hence, graphene nanopatch can be modeled as a nano-inductor  series with a nano-resistor (imaginary part of permittivity). The substrate materials are modeled as a nano-capacitor  and the fringing field effect is modeled using a fringing field nano-capacitor (positive background permittivity). All these aspects are considered in designing nano-circuit model for the proposed plasmonic graphene nanopatch antenna.Vdg and differential current Idg using ejωt time convention.E, L and A denote the incident field, the length of the hexagon and the cross-section area of the nanostructure, respectively. The nano-impedance of the proposed hexagonal-shaped nanostructure is derived using Eq.  comes parallel with the nano-impedance of the nanoptach antenna.This change will shift the resonance frequency slightly towards lower frequency region and reduces the reflections in the antenna. This leads to the change in the resonance characteristics of the nano-antenna. The proposed nano-antenna is designed on the substrate so that it will couple the graphene nanoptach efficiently. This makes the enhancement and localization of the optical signal in nanopatch antenna. The fringing nano-capacitance , zinc oxide , zinc oxide (W = 20 nm) and silicon substrates, respectively. The hexagonal-shaped graphene nano-antenna has a gain of 4.9 dBi, 2.46 dBi, 14.99 dBi, 8.25 dBi, 5.15 dBi, 10.87 dBi and 2.4 dBi at 29.87 THz, 30 THz, 35 THz, 113.5 THz, 132.5 THz, 85 THz and 24 THz, respectively . The study demonstrates that a hexagonal-shaped graphene nanoptach antenna exhibits minimum reflection and high gain on a silicon substrate. Further, graphene nano-antenna on a zinc oxide substrate has more bandwidth compared to other substrates. The field enhancement factors are calculated using the formula E0/Ei, where Ei is the incident field and E0 is the output enhanced field. It is observed that a maximum enhancement factor of 794 is obtained for graphene nanoptach on SiO2 substrate. This feature of nano-antenna can improve the sensitivity of the biosensor in biomedical applications.The optimum characteristics of the proposed hexagonal-shaped graphene nanopatch antenna on silicon dioxide, zinc oxide and silicon dieletric substrates are shown in Table R (radius of the hexagon), Tp (thickness of the hexagon) and µc . The study demonstrates that the proposed hexagonal-shaped graphene nano-antenna exhibits minimum reflection and high gain on a silicon substrate. Further, the graphene nanopatch antenna on a zinc oxide substrate has more bandwidth compared to other substrates. The optimum characteristics of the graphene nanopatch antenna occur at 29.87 THz with − 37.41 dB, 30 THz with − 24.75 dB, 35 THz with − 24.25 dB, 113.5 THz with − 22.78 dB, 132.5 THz with − 22.78 dB, 85 THz with − 41.84 dB and 24 THz with − 46.86 on silicon dioxide, zinc oxide and silicon substrates, which are appropriate for quantum plasmonic biosensing applications.A hexagonal-shaped graphene nanopatch antenna is designed and analyzed on silicon dioxide, zinc oxide and silicon dielectric substrates. Dispersive properties of graphene, such as permittivity and conductivity are studied using the Kubo model. The study demonstrates that graphene exhibits negative permittivity to support plasmon resonance at terahertz frequency. The nano-circuit model for the hexagonal-shaped nanopatch antenna is proposed and validated using CST simulations. The detailed analysis of the proposed hexagonal-shaped nanoptach antenna is performed using antenna parameters


Sarcoidosis affects approximately 1% of the population. Of these, up to one-third of patients will develop cutaneous manifestations. There are two situations where sarcoidosis can become life threatening; seizures secondary to neuro-sarcoidosis and arrhythmias due to cardiac infiltration. Currently, there are limited licensed therapies for the treatment of sarcoidosis. We describe a case of multi-system sarcoidosis whose extensive skin and pulmonary manifestations did not respond to conventional DMARDs and biologics requiring targeted therapies, in the context of recent military service.A 46-year-old male who worked as a Corporal in the Army presented with shortness of breath, blurry vision and painful red eyes to his local hospital. His symptoms had been ongoing for a few weeks prior to presentation. On examination he had reduced air entry in his lungs. His heart sounded regular; however, it was noted that he had erythema nodosum on his shins alongside tender nodular skin lesions on his elbows, feet and arms. He had synovitis in his MCPs and PIPs bilaterally. A chest X-ray demonstrated bilateral hilar lymphadenopathy and routine blood work was unremarkable. He was referred to the ophthalmologists who noted he had bilateral panuveitis with retinal vasculitis. His QuantiFERON, HIV and syphilis serology were negative; however, his hepatitis B core antibody was positive with a negative DNA. A skin biopsy confirmed non-necrotising granulomas and a subsequent HRCT demonstrated small volume bilateral pleural effusions, central mediastinal and hilar lymphadenopathy with micronodular changes. An EBUS confirmed non-necrotising, non-caseating epithelioid granulomas consistent with a diagnosis of sarcoidosis. He was commenced on a tapering dose of prednisolone (30mg to start with), hydroxychloroquine (400mg) and methotrexate (15mg). He remained well until the return of his lesions 24 months later. On assessment, his skin lesions had returned with dactylitis in his toes and synovitis in his wrists. Investigations revealed no improvement in his vital capacity and a markedly raised Il2Ra of 5739ng/L which was previously 2812ng/L (reference range 423-1843ng/L). A decision was made to commence infliximab (5mg/kg) to manage his inflammatory arthritis and cutaneous sarcoidosis. He remained well on infliximab for 6 months with the recurrence of his skin lesions requiring repeated courses of prednisolone (30mg). A CT-PET demonstrated active pulmonary and skin lesions. A decision to escalate his treatment was made.Our patient had both biochemical and radiological evidence of cutaneous and pulmonary sarcoidosis affecting his quality of life both physically and psychologically. He has significant exertional breathlessness, skin lesions across his torso and abdomen, and scored highly for depression and anxiety on GAD7 and PHQ9 respectively. There are few therapies available for the management of sarcoidosis beyond TNF inhibitors; however, there are promising clinical trials for the use of tofacitinib in cutaneous sarcoidosis. Our patient demonstrated both a seronegative inflammatory arthritis alongside his sarcoidosis and a decision was made to commence tofacitinib 5mg twice daily. His baseline DAS-28 score was 5.81. His skin lesions have gradually resolved 8 weeks after starting treatment and he has had an improvement in his respiratory and musculoskeletal symptoms. This is a very promising initial result for a treatment currently undergoing clinical trial for use in sarcoidosis.It is likely that his occupational risk factors could have triggered the sarcoidosis initially. He worked as a Corporal in the Army including 3 tours to Iraq over a 5-year period and would have been exposed to unspecified particular matter. Presentations of sarcoidosis following military service have been described previously. Similarly, there was a large spike in incidence of pulmonary sarcoidosis reported following the collapse of the Twin Towers. This is thought to be related to the increase in aerosol particular matter following the disaster, triggering an immune response when inhaled in susceptible adults. This has the potential to have financial implications for the patient.Sarcoidosis remains a challenging disease to diagnose and manage given its heterogenicity, but has potentially fatal consequences if left untreated. There is no single diagnostic test. Presentation varies from innocent bystander with isolated lymph node disease, to developing high disease burden with systemic involvement. This case highlights the potential for environmental factors to trigger sarcoidosis in susceptible patients. JAK inhibitors are the latest development in the field of sarcoidosis. Currently the only recognised JAK inhibitor for the treatment of sarcoidosis is tofacitinib as it inhibits both interferon by JAK1 and G-CSF through JAK2/3. STAT1 has been isolated during immunohistological analysis of skin lesions in sarcoidosis which is inhibited by tofacitinib and forms the basis of its use in cutaneous sarcoidosis. The management of sarcoidosis in the acute setting is commonly prednisolone; however, steroid sparing therapies should be considered in patients needing recurrent, prolonged courses of corticosteroids or those with evidence of critical organ involvement such as CNS, cardiac or lung disease. Life threatening disease or disease refractory to DMARD therapy should be considered for anti-TNF therapy with infliximab or adalimumab. More recently, tofacitinib has been promising as a treatment for sarcoidosis as seen in this case with clinical trials underway in Yale and Portland. Newer therapies in development include the GM-CSF inhibitor namilumab, and efzofitimod, a neuropilin 2 inhibitor.


Graphene is a carbon-based nanomaterial used in various industries to improve the performance of hundreds of materials. For instance, graphene-like materials have been employed as asphalt binder modifying agents in pavement engineering. In the literature, it has been reported that (in comparison to an unmodified binder) the Graphene Modified Asphalt Binders (GMABs) exhibit an enhanced performance grade, a lower thermal susceptibility, a higher fatigue life, and a decreased accumulation of permanent deformations. Nonetheless, although GMABs stand out significantly from traditional alternatives, there is still no consensus on their behavior regarding chemical, rheological, microstructural, morphological, thermogravimetric, and surface topography properties. Therefore, this research conducted a literature review on the properties and advanced characterization techniques of GMABs. Thus, the laboratory protocols covered by this manuscript are atomic force microscopy, differential scanning calorimetry, dynamic shear rheometer, elemental analysis, Fourier transform infrared spectroscopy, Raman spectroscopy, scanning electron microscopy, thermogravimetric analysis, X-ray diffraction, and X-ray photoelectron spectroscopy. Consequently, the main contribution of this investigation to the state-of-the-art is the identification of the prominent trends and gaps in the current state of knowledge. Transportation infrastructure is a crucial element for the socioeconomic development of communities ,2,3,4. TOverall, high-performance asphalt binders are achieved by mixing the virgin/pristine binder with an additive agent ,24,25,26In the last two decades, nanomaterials have been used with great notoriety to modify a broad type of materials, including asphalt binders ,40,41,42Unlike neat asphalt binders, GMABs have such a complex chemical structure that they cannot be characterized only with traditional laboratory tests  ,32,46,49The preceding situation has motivated this manuscript to develop a comprehensive literature review on the properties and characterization techniques of GMABs. In this way, the main objective of this manuscript is to identify the main trends and gaps in the current state-of-the-art. Thus, it is expected that this investigation encourages researchers to improve their current practices and address those aspects that are still susceptible to improvement in the future. It is important to note that the scope of this review is limited to three graphene-like materials, i.e., graphene, GO, and GNPs.Following, the structure of the subsequent sections of this paper is described. Asphalt binder is one of the derivatives obtained from refining crude oil  ,60,61,62Due to the variety of petroleum sources, each asphalt binder has a distinct chemical composition and a distinct performance for engineering applications ,69. BeyoIf the amount of resins and asphaltenes increases, the asphalt binder exhibits a more solid-like state. As a result, it causes an increase in stiffness.If the amount of saturates and aromatics increases, the asphalt binder exhibits a more liquid-like state. As a result, it causes a decrease in stiffness.If the amount of aromatics and asphaltenes increases, the asphalt binder reaches better properties at high temperatures.If the resin content increases, the viscosity of the asphalt binder augments proportionally.Adequate amounts of saturates, aromatics, and asphaltenes provide an appropriate fluid state.A low asphaltene content improves the temperature sensitivity behavior.The equilibrium of SARA fractions is essential in the behavior of asphalt binder because the maltenes allow the proper state of fluidity/workability, and the asphaltenes ensure adhesion with aggregates ,84,85. SIn this way, it is evident that a delicate chemical equilibrium in the SARA fractions widely controls the engineering performance of asphalt binders. The preceding is particularly important because incorporating graphene-like materials alters the SARA fractions. Thus, it explains why GMABs exhibit such complicated behavior, which is difficult to understand only by using traditional laboratory protocols. Subsequent sections of this manuscript detail the characteristics and properties of GMABs.The further substantial effect that graphene-like materials have in asphalt binders is that the introduction of these additives prevents the oxidation aging mechanism (by extending the diffusion path of the gas) ,89,90,91Consequently, it is notorious that the oxidation mechanism generates a complicated chain reaction of molecular rearrangements ,105,106.Despite being (correctly) isolated less than two decades ago , graphene is a widespread substance with applications in dozens of heterogeneous industries  ,114,115.The GO is the 2D material formed by oxidizing graphite, which is usually achieved with chemicals, such as sulphuric acid ,51,88,91GNPs are platelet-like graphite nanocrystals formed by several graphene layers  ,133,134.Some main preparation methods for graphene-like materials are anodic bonding, chemical synthesis, using benzene as the building block, chemical vapor deposition process, growth from SiC, liquid phase exfoliation, micromechanical cleavage, molecular beam epitaxy, photoexfoliation, and precipitation from a carbon-containing metal substrate ,145,146.GMABs have remarkable resistance to different aging processes, i.e., thermal, ultraviolet, and even water aging ,90,161. Direct addition method: the graphene-like materials are directly added into the asphalt binder (previously elevated to a high temperature).Indirect addition method: the graphene-like materials and asphalt binder are simultaneously dissolved into a medium solution to subsequently form a uniform solution.Auxiliary addition method: the graphene-like materials are first altered by specific functional groups, and then, the new modifying agent is melted into the asphalt binder.Although the effects described in The graphene like-materials exhibit a large shape ratio , increased specific surface area, and scalable pore dimension ,147,165.GMABs are usually initially characterized with a traditional test protocol, that is, the set of evaluations for density, penetration, softening point, dynamic viscosity, ductility, flash point, and solubility ,32,46,49The AFM is a microscopy designed to record the topography of materials at a sub-nanometric scale for liquid and air media ,175,176.The DSR is an apparatus utilized to measure the rheological response of some fluids under a wide range of temperatures, frequencies, and shear stress ,188,189.The set of techniques employed to estimate the proportion of the chemical elements that contain a material is denominated EA ,197,198.The incorporation of graphene-like materials within the asphalt binder causes the asphalt matrix to alter the functional groups, i.e., some are transformed into new ones ,37,169. FTIR is a spectrometry method to estimate the capacity of a material to absorb light (infrared radiation) regarding a characteristic wavelength range ,216. The−1 [As its name indicates, the RS is a spectroscopy technique that emits/relies upon Raman scattering  in a specific (monochromatic) electromagnetic spectrum ,231. Usu−1 ,169. Und−1 ,243.The SEM is a method for scanning and analyzing the microscopic morphology of organic and inorganic materials ,245. TheThe GMABs can develop complex thermal stability, varying over time ,166. AltAfter modifying an asphalt binder with graphene-like materials, the asphalt matrix develops high quantities of new crystal structures ,49,158. Current investigation trends show that the researchers focus on applying optical techniques to study GMABs. Although these techniques are extremely useful for performing the physical–chemical characterizations of different materials, these laboratory protocols also can present significant disturbances and errors in the result reports ,294. NotOn the other hand, another critical gap was evidenced in the literature: the tests on asphalt binders are usually conducted only before  and after  the modification processes. In this way, it is not common for the researchers to evaluate the properties of the GMABs over time, e.g., after several months of modifying the binder. The preceding implies that in the current state-of-the-art, there is no consensus on how the physical–chemical properties of the GMABs evolve in the long term. Consequently, this scenario is disturbing because the average service life of traditional asphalt pavements is 20 years, while it is 50 years in the case of perpetual pavements ,309,310.In light of the above, the current state-of-the-art about graphene-like materials and their applications to produce GMABs is susceptible to improvement. Therefore, some suggestions for future research lines are presented below: (i) implement machine learning to interpret the results obtained through advanced characterization techniques; (ii) research the physical–chemical change of the GMABs over the long term; (iii) develop mathematical–computational models to predict the effect of graphene-like materials on the asphalt binder performance; (iv) establish boundaries about the optimal geometric features that the graphene-like materials should have to guarantee the better possible behavior; and (v) evaluate the viability of other graphene derivatives, such as expanded graphite nanosheets, flake graphite, fluorographene, graphane, graphene nano-composites, graphene nanoribbon, graphene nanosheets, graphene quantum dots, graphone, graphyne, nanographite, and reduced graphene oxide.Neat asphalt binders are modified with graphene-like materials to produce high-performance binders.The prominent advantages of GMABs over traditional asphalt binders are decreased thermal susceptibility and increased resistance to aging, fatigue, rutting, and moisture damage.The primary graphene-like materials are used to modify asphalt binders are graphene, GO, and GNPs.The main processes for producing GMABs are the direct addition method, indirect addition method, and auxiliary addition method.3+-TA, glutaraldehyde, nickel, polyethylene, polystyrene, polyurethane, polyvinylpyrrolidone, and SBS.GMABs are highly compatible with a wide variety of additional modifying agents, such as carbon fibers, cross-linked chitosan, crumb rubber, electric arc furnace slag, epoxy resins, ethylene bis(stearamide), FeConventional laboratory tests  are insufficient to characterize the complex behavior of GMABs. Therefore, it is necessary to employ advanced characterization techniques.In order to properly characterize the GMABs, it is essential to conduct assessments regarding chemical, rheological, microstructural, morphological, thermogravimetric, and surface topography properties. Notably, AFM, DSC, DSR, EA, FTIR, RS, SEM, TGA, XRD, and XPS are the leading tests for these purposes.There is still no consensus in the literature on the physicochemical properties of GMABs and their performance as a material for the road infrastructure industry. Regardless, all cutting-edge evaluation techniques indicate an improvement in low- and high-temperature performance .Two primary gaps in the literature were identified: (i) although most of the advanced characterization techniques for GMABs are based on optical methods, researchers do not use computational approaches  to automatize the data interpretation process, and thus, reduce the inaccuracies associated with these observations/measurements; and (ii) not enough research efforts have been carried out to understand the behavior of the GMABs in the long-term.In this research, a comprehensive literature review on the properties and characterization techniques of GMABs was carried out. Likewise, the main physicochemical aspects of asphalt binders and graphene-like materials were explored and discussed. In this way, it was possible to draw the following conclusions:


Z|/|Z| up to 638,800%), a lack of stability and repeatability has been observed. GO/MWNT-based humidity sensors are more conductive than GO sensors and relatively less sensitive . However, they are more stable in harsh humid conditions, repeatable, and reproducible even after several years of shelf-life. In addition, they have fast response/recovery times of 10.7 s and 9.3 s and an ultra-fast response time of 61 ms when abrupt humidification/dehumidification is applied by respiration. All carbon-based sensors’ overall properties confirm the advantage of introducing the GO/MWNT hybrid and laser direct writing to produce stable structures and sensors.In this paper, the relative humidity sensor properties of graphene oxide (GO) and graphene oxide/multiwalled nanotubes (GO/MWNTs) composites have been investigated. Composite sensors were fabricated by direct laser scribing and characterized using UV-vis-NIR, Raman, Fourier transform infrared, and X-ray photoemission spectroscopies, electron scanning microscopy coupled with energy-dispersive X-ray analysis, and impedance spectroscopy (IS). These methods confirm the composite homogeneity and laser reduction of GO/MWNT with dominant GO characteristics, while ISresults analysis reveals the circuit model for rGO-GO-rGO structure and the effect of MWNT on the sensor properties. Although direct laser scribing of GO-based humidity sensor shows an outstanding response (|Δ Since the first humidity sensor was invented in the 15th century ,3,4. FroGenerally, several principles of humidity measurement, such as impedance, capacitance, resistance, gravimetric (frequency), optics, fluorescence, and power generation/self-powered, utilize different kinds of conventional and novel materials, e.g., ceramics, polymers, and nanomaterials, as summarized in different reviews ,5,6,7,8.Based on the analysis of the different types of sensors, the ability of a material to interact with water molecules is an essential part of how sensitive it is to humidity. Thus, one area of research for humidity-sensitive materials is synthesizing materials better at absorbing water molecules or finding materials with a high water-molecule-adsorption capacity, hydrophilicity, response/recovery time, less hysteresis, time stability, etc. These are the bottlenecks for the development of humidity sensor materials. Recently, a wide spectrum of materials, material hybrids, and composites have been developed based on different kinds of principles such as paper-based , cellulo3O+) as charge carriers [Graphene has attracted more attention due to its excellent electrical, mechanical, and chemical properties . Unlike carriers . This cacarriers . 2O3/GO composites [Many researchers have paid attention to GO and rGO due to their low cost and the possibility of large-scale production to fabricate high-performance humidity sensors ,32,33,34mposites .Combined with direct laser writing, GO structured sensors have the advantages of high performance, low cost, miniaturization, patterning, low-temperature operation, non-toxic, and no-photomasks . Apart f−4 S/m) [Because the interaction between the water molecules and GO oxygen functional groups is strong, GO-based humidity sensors exhibit significant hysteresis and inconsistent behavior . In addi−4 S/m) , high po−4 S/m) ,33. Carbon nanotubes (CNTs), materials with intrinsic mechanical and electronic properties, can improve composites’ mechanical and electrical conductivity and are often used as reinforcing agents for composite materials . CNTs arIn this paper, we investigate GO and GO/MWNT composites reduced and structured by laser scribing. rGO/MWNT-GO/MWNT-rGO/MWNT structure is proposed where rGO/MWNT works as interdigitated electrodes and GO/MWNT works as a water vapor-sensitive layer. The sensitivity toward humidity is compared to different structured sensors. The repeatability, response time, and long-term stability have been investigated. Compared to many literature sources discussing humidity sensors based on GO, highly sensitive GO-based sensors can be obtained and further improved by GO/CNT composite to obtain all carbon-based humidity sensors. It has several advantages over GO-based sensors. In addition, ultra violet-visible near-infrared, Raman, Fourier transform infrared, and X-ray photoemission spectroscopies, and electron scanning microscopy coupled with energy-dispersive X-ray analysis have been utilized to investigate the physical characterization of the composites and laser-based structures. Impedance spectroscopy measurement investigation was also carried out to explore the equivalent circuit model for the laser direct written sensors.GO aqueous dispersion (0.4 wt.%) was purchased from Graphenea Inc., San Sebastián, Spain. The GO dispersion was diluted with deionized water to 0.3 wt.% GO. A total of 12 mL of 0.3 wt.% GO solution was taken out for GO humidity sensors. A total of 12 mL of 0.3 wt.% GO solution was used for the preparation of GO/MWNT dispersion. A total of 2.4 mg of MWNT powder  was added to the GO solution. The solution was placed for sonication and treated at 30% power for 45 min using a Bandelin sonication device (200 W)  to disperse the CNTs in GO. Thus, a 12 mL MWNT/GO (GO/MWNT) solution was obtained, having a mass fraction of MWNT of 0.02 wt.% and a mass fraction of GO of 0.3 wt.%, 2, which is the area available for scribing by Lighscribe DVD disc covered by polyethylene terephthalate (PET) substrate, 2. To ensure the uniformity of the film, drop coating and drying processes were carried out in a compartment with a self-balanced workbench. After 24 h of drying process at room temperature, discs with GO and GO/MWNT films were obtained. The digital design as a rectangular film/interdigitated electrode (IDE) was made by laser scribing software . The DVD laser with a wavelength of 785 nm was used to directly write the electrodes. The laser reduction was based on DVD driver (HP DVD AD-7721H) with 5 mW power focused on a spot size of 50 µm [For the preparation of the sensors based on DVD laser scriber, every solution was drop-casted on an area of 90 cmof 50 µm . In addi−1 grating. Laser power on the sample was kept at 1 mW to prevent thermal degradation/reduction of the samples. FTIR measurements were performed using spectrometer INVENIO S . All the measurements were recorded in attenuation total reflection (ATR) mode on Germanium crystal from 550 to 4000 cm−1. The baseline corrections and compensation of peaks due to the atmosphere were performed in OPUS software provided with the instrument.The optical absorption properties of the deposited GO and GO/MWNT dispersions were investigated by UV-vis-NIR measurements using a Cary 60 spectrophotometer . Raman spectra, excited with a diode-pumped solid-state high brightness laser (excitation wavelength 532 nm), were collected on a DXR Raman microscope  equipped with an Olympus optical microscope and a CCD detector. The laser beam was focused on the sample using an objective magnification of 10×. The scattered light was analyzed by the spectrograph with 900 lines mmSEM-EDX characterization was performed using Phenom ProX scanning electron microscope . Before the analysis, samples were coated with a thin Cu layer to improve the visibility of carbon structures. −9 mbar. SPECS FG15/40 electron flood gun was used for charge neutralization to minimize the effects of charging at the samples. All the peak positions were referenced to C1s at 284.8 eV. Spectra were acquired using the manufacturer’s SpecsLab data analysis software and processed using the commercial CasaXPS software suite.XPS was performed to investigate the chemical speciation on the GO based sample’s surfaces. SPECS Systems with XP50M X-ray source for Focus 500 and PHOIBOS 100/150 analyzer were used to evaluate the samples. Al Kα source (1486.74 eV) at 12.5 kV and 32 mA was used for this study. Survey spectra (0–1000 eV binding energy) were recorded with a constant pass energy of 40 eV, step size 0.5 eV, and dwell time of 0.2 s in the FAT mode. High-resolution spectra of O 1s and C 1s peaks were recorded with a constant pass energy of 20 eV, step size of 0.1 eV, and dwell time of 2 s in the FAT mode. Spectra were obtained at a pressure of 9 × 102, Mg(NO3)2, NaCl, KCl, K2SO4) were reagent grade with at least 99% purity . The fixed points provided by the binary aqueous solutions were checked by a calibrated sensor and amounted to 11.3%, 32.78%, 52.89%, 75.29%, 84.34%, and 97.3% relative humidity. An impedance analyzer 4294A  was used to measure the impedance spectra in the range 40 Hz–110 MHz, as shown in For the acquisition of impedance spectra, saturated salt fixed humidity points were used. The used salts  for both rGO and rGO/MWNT composite, as seen in the spectra, confirming the reduction of GO [−1 are observed for the reduced films, which can be attributed to symmetric(sCH2) and asymmetric stretching (aCH2) of the C-H bonds in the lattice [After the laser irradiation, the bands related to -OH functional groups at 3316 cmon of GO ,62. Neve lattice ,64.SEM analysis of GO and GO/MWNTs composites showed similar morphologies before reduction. The layers are relatively smooth, with wrinkle networks that likely formed during the drying process  and rougThe chemical composition of the studied sensing layers was investigated using EDX and XPS. According to EDX analysis, C and O are uniformly distributed in all the samples , while sTo investigate the moisture-sensitive properties of GO materials, impedance measurements were performed under different humidity environments provided by the humidity-fixed points of binary saturated aqueous solutions. Four sensors based on GO and GO/MWNT have been investigated. They are the laser-scribed rectangular shape (rGO and rGO/MWNT sensors) and the interdigitated (IDE) structures having (rGO-GO-rGO and rGO/MWNT-GO/MWNT-rGO/MWNT forms), abbreviated as GO IDE and GO/MWNT IDE sensors, as shown in To evaluate the sensitivity to relative humidity, the impedance modulus is plotted against relative humidity at different frequencies, For IDE sensors, To highlight the reasons for differences in sensitivity at low humidity, on the one hand, rGO rectangle shape sensors, as stated above, have behavior similar to a large extent to the behavior of carbon nanotubes and graphene, where the increase in humidity results in the rise of resistance. They are highly reduced GO and GO/MWNT as the initial resistance at 11% RH is very low. This means they are of hydrophobic nature. So, at low humidity, water molecules are only adsorbed on the surface and as having donating properties, while the rGO and MWNT films are of p-type nature, and thus the resistance increases due to the increase in holes . As the The capacitance measurements versus humidity at different frequencies are plotted in To summarize, MWNT reduces the high impedance of GO material at a low-humidity range, and thus, the conductivity of GO/MWNTs materials has been significantly improved below RH 50%. Under normal indoor conditions, the rGO/MWNT sensor impedance is only 16% of the GO sensor at 1 kHz. MWNTs, fill the gaps between GO material by connecting independent 2D GO films to form a 3D structure, which further increases the specific surface area of the material and makes the charge transport between GO and/or rGO flakes more effective . It helpTo test the humidity sensor and construct calibration curves for the developed sensors, the humidity chamber base on two gas flows was used to obtain the GO and GO/MWNT IDE sensors’ properties. Given the link between absolute humidity, relative humidity, and temperature, the recorded temperature was kept constant at 23 °C to ensure the accuracy of the measurements. The results are shown in Z0 is the impedance of the humidity sensor at RH 10%, and xZ is the impedance of the humidity sensor at a particular RH. The results are shown in 5% at 97% (100 Hz). However, for GO/MWNT, the maximum relative change is 1.63 × 105% (100 Hz) with better linearity in the semi-log scale, and impedance ranges from 7.3 kΩ to 7.7 MΩ. The high impedance of GO IDE will increase the difficulty of subsequent sensor circuit design and increase power consumption [sumption . It can be concluded that two factors control the overall sensitivity between the lowest/highest humidities. The first is the initial impedance of the unreduced GO-based sensitive area, and the second is the final resistance induced by proton hopping, in addition to the rGO fingers’ impedance. It is noticed that the final resistance is almost in the same order of magnitude for both materials GO and GO/MWNT. The resistance of the water film itself depends on the geometrical shape  and gap Based on the discussion so far, we choose mainly GO/MWNT for subsequent sensor characteristics and at a test frequency of 1 kHz.DZ and AZ are the impedances modulus measured during desorption and adsorption, and S is the sensitivity calculated based on Equation (3), where |xZ| and |yZ| are the impedance modulus obtained at humidity points xRH% and yRH%.The hysteresis test result of the humidity sensor is shown in In this section, the developed structured sensors were put in different humidities for a long time to study the stability of the sensors. Due to the long experimental period of the stability test, saturated salt solutions were used for this test. Five saturated salt solutions with five humidities, i.e., 11, 32, 52, 75, and 97%, were placed in sealed bottles (at ambient room temperature ~23 °C), and in each solution, four different sensors were put, i.e., rGO and rGO/MWNT films, and the IDE sensors (five each). The IDE GO and GO/MWNT-based sensors showed excellent stability in the humidity range of 11–97%, In addition, Following that, the repeatability of the GO/MWNT IDE sensor was examined, where the step response procedure was carried out by measuring the sensor impedance over time for 8 h following the humidity profile inside the chamber. A staircase-wise profile from 10 to 90%RH and back to 10% at an almost constant temperature of ~22.4 °C was applied, as shown in 63 and t90 and amounted to 4.1 s and 10.7 s, respectively. The recovery times, t37 and t10, were 3.0 s and 9.3 s, respectively. In order to have a more abrupt change, breathing  was applied, 2 gas, where the effect of humidity is pronounced, To test the humidity response and recovery time of the sensor, humidifying processes (wet air) and drying processes (dry air) were performed in a small chamber controlled by valves where humidifying will continuously inject highly humid nitrogen into the surface of the sensors under a constant flow speed to have a sudden increase in humidity. To ensure the humidity’s stability on the sensor’s surface, we fixed the materials/sensors very close (5 mm) to the gas supply port. After a preset time, the drying valve was opened, and the drying process was applied using dry nitrogen. Therefore, the humidity on the surface of the sensor is supposed to drop immediately from 80% to 10% and remain at 10%. By applying the procedure, the measured impedance at 1 kHz is plotted versus time a. The reThe obtained results provide a suitable picture of the sensor characteristics indicating its suitable performance over many recent materials for humidity sensors. In comparison with many papers, high sensitivities are demonstrated  with proIn view of several works, GO-based humidity sensors have been utilized to detect water vapor from different biological exertion, such as exhalation, skin humidity, and no-contact sensing ,76,77. OThe impedance spectroscopy was performed in the frequency range of 40 Hz to 110 MHz. In 1) corresponding to rGO resistance in parallel with a constant phase element (CPE), denoted as CPE1, for the interface between the electrode and rGO reflecting the surface irregularities and the remaining unreduced GO areas [n is a dispersion parameter and Q in F/s(1−n); as can be seen, the resistance varies little with RH, having a significant drop only at the highest RH, and the resistance is around 10 kΩ. The same explanation can be applied to rGO/MWNT material ZCPEmaterial b. This CFor the IDE structure of both GO and GO/MWNT c,d, highf − CPE/Rf + CPE, corresponding to low-medium-high humidity, which may be suitable for GO with metallic electrodes [f/Cf − Cf/(Rf + wZ)) uses the Warburg diffusion element [f + wZ)/Cf), where wZ represents the water diffusion impedance, to interpret the humidity sensing mechanism [Most GO-based humidity sensors show evolving equivalent circuit models as CPE − CPE/Rectrodes ,69,76. A element ,77,86. H element ,68,71,75echanism ,76. Howeechanism  by simul1/R1 as the applied potential (50 mV) is slightly greater than the open-circuit potential [1/R1, as shown in 2/CPE2). This applies to the humidity range of 11–52%, where fewer water ions are available.Here, the complex impedance spectra show two partial semi-circles arcs. The small one is visible in the high-frequency range at ~7 MHz, otential ,87. Thus1) corresponds to the GO-rGO double-layer capacitance, the resistance (R2) is the contact resistance of the rGO fingers, and C1/R1/CPE1 is the total for GO + rGO or GO/MWNT + rGO/MWNT, which likely assembles the complex impedance of the interfaces with different reduction levels along the sensing layer depth profile at the contact between the reduced and non-reduced part of the sensor. Thus, we suggest that such an evolution of the equivalent circuit is due to the gradient of the reduction degree in the IDE structure and penetration of water molecules in the deeper parts of the sensing layers. These water molecules adsorbed through the oxygen-containing functional group will be ionized into H3O+ under the action of electric field force, which reduces the sensor’s resistance. The proton hopping transport is easily formed in the water layers formed via Grotthuss reaction (H2O + H3O+ → H3O+ + H2O) [1 reflects more capacitive behavior, while n3 reflects ionic behavior with a tendency to resistive behavior with an RH% increase [1 and CPE2, 2 corresponds to rGO resistance increase with humidity with the rise of humidity [At higher humidities, 75–97% RH, by the increase in water content, the equivalent circuit can be represented as a heterostructure-like circuit model for electrode interface in corrosion or solar cells with several stacked materials layers . In our + + H2O) . Additioincrease . While Chumidity . 4%, while the range of impedance is from 7.3 kΩ to 3.2 MΩ. Moreover, it also has an excellent performance in terms of hysteresis, repeatability, and several years of stability. The measured response/recovery times in the humidity chamber were 10.7 s and 9.3 s. However, by respiration test followed by dry air dehumidification, they measured as low as 61 ms and 2.3 s. By direct laser scribing, a humidity-insensitive film can be produced where no additional coating is required, which is beneficial for other sensor applications. GO/MWNT material has potential in the future of the humidity sensors field. This expectation is not only based on excellent sensing characteristics but also on the exceptional stability of such sensors.In this work, direct laser-structured GO-based humidity sensors were characterized. GO moisture-sensitive properties of the GO-based humidity sensor were additionally enhanced, using MWCNT to form GO/MWNT sensing layers. Based on the obtained results, MWNTs interact with GO to form a stable composite, enhancing the adsorption properties and reducing the high impedance of GO at low humidity, making the impedance measurement easier and more reliable. GO/MWNT-based sensor has a humidity response in the tested range of RH 10% to 97%. At the working frequency of 1 kHz, its sensitivity is 4.6 × 10