TDP-43 is a nuclear protein involved in pivotal processes, extensively studied for its implication in neurodegenerative disorders. TDP-43 cytosolic inclusions are a common neuropathologic hallmark in amyotrophic lateral sclerosis (ALS) and related diseases, and it is now established that TDP-43 misfolding and aggregation play a key role in their etiopathology. TDP-43 neurotoxic mechanisms are not yet clarified, but the identification of proteins able to modulate TDP-43-mediated damage may be promising therapeutic targets for TDP-43 proteinopathies. Here we show by the use of refined yeast models that the nucleolar protein nucleolin (NCL) acts as a potent suppressor of TDP-43 toxicity, restoring cell viability. We provide evidence that NCL co-expression is able to alleviate TDP-43-induced damage also in human cells, further supporting its beneficial effects in a more consistent pathophysiological context. Presented data suggest that NCL could promote TDP-43 nuclear retention, reducing the formation of toxic cytosolic TDP-43 inclusions. Neurodegenerative diseases (NDs) represent a huge health problem worldwide. In most cases, the origin of neurodegeneration remains largely unknown, but it is now commonly accepted that many of these disorders share the accumulation and aggregation of misfolded proteins as a major hallmark of neuropathology (for which they are often referred to as proteinopathies). Despite the role of such a protein misbehavior and the formation of protein assemblies may differ in distinct NDs, possibly also in different subtypes of the same ND, in all cases disease-associated misfolded proteins are supposed to either acquire toxic properties or lose their normal functions, or both, thereby driving neuropathogenic mechanisms .Among NDs, the vast majority of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and also a subset of late-onset Alzheimer’s disease (AD) cases are characterized by the presence of neuronal cytoplasmic inclusions mainly composed of an ubiquitinated and hyper-phosphorylated form of the transactive response DNA binding 43 kDa protein (TDP-43) . TDP-43 The above concept is further reinforced by acknowledging that intracellular TDP-43 aggregates are a major histopathological hallmark of ALS, found in post-mortem samples of 97% ALS patients . AlthougSaccharomyces cerevisiae , and Huntington’s disease (HD), have been successfully modeled in the yeast revisiae .In the last decade, different yeast models have been generated ectopically expressing TDP-43 at either high or low levels, mainly by the use of multicopy or centromeric plasmids, respectively . IntegraDrosophila and human cells) supported the linkage between ALS/FTD and the function of intra- and extra-nuclear membrane-less organelles, identifying some nucleolar proteins, cytosolic stress granules components and nucleocytoplasmic trafficking factors as genetic modifiers  of the cytotoxicity triggered by ALS-related proteins, such as C9orf72 DPRs  in light of multiple aspects, including the following structural and functional features of the protein that are reminiscent of TDP-43 attributes: (i), NCL is able to bind DNA/RNA molecules thanks to multiple RNA recognition motifs (RRM) domains; (ii), NCL carries an intrinsically disordered C-terminal region able to interact with different functional partners; (iii) NCL is one of the most abundant non-ribosomal proteins in the nucleolus, where it plays fundamental roles in nucleolar assembly and function, rRNA and ribosome metabolism , which aWe are here providing evidence that NCL is able to act as a potent suppressor of TDP-43 toxicity, relieving the lethal damage induced by WT or ALS-mutant TDP-43 overexpression in yeast cells. Additional experiments in mammalian HEK293T cell models further support that the NCL overexpression can prevent TDP-43-dependent toxicity, reducing the formation of detergent-insoluble assemblies and restoring cell viability, possibly by avoiding the non-physiologic translocation of TDP-43 out of the nuclear compartment.All yeast strains and plasmids used in this study are listed in Procedures for yeast growth and manipulation were performed according to standard protocols .Aldrich. Induction of gene expression under Gal1 promoter control was obtained by growing transformed yeast cells in raffinose containing medium for 16 h, followed by dilution of the cultures to an optical density at 600 nm light adsorption (OD600) of 0.1 and growth in galactose containing medium. Cells were routinely incubated at 30°C.Yeast cells were grown in standard rich media  supplemented with 20 g/L of glucose or galactose as carbon source, or in synthetic minimal media  containing 20 g/L of glucose, raffinose or galactose and lacking the specific nutrients allowing for selection of transformed yeast clones. Media components were from Difco (Thermo Fischer Scientific), and auxotrophic requirements were from Sigma-The PEG/lithium acetate method was used to transform yeast  and then600 culture turbidity measurements). In particular, for spotting assays, yeast cells were grown overnight at 30°C in liquid media containing raffinose. Cultures were then normalized to OD600 = 1, serially diluted and spotted onto synthetic solid media containing glucose or galactose and the auxotrophic requirements as needed. Plates were maintained at 30°C for different days, as indicated. Conversely, OD600 measurements were performed in a Microplate Reader spectrophotometer (TECAN) at a 600 nm wavelength (bandwidth 9 nm) with 15 flashes and 200 μL of culture per well. Briefly, three yeast clones were grown overnight in a raffinose containing medium (non-inducing condition), diluted in triplicate to OD600 = 0.1 in a galactose containing medium (inducing condition), and grown for 24 h. OD600 was then recorded immediately after the transfer (t0) or after 24 h of growth (t24), in the galactose medium, and the OD600 ratio (t24/t0) was calculated. Data were then normalized to the mean ratio of control cultures CEN.PK IMX672 (hereafter abbreviated as CENPK).Yeast cell viability was performed as previously described  by measuex = 488 nm for the excitation of GFP) and a computer-assisted charge-coupled device camera (Hamamatsu Orca flash 4.0), which allowed the acquisition of digital micrographs for either fluorescence or brightfield microscopy.For fluorescence microscopy observations, cells grown for 6 h in a galactose-containing liquid medium were used, while for cell shape analysis by brightfield microscopy cells were cultured on galactose containing solid substrate for 2 days. In both cases, before microscopy analysis, harvested cells were washed and resuspended in PBS, and then mounted on a coverslip with a thin agar slab as described previously . Images For morphometric measurements, the cell length-to-width ratio was calculated on digitalized images using the Fiji ImageJ software.For confocal microscopy observations, 1 mL of yeast cultures grown 24 h in galactose-containing medium were fixed in 4% formaldehyde . To stain nuclei, DAPI  was added during cell fixation. After fixation, cells were washed twice with sterile water and then resuspended in 1 M sorbitol. Cell suspension was dropped onto a slide  and covered with a coverslip . Cells wg, 10 min, 4°C). Cells were then resuspended in 0.1 M NaOH and incubated at room temperature for 20 min. After pelleting, cells were finally lysed by vortexing (2 min) in a buffer containing 62.5 mM Tris-HCl (pH 6.8), 2.3% (w/v) SDS and 10% (w/v) glycerol (buffer O). Lysates were boiled (3 min) and, after centrifugation to remove cell debris, protein quantification was performed in the supernatant using a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific), according to the manufacturer’s instructions.The extraction of proteins from yeast cells for Western blot (WB) and MS analyses was performed as described elsewhere  with som–1 in 100 mM TEAB, pH 8.0) to each filter . After digestion, filters were subjected to two subsequent centrifugation step  adding 50 μL of 100 mM TEAB for peptide recovery. Peptide mixtures were then labeled using 6-plex TMT reagents (Thermo Scientific), and subjected to fractionation and purification as previously described (18 BioPure spin columns (The Nest Group). To reduce sample complexity, TMT-labeled peptides were fractionated by strong cation exchange fractionation (SCX) before liquid chromatography (LC) and MS. Briefly, labeled samples were diluted with four volumes of 0.1% formic acid and then applied to the SCX macro spin columns (The Nest Group). After two washings, retained peptides were stepwise eluted in five fractions containing increasing concentrations of ammonium formate . Prior to sample injection into the high-performance liquid chromatography-high resolution tandem mass spectrometry (HPLC-HRMS/MS) workstation, each fraction was desalted by means of the C18 BioPure spin columns. The five labeled fractions of each peptide extract were finally dried using a stream of nitrogen (RT) and suspended in 100 μL of 0.1% formic acid for the subsequent MS analysis.For tandem mass tag (TMT)-based quantitative proteomic analysis, protein extracts (50 μg) from three biological replicates of parental CENPK and TDP 1C yeast strains were processed according to the filter-aided sample preparation method , using fescribed . The thr18, 150 mm × 2.1 mm, 2.6 μm, Phenomenex) kept at 30°C.Relative quantification of yeast proteins was achieved using a hybrid quadrupole-orbitrap Q-Exactive (Thermo Fisher Scientific) mass spectrometer, coupled to a UHPLC system Ultimate 3000 (Thermo Fisher Scientific). Each SCX fraction was separated on a reverse-phase analytical column (Aeris peptide C–1 (35 min) using a gradient elution with the following composition (expressed as A:B ratio): 97.5:2.5 for 1 min; from 97.5:2.5 to 70:30 in 19 min (following a linear gradient), from 70:30 to 50:50 in 4 min; from 50:50 to 5:95 in 2 min and maintained for 4 min to wash the column; then back to 97.5:2.5 (to re-equilibrate the system) in 0.5 min and maintained for 4.5 min. The injection volume was 10 μL.Elution solvents for peptide separation were water (A) and acetonitrile (ACN) (B), both containing 0.1% (v/v) formic acid. The chromatographic separation was carried out at a flow rate of 200 μL × min6 ions, an injection time (IT) of 250 ms, an isolation window of 2.0 Th, and a scan range from 300 to 2000 Th. Higher energy C-trap dissociation (HCD) was performed with a normalized collision energy of 30, AGC target of 2 × 105 ions, an IT of 120 ms, a dynamic exclusion of 30 s and a resolution of 17,500. Fragmentation spectra were used for peptide identification and quantification, setting a fixed starting mass of 100 Th. To increase the number of identified peptides, each SCX fraction was analyzed twice. To this purpose, the m/z values of peptides positively identified in the first analysis (as described in details in the next section) were used to create a static exclusion list that was then applied to a second HPLC-HRMS/MS analysis  for each sample fraction.Ion source capillary temperature was 325°C, the sheath gas flow rate 35 (arbitrary instrument units), auxiliary gas flow rate 10 (arbitrary instrument units), S-lens voltage 55 V, heater temperature 325°C, and the spray was optimized at 3 kV. The instrument operated in data-dependent mode with a top-7 acquisition method . Full scan spectra were acquired using an automatic gain control (AGC) target of 3 × 10S. cerevisiae database (3AUP000002311). Enzyme specificity was set to trypsin, and a maximum of one missed cleavage was allowed. The precursor and product ion mass tolerances were set to 10 ppm and 0.2 Da, respectively. Oxidation of methionine was selected as variable modification, while 6-plex TMT at N-termini and at lysine residues, and carbamidomethylation of cysteines were set as fixed modification. Relative quantification was performed directly by the Proteome Discoverer software and only unique peptides were considered for quantification purposes. Based on the Percolator algorithm, proteins were considered as correctly identified if at least two unique peptides were quantified with an individual q-value < 0.05. Proteins were then grouped according to the principle of maximum parsimony. For quantification, the reporter mass tolerance was set to 20 ppm. The software detected the reporter ions , and performed the quantification of relative peptide abundance normalizing the intensity of each reporter ion to that of CENPK 1 sample (126.1 Th). Normalized intensity values of proteins derived from Proteome Discoverer were exported in an Excel spreadsheet, and the matrix was arranged for further analysis. The final fold-change of a given protein was calculated as the mean value of the normalized ratios (TDP 1C/CENPK) of the three replicates. Finally, a two-tailed t-test was performed, and only proteins with a ratio > 1.33 or <0.77, and a p-value < 0.05, were considered over-expressed or under-expressed, respectively.Raw files derived from HPLC-HRMS/MS runs were analyzed with a MudPIT protocol using the Proteome Discoverer 1.4 software (Thermo Fisher Scientific) interfaced to a SEQUEST HT search engine (Thermo Fisher Scientific). All MS/MS data were searched against the UniProt 18 spin columns (The Nest Group), following manufacturer’s instructions. Briefly, samples were loaded in pre-activated C18 spin columns, that were washed twice with 200 μL of 0.5% formic acid (v/v), and then peptides were eluted using 75% ACN containing 0.1% formic acid (v/v). Peptide extracts were dried under a stream of nitrogen and, immediately before HPLC-HRMS/MS analyses, dissolved in a solution (100 μL) containing 5% (v/v) ACN and 0.1% (v/v) formic acid to obtain a final concentration of 0.5 μg × μL–1 of protein digest. Confirmatory analyses were performed using the same HPLC-HRMS/MS apparatus described above operating in the PRM mode. Peptides were separated using the same chromatographic gradient described above. The scheduled PRM method was developed by recording and selecting the most intense charge state of the considered peptides and four diagnostic precursor-to-product ion transitions in serial injections of a representative yeast digest sample. The same was done for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which was chosen as housekeeping protein. PRM acquisition parameters were as follows: HCD fragmentation with normalized collision energy (NCE) of 27, AGC target of 5 × 105 ions, an IT of 120 ms, an isolation window of 1.6 Th with an offset of 0.4 Th. Two proteotypic peptides were selected for each protein included in the PRM analysis. The selected precursor-to-product ion transitions, together with the retention time and the instrumental settings used in the acquisition method are reported in The targeted MS-based parallel reaction monitoring (PRM) analysis was carried out in WT CENPK, and TDP-43 1C and 2C strains expressing, or not, NCL. To this end, 50 μg of yeast lysates from nine different cultures for WT CENPK and for each of the four different TDP-43 clones  were prepared and processed according to the filter-aided sample preparation (FASP) method as described above. After digestion, peptide mixtures were acidified (pH < 3) by adding formic acid and desalted using BioPure CTo ensure that no instrumental drift occurred during PRM analysis, a pooled sample prepared by mixing together equal amounts of peptide digests was injected and analyzed at the beginning and at the end of the analytical sequence. The Skyline software (version 3.5.0.9191)  was usedThe MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository  with theL-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin and maintained at 37°C in a humidified incubator with 5% CO2. HEK293T cells were seeded at 25% of confluence in multi-well culture plates 24 h before transfection. Cells were (co-)transfected using the Lipofectamine 3000 transfection kit (Invitrogen) following the manufacturer’s instructions and analyzed 48 h after transfection. Plasmids used to transfect HEK293T cells are reported in Human epithelial kidney HEK293T cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) foetal bovine serum (FBS), 2 mM Cell viability was assessed using the MTS  assay , based on the reduction of MTS by viable cells, following the manufacturer’s instructions. Briefly, cells were seeded in 96-wells and co-transfected with the desired plasmids (as indicated in the figure legends), and – 48 h after transfection – the cell culture medium was removed and the MTS reagent [20 μL in 100 μL of phosphate-buffered saline (PBS)] was added to each culture well. Cells were then incubated at 37°C (90 min), after which the absorbance of reduced MTS (λ = 490) was determined using a Microplate Reader (TECAN) spectrophotometer.For confocal microscopy observations, HEK293T cells were seeded onto 13 mm coverslips and co-transfected using plasmids coding for the desired fluorescent constructs. 48 h after transfection, cells were rinsed twice with PBS, fixed  with paraformaldehyde [2% (w/v) in PBS], rinsed again and permeabilized  with Triton-X100 [0.1% (w/v) in PBS]. Cell nuclei were counter-stained with Hoechst 33342 , and coverslips were finally washed in PBS and mounted in Mowiol 40–88 (Sigma-Aldrich) [8% (w/v) in glycerol:PBS ]. Images were collected with a Leica SP5 confocal microscope using 40X or 63X HCX PL APO  oil-immersion objectives. Settings of confocal microscope and image acquisition and analysis were performed as described above. For quantitative analyses reported in g, 10 min, 4°C) to precipitate cell debris, the total protein content in the supernatant was determined by the BCA assay kit (Thermo Fisher Scientific).For protein extraction, HEK293T cells, grown onto 12-wells plates and transfected as described above, were washed twice with ice-cold PBS and lysed with buffer O (60 μL/well). After centrifugation ] or Mini-Protean TGX precast gels ] and electroblotted onto polyvinylidene difluoride (PVDF) membranes . Membranes were stained with Coomassie brilliant blue (Sigma-Aldrich) to check for even protein loading, and digitalized images were collected for subsequent densitometric analysis. After Coomassie destaining with methanol, membranes were incubated  in a blocking solution[5% (w/v) non-fat dry milk (Bio-Rad Laboratories) in TRIS-buffered saline (TBS) added with 0.1% (w/v) Tween-20 (TBS-T)], and then probed  with the desired primary antibody diluted in TBS-T containing 1% (w/v) bovine serum albumin (BSA). After three washings with TBS-T, membranes were incubated  with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibody  , depending on the primary antibody. Immunoreactive bands were visualized using an enhance chemiluminescence reagent kit (EMD Millipore) and digitalized by means of an UVItec imaging system (Eppendorf). For densitometric analyses, the intensity of each immunoreactive band was normalized to the optical density of the corresponding Coomassie blue-stained lane  bromophenol blue] were subjected to SDS-PAGE . Cells were lysed by vortexing  in a MagnaLyser apparatus (Roche Diagnostics). The protein fraction was obtained after precipitation of cell debris by centrifugation , and proteins were quantified by a Lowry assay kit (Sigma-Aldrich). 1 mg of total proteins (in IP buffer) was added with either 1 μg of anti-NCL rabbit pAb , or anti-GFP mouse mAb , or anti-RFP mouse mAb  antibody and incubated  under continuous gentle inversion mixing. As negative control, the same amount of lysate was incubated in the absence of antibody.For protein co-immunoprecipitation (co-IP) assays from 2HPO4, and 2 mM KH2PO4, 0.2% NP-40, 10% glycerol (w/v), 5 mM EDTA and Roche complete EDTA-free protease inhibitor cocktail), and maintained on ice for 15 min. Cell lysates were then passed (five times) through 21-gauge needles and centrifuged . The total protein content in the supernatant was determined using the BCA assay kit (ThermoFisher Scientific), and 200 μg of total proteins were incubated  with 0.2 μg of the rabbit polyclonal anti-NCL  in lysis buffer under continuous gentle inversion mixing. When needed, cell lysates were treated with RNAse A  prior to immunoprecipitation. Non-transfected cells were processed as described above as negative control.The co-IP protocol for HEK293T cells was adapted from g, 4°C), washed three times with lysis buffer and once with 50 mM Tris–HCl (pH 7.5), and finally boiled (3 min) in reducing sample buffer. Immunoprecipitated proteins were separated onto 10% SDS-PAGE gel, electroblotted onto PVDF membranes (Millipore) and analyzed by WB with antibodies to target proteins.For both yeast and HEK293T samples, protein-antibody complexes were precipitated by adding protein A-Sepharose . After incubation  under gentle shaking, sepharose bead-bound immunocomplexes were collected by centrifugation  to precipitate cell debris. Fractionation was performed by centrifuging  the cleared lysate, the supernatant (“detergent-soluble” fraction) was saved, and the pellet was resuspended in lysis buffer and centrifuged again as above. The final pellet (“detergent-insoluble” fraction) was resuspended in a urea-based buffer containing 7 M urea, 2 M thiourea, 4% CHAPS and 30 mM Tris (pH 8.5).The protocol for detergent-soluble and -insoluble protein fractionation from yeast cells was adapted from g, 5 min, 4°C). Cells were then resuspended in 300 μL of radio-immunoprecipitation assay (RIPA) buffer  supplemented with 2 mM EDTA, 1 mM EGTA and complete EDTA-free protease inhibitor cocktail (Roche). Following incubation on ice (15 min), cells were sonicated (10 s) and the obtained lysate was cleared by centrifugation  to remove cell debris. A small amount of the lysate  was kept for subsequent analysis, while the remaining lysate was centrifuged . The resulting supernatant (“detergent-soluble” fraction) was saved and the pellet, after washing in RIPA buffer, was centrifuged again as above. The final pellet (“detergent-insoluble” fraction) was resuspended in the same urea-based buffer used for yeast samples.Detergent solubility fractionation of proteins from HEK293T cells was performed according to N,N’-methylenebisacrylamide 37.5:1 (w/w)] and WB for the detection of the target proteins as described previously. To determine the amount of RIPA-soluble (or insoluble) TDP-43, the percentage ratio between the intensity of TDP-43 soluble (or insoluble) immunoreactive bands and the sum of the intensity of TDP-43 bands in the soluble and insoluble fractions were calculated. The reliability of the fractionation protocol was confirmed by evaluating the absence of GAPDH in the insoluble fractions.The quantification of the protein content in all fractions of both yeast and HEK293T samples was carried out by the Lowry assay kit (Sigma-Aldrich). Protein fractions were then analyzed by SDS/PAGE [using 10% (w/v) acrylamide-The protein–protein interaction network and gene ontology enrichment analysis was performed using the publicly available Cytoscape 3.7 software  integratp-value < 0.05 was considered statistically significant .Data were analyzed using Prism 7 (GraphPad Software) and Microsoft Excel (Microsoft Corporation) software. Data are reported as mean ± standard error of the mean (SEM). The number of biological replicates (n) reported in figure legends indicates the number of cultures/transformations, or the number of transfection experiments performed in different days, for yeast and HEK293T cells, respectively. Depending on the experiment, the statistical analysis was carried out as indicated in each figure legend. A S. cerevisiae yeast strains in the CEN.PK IMX672 genetic background , or strongly overexpressing the protein by means of an ectopic galactose-inducible multi-copy (MC) plasmid. The genome editing correctness in the obtained yeast strains was verified by PCR , although the molecular mechanisms underlying such a defect remained unsolved . We thusA strict correlation between the elongated phenotype and cell death, however, is ruled out by the observation that such morphological defect also appears in yeast cells expressing non-toxic levels of TDP-43  human TDP-43 bearing the ALS-related Q331K and M337V missense mutations that are known to exacerbate TDP-43 cytotoxicity . These mThe above data demonstrated that expression of WT or ALS-associated mutant human TDP-43 markedly affects cell viability and growth in yeast strains carrying two or more copies of the transgene, while the expression of TDP-43 (either WT or mutated) from a single genomic copy was not sufficient to cause cell death, pointing to a saturating effect of two transgene copies for TDP-43 cytotoxicity.This experimental evidence validates the use of the here presented yeast strains as valuable cell models for the identification of biological factors and/or chemical compounds that can rescue TDP-43-dependent harm.Once established, we used such yeast models for TDP-43 proteinopathies to test the possible effects of NCL, which – as detailed in the Introduction – could be a promising player in the mechanisms of TDP-43 cytotoxicity. To this purpose, we overexpressed NCL (as a fusion construct with the red fluorescent protein mKate2) in the yeast strains bearing two or three integrated chromosomal copies of the TDP-43-GFP construct (WT or mutated), by means of an ectopic MC plasmid driven by a galactose-inducible promoter.Both cell viability  and growNotably, we also observed that the genetic 1:1 stoichiometry of NCL with respect to TDP-43 was not sufficient to prevent TDP-43 cytotoxicity, as demonstrated by the loss of viability of cells carrying two integrated chromosomal copies of both TDP-43-GFP and NCL-mKate2 constructs, driven by the same galactose-inducible promoter .In the attempt to shed light on the ways by which NCL prevented TDP-43 toxicity, we next investigated the effects of NCL overexpression on TDP-43 behavior and the TDP-43-related morphologic phenotype in the TDP 2C strain. By biochemical fractionation assays, we found that the co-expression of NCL significantly increased the detergent-soluble fraction of WT TDP-43 . Quite sNotably, untagged NCL, expressed by a MC plasmid, still retained the capability to counteract the toxicity of untagged TDP-43, as demonstrated by cell viability assays , ruling Collectively taken, the above data indicated that NCL overexpression was able to potently counteract the toxic effects of TDP-43 in yeast cells, and that such beneficial effect could be related to the aggregation dynamics of TDP-43, i.e., NCL reduced the detergent-insoluble fraction of TDP-43.NSR1 gene. We found that NSR1 deletion did not impinge on the toxic phenotype induced by TDP-43 overexpression and that the ability of NCL to suppress TDP-43 toxicity was unperturbed in the ΔNSR1 genetic background  by multicopy plasmids , or the ckground .We then applied different approaches aimed at exploring the mechanisms by which NCL was able to counteract TDP-43-related toxic phenotypes.p-value < 0.05) up-regulated  or down-regulated  in the TDP 1C strain , both grown to log phase in raffinose medium (non-inducing) and transferred to galactose medium for 24 h to induce transgene expression. We chose to run the preliminary untargeted MS analysis on the TDP 1C strain, in which TDP-43 expression levels were sub-lethal, in order to possibly identify cell pathways induced by TDP-43 (and that NCL is able to counteract) whilst avoiding proteome alterations due to secondary events in downstream cell death pathways. By this assay, a total of 631 proteins were properly identified and quantified in all biological replicates of both strains. Interestingly, only 38 proteins were either significantly  and that the vast majority of them belonged to discrete clusters. Notably, a gene ontology (GO) analysis showed that the most enriched cluster of up-regulated proteins included proteins involved in cellular response to stress, such as heat and oxidative stress, and oxidant detoxification  in both transgenic strains  on the levels of the subset of 19 proteins that were mostly deregulated by TDP-43 expression (in both the 1C and 2C strains) according to the unbiased proteomic analysis, and whose alteration was confirmed by the validation study of  strains  and that strains .In summary, the MS data indicated that the expression of TDP-43 (in the TDP 1C strain) triggered weak changes (∼6%) of the whole yeast proteome, resulting, however, in the significant up-regulation of specific proteins implicated in the response to oxidative and heat stress, and in protein folding and refolding processes that may be related to a proteostatic stress induced by TDP-43 expression. Most importantly, we also found that NCL brought the levels of some of these proteins in TDP-43-expressing strains toward the values observed in control cells, suggesting that it could decrease in some ways the proteostatic stress associated to TDP-43 intoxication.As mentioned, human NCL prevalently localizes in the nucleolar compartment where it plays multiple roles , but it may also translocate outside the nucleus taking part to nucleocytoplasmic trafficking . We therThe above described genetic/functional interaction between TDP-43 and NCL might also be related to a physical interaction between the two proteins, considering that both proteins, although being mainly nuclear, may also localize in the cytosol. This possibility was suggested by a previous study showing – by immunoprecipitation of overexpressed TDP-43 followed by identification of co-purified proteins by MS – that NCL was a physical interactor of TDP-43 in HEK293T cells . We thusWe next asked if NCL was able to suppress the TDP-43-induced phenotypes observed in the yeast models also in mammalian cells. To answer this question, we overexpressed human TDP-43 and NCL in the immortalized human cell line HEK293T by transient transfection with plasmids encoding fluorescence-tagged TDP-43-mKate2 and NCL-GFP chimeric constructs see .We firstly assessed the viability of transfected cells by the MTS assay. Cells overexpressing the TDP-43 (either WT or bearing the Q331K missense mutation) chimera were significantly less viable (around 50%) compared to control cells  was toxic to, and formed detergent-insoluble aggregates in, HEK293T cells and that NCL was able to attenuate both phenotypes, facilitating the nuclear localization of TDP-43. These data substantiated a functional role of NCL in antagonizing TDP-43 proteinopathies.The finding that extranuclear NCL is prominent in abolishing TDP-43 toxicity in yeast , prompteFinally, following the co-IP observations in yeast cells  and the TDP-43-mediated neurodegeneration is characterized by the depletion of nuclear TDP-43 and the concomitant accumulation of the protein in aberrant cytoplasmic assemblies. These two hallmarks suggested that TDP-43 proteinopathies arise from either a loss of function, by impairing the normal function of nuclear TDP-43 in regulating transcription and mRNA processing, or a gain of toxicity of the cytosolic fraction of the protein, or both . Whatevein vitro cell models.Here we showed that NCL, sharing different structural and functional attributes with TDP-43, is able to revert cell death and other phenotypes related to TDP-43 overexpression in both yeast and human S. cerevisiae yeast model already provided important hints into NDs caused by protein misfolding and aggregation, including TDP-43 proteinopathies , that are known to increase TDP-43 toxicity in yeast cells . HoweverOur models also recapitulated recently described alterations of yeast cell morphology caused by TDP-43 expression , but theTaken together, the above findings demonstrated that the novel models here generated were able to recapitulate in a more stable genetic context the major effects of TDP-43 expression in yeast cells, i.e., cytotoxicity, increased propensity to protein aggregation/condensation, and specific morphological alterations, and were therefore suited for the identification of possible antagonists of TDP-43-induced cellular damage and to investigate the cell pathways thereof.Drosophila model  the most abundant nucleolar protein and is the first identified RNA binding protein able to shuttle from nucleus to cytoplasm, being involved in the transport of RNA molecules and proteins across the nuclear membrane . Importala model , althougla model .Another link between NCL and ALS/FTD was suggested by the co-segregation of NCL and some RNAs  or RNA binding proteins (including TDP-43 and FUS/TLS), in RNA foci accumulated in ALS patient tissues, resulting in the altered expression of specific RNA targets, RNA misprocessing events and disruption of nucleolar functions .Moreover, NCL was also reported to play a role in the long-term maintenance of mature neurons , and hasImportantly, we confirmed in mammalian cells the evidence that NCL can potently counteract TDP-43 (WT or bearing ALS-related missense mutations) cytotoxicity and TDP-43 detergent-insolubility observed in our yeast models. Indeed, data from human HEK293T cells indicated that NCL overexpression markedly decreased the number of cells with cytoplasmic TDP-43 aggregates, while concurrently increasing the number of cells displaying proper TDP-43 nuclear localization. We further observed that the TDP-43-dependent activation of apoptotic cell death in human cells was significantly reduced by NCL co-expression, suggesting that NCL could suppress TDP-43 toxicity also by directly acting as an antiapoptotic factor, in light of its reported ability to stabilize the mRNA of the Bcl-2 anti-apoptotic factor , and to However, in yeast cells, NCL was not able to counteract the cytotoxicity induced by the overexpression of FUS and C9orf72 DPR, both of which were recognized – similarly to TDP-43 – to induce cell death via the mitochondrial apoptotic pathway , suggestFurther studies are needed to establish the exact mechanisms by which NCL specifically target TDP-43 pathology. However, the observation that  the removal of the NLS did not influence NCL ability to rescue TDP-43 toxicity, which was conversely abrogated by introducing an additional NLS signal forcing NCL retention in the nuclear compartment, strongly suggested that the extranuclear localization of NCL, possibly linked to its nucleocytoplasmic shuttling role, is required to suppress TDP-43 proteotoxicity.According to the evidence provided by this work, a possible scenario could be proposed, whereby the extranuclear fraction of NCL interacts with TDP-43 in the cytosol, restoring its correct movement across the nuclear membrane and promoting its nuclear localization and solubility, therefore preventing the formation of cytosolic TDP-43 aggregates, and finally alleviating the proteostatic stress induced by TDP-43 overexpression and/or disease-related mutations.An emerging theme in TDP-43 proteinopathies is the ability of the protein to undergo phase transitions, forming liquid-like condensates that may be more harmful to neurons than insoluble aggregates. The nature  and the cell localization  of TDP-43-containing structures that NCL is able to cure may thus worth further investigation.Concurrently, another sensible hypothesis entails that NCL exerts a chaperone-like activity for TDP-43, as already suggested for histones, whereby NCL helps histone nuclear import and storage when not assembled with DNA . By thisThis hypothesis is supported by the MS analysis performed in yeast cells providing evidence that among numerous altered proteins by TDP-43 expression, the presence of NCL restored the normal levels of some HSPs. Interestingly, NCL restored the levels of HSP70 and of HSP82 (the yeast homolog of HSP90), both previously suggested to functionally interact with TDP-43 . PossiblThe targeted proteomic approach was based on a preliminary untargeted large-scale MS analysis of yeast cells, which identified several proteins dysregulated by TDP-43 expression. Although we may possibly have identified a higher number of dysregulated proteins in the toxic TDP 2C strain, we chose to run such a first large-scale study in the non-toxic TDP 1C strain in order to avoid foreseeable secondary whole proteome alterations related to cell death pathway activation. Such a choice was corroborated by the subsequent PRM targeted analysis, demonstrating that most of proteins involved in cell stress response, protein folding, and proton transport that we found to be modified in TDP 1C cells were equally altered in the (toxic) TDP 2C strain.Specifically, we found in both strains the upregulated expression of proteins involved in the response to oxidative stress or also of some chaperone proteins belonging to the HSP subfamily involved in protein folding and stress granule disassembly, which may speculatively be associated to a compensative response to TDP-43-induced protein misfolding stress. Conversely, among the MS identified proteome no evident alteration was observed for proteins directly involved in nucleocytoplasmic trafficking, while – unfortunately – our analysis failed in detecting any protein possibly implicated in the filamentous yeast growth or autophagy. Importantly, however, we also found that TDP-43 expression in yeast is associated to a down-regulation of several subunits of ATP synthase, supporting the idea that TDP-43-induced damages may also involve mitochondrial disfunctions, as reported previously .in vivo ALS experimental settings, NCL seems a promising means to understand the mechanisms of TDP-43-associated proteinopathies and a possible therapeutic target for ALS and related disorders.Here, reported findings clearly identified NCL as a powerful modifier of TDP-43 proteotoxicity in both yeast and mammalian cells. Although studies are needed to assess if the above proposition still stands in ALS selectively vulnerable MNs and in The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD022432.RL, AB, and CP: conceptualization. RL, RS, and CP: methodology. CP, RS, and RB: formal analysis. CP, RB, RS, MM, JA, and AM: investigation. FT, GS, and MM: resources. RL, AB, and CP: writing – original draft. RL, AB, CP, FT, MM, GS, and RS: writing – review and editing. RL and AB: supervision and funding acquisition. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Traumatic brain injury (TBI) is a disabling disorder and a major cause of death and disability in the world. Both single and repetitive traumas affect the brain acutely but can also lead to chronic neurodegenerative changes. Clinical studies have shown some dissimilarities in transactive response DNA binding protein 43 (TDP-43) expression patterns following single versus repetitive TBI. We explored the acute cortical post-traumatic changes of TDP-43 using the lateral fluid percussion injury (LFPI) model of single moderate TBI in adult male mice and investigated the association of TDP-43 with post-traumatic neuroinflammation and synaptic plasticity. In the ipsilateral cortices of animals following LFPI, we found changes in the cytoplasmic and nuclear levels of TDP-43 and the decreased expression of postsynaptic protein 95 within the first 3 d post-injury. Subacute pathological changes of TDP-43 in the hippocampi of animals following LFPI and in mice exposed to repetitive mild TBI (rmTBI) were studied. Changes in the hippocampal TDP-43 expression patterns at 14 d following different brain trauma procedures showed pathological alterations only after single moderate, but not following rmTBI. Hippocampal LFPI-induced TDP-43 pathology was not accompanied by the microglial reaction, contrary to the findings after rmTBI, suggesting that different types of brain trauma may cause diverse pathophysiological changes in the brain, specifically related to the TDP-43 protein as well as to the microglial reaction. Taken together, our findings may contribute to a better understanding of the pathophysiological events following brain trauma. Traumatic brain injury (TBI) is one of the most common causes of disability and death in younger individuals ,2,3. It Both single and repetitive exposures to traumatic injury may affect the brain acutely and can lead to chronic neurodegenerative changes, such as chronic traumatic encephalopathy (CTE), a degenerative brain disorder found in individuals with a history of repetitive brain traumas and associated with behavioral problems and the development of dementia . Even thA315T mice, the synaptic function is impaired due to the development of mushroom spines with lower efficiency . A decrease in the nuclear expression of this protein was detected at day 3 following LFPI but only compared to the day 1 level . Further, we wanted to determine whether these TDP-43 expression changes are cell type specific, i.e., if the increased cytoplasmic presence of this protein could be detected in the ipsilateral cortical neurons, microglia, or astrocytes after LFPI. Immunofluorescence analysis of the ipsilateral cortices of injured mice . Next, we investigated the TDP-43 expression patterns following rmTBI. The Western blot analyses showed no difference in cytoplasmic . Levels of the phosphorylated TDP-35 (p = 0.023) in the ipsilateral hippocampus after single moderate LFPI were also higher in comparison to the related control group, as seen in representative immunoblots .Next, we wanted to test whether the LFPI or rmTBI may trigger other post-translational changes of the investigated protein, specifically phosphorylation of TDP-43 and its fragments. As revealed in d TDP-35 I . After detecting the differences in the presence of the pathological TDP-35 fragment as well as in the appearance of P-TDP-43 and P-TDP-35 in the mouse hippocampi after LFPI in comparison to rmTBI animals, at 14 d following the experimental procedures, we wanted to determine whether these changes are related to microglial dysregulation in the same brain region. Namely, it has been suggested that the immune activation, including the activation of the microglia, the brain’s immune system cells, could result from an initial pathological insult. This triggering event could consequently cause an ongoing cytotoxic response, including the accumulation of TDP-43 , and resIn order to test our hypothesis, we evaluated the hippocampal levels of some protein markers of microglia activity by using Western blotting. Densitometric analyses of the protein blots revealed that, both in the LFPI and the rmTBI experiments, there were no changes in the hippocampal levels of the investigated microglial markers, specifically Iba1, iNOS, TLR-2, CD86, and CD206, at 14 d after both brain trauma protocols in relation to their designated control groups . Considering that this molecular approach to phenotypic characterization of microglia activation has garnered much criticism in recent years as being too simplistic and reductive , we alsop = 0.679; rmTBI: p = 0.299) (p = 0.020) (p = 0.744)  (In = 0.299) D,H. Howe= 0.020) G, which = 0.744) C. In the= 0.792) B,F. In cGiven the fact that no change in the hippocampal expression of the pathological TDP-43 species was observed in the rmTBI group of animals, we argue that, at this subacute time point, changes in the microglia activation state are unrelated to TDP-43 protein function and changes. This is also supported by the absence of correlation between the presence of the pathological TDP-35 fragment, the appearance of P-TDP-43 and P-TDP-35, and the microglia state in the hippocampi of mice subjected to LFPI.In this study, we provide evidence from in vivo experiments on some changes in the cortical TDP-43 expression patterns as well as in the levels of the p65 subunit of NF-κB and in the postsynaptic protein PSD-95 in a mouse model of single TBI of moderate severity. We used a model that resulted in brain trauma with characteristics similar to the injuries suffered in traffic accidents or falls. We also detected, in the subacute post-injury period, some differences in the hippocampal expression of the pathogenic TDP-35 fragment, levels of phosphorylated full-length TDP-43 and its 35 kDa cleaved fragment, as well as in the microglial reactivity between the aforementioned model and a TBI model representative of the type of repetitive brain trauma, typical for contact sports or in the military personnel. The brain trauma models we chose for this study represent one of the most used TBI models for their respective types of brain injury and, in both of them, neurobehavioral and cognitive deficits, regularly observed in patients with brain trauma, are detected ,55. HoweIn the animals subjected to a single moderate LFPI, we detected significant upregulation of the cytoplasmic TDP-43 in the ipsilateral cortices of injured animals at 1 and 3 d after TBI, with decreased nuclear TDP-43 content only on the third post-injury day. TDP-43 cellular mislocalization has also been previously described in several studies in which different models of TBI, varying injury severities, and experimental animal species were used ,48,49,50What we also detected, in the ipsilateral cortices of mice following LFPI, is that the TDP-43 cytoplasmic accumulation was present not only in neurons, as was reported by Wright et al. , but alsNF-κB is a major transcription factor and its activation is important for neurogenesis and synaptic plasticity in neurons ,27; howeOur results regarding the TDP-43 and NF-κB cytoplasmic expressions in the animals with a single moderate LFPI also correlate to the findings by Thammissety et al. , in whicSynaptic dysfunction is one of the pathophysiological hallmarks of TBI and numerous chronic neurodegenerative disorders, such as AD, in which synapse loss is not always concomitant with neuronal death, but can also happen in surviving neurons . PSD-95,The victims of TBI often suffer from lasting cognitive deficits, memory difficulties, and behavioral disturbances, and increasing evidence suggests that, even without the presence of evident morphological changes, subtle physiological changes that occur in the hippocampus after TBI are of potentially great importance . Among tIn our experiments, at 14 d following LFPI induction, the presence of the pathological TDP-43 species in the hippocampi of injured animals was not accompanied by changes in the microglial activation. Conversely, in mice subjected to repeated concussions and sacrificed at 14 d after the last mild trauma, we discovered some morphological changes pointing to microglial activation, but with no signs of TDP-43 proteinopathy. Taken together, these results indicate that, in this brain region and at the investigated time point, the TDP-43 protein expression pattern is not related to neuroinflammation. In this study, we did not consider the effects of the biological variables, such as the animal sex, or the investigation bias, e.g., regarding the judgment in cell counting. Moreover, we did not observe behavioral effects and differences in these subacute time points.In conclusion, the present study demonstrated that single moderate TBI causes acute changes in the subcellular TDP-43 expression levels in the ipsilateral cortices of injured animals and a decrease in the cortical PSD-95 levels. PSD-95 expression correlated with the presence of increased cytoplasmic TDP-43 expression in the surviving cortical neurons acutely following LFPI. At 14 d post-LFPI, we also detected some pathological changes of the hippocampal TDP-43, which was not detected in the same brain region in mice following repeated mild brain traumas. Variances in the microglia activity in the hippocampus between the two models of TBI also indicate that there are some differences in the pathophysiological processes employed after different types of brain injury, which might in turn prove to be important for the prognosis and the treatment of TBI.In our future investigations, we plan to examine the molecular background of observed TDP-43 mediated changes, specifically in relation to the expression of TBI specific inflammatory response, changes in different miRNAs expression profiles and the activation of MAPK signaling pathways.n = 50). Male animals were chosen for this study in order to exclude the possible effects of different levels of female sex hormones released during the estrus cycle [Experiments were performed on adult male wild type C57BL/6 mice  apparatus, adjusted to the mouse ,79. In bRepetitive mild brain trauma was induced in mice using the weight drop model as described by Kane et al.  and usedAnimals from the LFPI experiments were sacrificed at 1, 3, or 14 d after the single moderate TBI while the mice from the rmTBI protocol were euthanized at 14 d after the last mild brain trauma. All the sham injured mice, used as the respective control groups for the LFPI and the rmTBI experiments, were sacrificed 1 day after the end of the experimental procedures. We decided to use this single time point after sham TBI procedures by considering the results of our previous research using the LFPI model in rodents as well as the results of our preliminary studies of rmTBI in mice. Namely, in our previous experiments, we found no significant differences between the control animals sacrificed at different time points following sham procedures, which is something that can be also supported by a number of previously published studies ,81,82. FBrains from the separate cohorts of mice were used for immunohistological assays. For that purpose, at the same time points , mice we3, 1 mM PMSF, 10 µg/mL aprotinin, 10 µg/mL leupeptin and 10 µg/mL NaPP), homogenized in Dounce homogenizer and incubated for 30 min on ice. After centrifugation at 13,375 rpm for 5 min at 4 °C, the supernatant was collected as the cytosolic fraction and stored at −80 °C. Pellet was resuspended with lysis buffer B (buffer A without Triton-X) and centrifuged at 3000 rpm for 5 min at 4 °C. The supernatant was thrown away and the pellet was resuspended in the nuclear buffer (buffer B with 450 mM KCl and 50% glycerol) and frozen for 30 min at −80 °C. After thawing at RT, the sample was centrifuged at 13,375 rpm for 15 min at 4 °C, and the supernatant was collected as the nuclear fraction and, finally, stored at −80 °C. Cytosolic and nuclear fractions from the isolated cortices and the hippocampi were prepared for the detection of the designated proteins’ expression levels by Western blotting technique, as previously described by Yadavilli et al. . BrieflyEqual amounts of proteins per lane were loaded on a sodium dodecyl sulfate-polyacrylamide gel, separated by electrophoresis, and then transferred to nitrocellulose membranes. According to the antibodies’ manufacturers’ recommendations, membranes were blocked in 5% non-fat dry milk or bovine serum albumin (BSA) as the blocking buffers for one hour at RT. The incubation of the membranes with different primary antibodies, as shown in Frozen brain sections were left to achieve RT and then dried at 50 °C for 10 min. Permeabilization was done with tris-buffered saline (TBS) with 0.025% Triton X-100, for 10 min at RT. Blocking buffer made of 5% normal serum  was prepared in 1% BSA/TBS-Triton X-100 (0.025%), and applied for 2 h at RT. Primary antibodies, according to Cresyl violet staining (Nissl staining) was used for identifying the basic neuronal structure and the brain injury extent. Frozen sections were left to achieve RT and afterwards were dried at 50 °C for 10 min. Slides were immersed directly into 1:1 alcohol/chloroform overnight and were then rehydrated through 100% and 95% alcohol to distilled water. Staining of the slides was performed with warmed acidic 0.1% cresyl violet solution for 10 min. Slides were then rinsed in distilled water and, after differentiation in 95% ethyl alcohol, dehydrated in 100% alcohol and cleared in xylene. Mounting was performed with Entellan .2) were selected. Images were converted to the binary format, processed using the appropriate ImageJ plugins , and then used for the determination of the process length and the number of endpoints/processes per Iba1 positive cell by blind investigator. In the same microphotographs, the number of Iba1 positive cells in the dentate gyrus of the hippocampus was counted. In the animals from the LFPI and the rmTBI experiment, microphotographs of hippocampal sections immunolabeled with a primary antibody raised against ionized calcium-binding adaptor molecule (Iba1) were used for the quantitative analysis of microglia morphology, as previously described by Morrison et al. , in whict-test and comparisons between multiple groups were done using one-way analysis of variance (ANOVA) followed by Duncan’s post-hoc multiple comparison test. Results are expressed as mean ± standard deviation (SD). In all the comparisons, p < 0.05 was considered to indicate statistical significance. To remove the variations between the Western blotting sessions, results of the densitometric analyses were corrected with the Factor Correction program version 16.1 [Data were collected using Microsoft Excel 2016 . For the densitometric analyses of the expression levels of different proteins, the values of the relative optical densities of the bands were corrected for the corresponding β-actin (cytosolic samples) or histone-H3 (nuclear samples) contents and were then expressed as the % of the values from the related control groups. For all the statistical analyses, Statistica software version 13.0  was used. Sample size, i.e., the number of animals per group for the Western blotting and the immunofluorescence analyses, was calculated by using the results of our previous research in which complimentary methods were used ,85. Nameion 16.1 .


We propose a concept of an electrically controllable plasmonic directional coupler of terahertz signal based on a periodical structure with an active (with inversion of the population of free charge carriers) graphene with a dual grating gate and numerically calculate its characteristics. Proposed concept of plasmon excitation by using the grating gate offers highly effective coupling of incident electromagnetic wave to plasmons as compared with the excitation of plasmons by a single diffraction element. The coefficient which characterizes the efficiency of transformation of the electromagnetic wave into the propagating plasmon has been calculated. This transformation coefficient substantially exceeds the unity  due to amplification of plasmons in the studied structure by using pumped active graphene. We have shown that applying different dc voltages to different subgratings of the dual grating gate allows for exciting the surface plasmon in graphene, which can propagate along or opposite the direction of the structure periodicity, or can be a standing plasma wave for the same frequency of the incident terahertz wave. The coefficient of unidirectionality, which is the ratio of the plasmon power flux propagating along (opposite) the direction of the structure periodicity to the sum of the absolute values of plasmon power fluxes propagating in both directions, could reach up to 80 percent. Two different methods of the plasmon propagation direction switching are studied and possible application of the found effects are suggested. As was shown in3, the momentum relaxation time of charge carriers in graphene reaches the value of 2 ps at room temperature. Hence, graphene is a promising platform for excitation and manipulation of plasmons in the THz frequency range4. Possibility of population inversion of free charge carriers in graphene at THz frequencies5 along with the possibility of excitation of highly confined graphene plasmons6 resulted in proposals of THz graphene lasers8 and plasmonic amplifiers10 which may be used for signal processing in THz integrated nanocircuits in the subwavelength regime11.Controlling the propagation the plasma waves (plasmons) as well as switching of plasmon propagation direction is important for designing integrated plasmonic nanocircuits. Noble metals are suitable for exciting and controlling plasmons in the visible and near-infrared frequency range. But at terahertz (THz) frequencies, plasmons on the surfaces of noble metals are weakly confined. Terahertz plasmonic devices can be implemented on other materials, graphene for example. Graphene is a unique material with a zero energy gap and linear spectrum of electrons at THz frequency range12, a single metal gate13 or a thin slit in a metal gate14 which are placed above graphene. One may use the diffraction of external waves on the cantilever of a scanning near-field microscope15. However, the diffraction of THz wave on a single diffraction element couples THz wave to plasmons in graphene with rather low efficiency. Another conventional method for exciting plasmon polaritons in graphene is using the prism coupling17 in the Otto or Kretschmann configurations19. In order to create plasmonic splitters or directional couplers one has to make the plasmon propagate in a desired direction. Diffraction of incident wave on a periodical grating gate with a symmetric unit cell demonstrates high efficiency of transformation of incident wave into standing plasma wave21 but does not allow for launching the unidirectionally travelling plasmon. Creation of travelling plasmon by using attenuated total reflection methods suffers from strong effect of the angle of incidence on the transformation of incident wave into travelling plasmon. To enhance the transformation efficiency of an incident electromagnetic wave into a propagating plasmon, a periodical graphene structure with a dual-grating gate having an asymmetric unit cell was proposed and theoretically investigated in23. Spatially asymmetric plasmon structures can detect THz radiation26. Excitation and amplification of THz plasmons using diffraction of incident wave on a periodical grating gate were experimentally observed in28.There are several methods for excitation of plasmons in graphene. It can be the excitation of surface plasmon polaritons propagating along the graphene using diffraction of incident THz wave on a grating gate29, where the method of switching the graphene conductance between high and low conductance states using appropriate bias voltage pulses was proposed. Trapping the plasmon polaritons on the graphene surface within a broadband frequency range was demonstrated theoretically and numerically in30. A tunable Y-branch graphene plasmonic switch operating at the wavelength of 1.55 mm was proposed and analyzed analytically and numerically in31. Proposed in that paper plasmonic switch supports transverse magnetic and transverse electric graphene plasmons whose propagation characteristics could be controlled by modulating the external electric field and the temperature of graphene. Plasmonic splitter based on two different metal–insulator-metal waveguides with periodic grooves and a subwavelength slit was proposed and theoretically studied in32. Terahertz tunable plasmonic directional coupler based on a graphene and a thin metal film with a nanoscale slit was proposed in14. The possibility of switching the plasmon propagation direction applying different voltages to the graphene layer was numerically shown in that paper.Tunable switches, splitters and couplers are of great importance in optical communication systems and integrated nanocircuits. Graphene-based switch in the near infrared frequency range and its fabrication and operation principles were demonstrated in33 or injection pumping34, or diffusion pumping proposed in36. Passive graphene (without population inversion) is overlayed upon the insulating layer separating the graphene layers. This passive graphene is screened by periodic metal dual grating gate that is placed over the structure. Incident THz electromagnetic wave with the electric field polarized across the grating gate fingers diffracts on the periodic gate and excites transverse magnetic (TM) plasmons (the magnetic field of the plasmon is polarized in the y-direction) in both graphene layers. We have studied the structure with equal widths of the grating-gate fingers in both subgratings comprising the dual grating gate. Thus, the structure under consideration has a geometrically symmetric unit cell. It is known that for effective excitation of the propagating plasmon one needs an asymmetry in the unit cell of the structure21. Applying constant dc voltage U or V between the gate and upper graphene layer, one can change the Fermi energy of the free charge carriers in the gated regions of the upper graphene by the value of eU or eV, respectively, where e is the electron charge. Change of the equilibrium electron concentration in graphene applying the constant voltage to the gate subgratings of the dual grating gate structures is experimentally and theoretically described in28. As was shown in37, the voltage applied to the gate almost does not affect the concentration of the free charge carriers in the lower graphene layer. Carrier concentration in the lower graphene could be changed if only the upper graphene concentration is depleted that was not realized for applied voltage values used in our calculations. Applying different dc voltages to different subgratings of the dual grating gate one can create spatial asymmetry in the unit cell of the studied structure (forming different Fermi energy levels under different gate subgratings) which allows for switching of plasmon propagation direction. Active graphene allows for amplification of the propagation plasmon.In this paper, we theoretically investigate electrically controllable THz plasmonic directional coupler based on a periodic graphene structure with a dual grating gate, termed dual grating gate graphene heterostructure (DGGGH), which is schematically shown in Fig. T = 300 K (assumed the same in both graphene layers), and free carrier momentum relaxation time is 38. The quasi-Fermi energy value in the lower (active) graphene  was taken to be 7. The frequency was fixed at 7.The coefficients of absorption of the electromagnetic wave and transformation of this incident wave into the propagating plasmon in the studied DGGGH are presented in Fig. entclass1pt{minimaU  are caused by the presence of the lower active graphene.The coefficient of absorption of the incident wave in the DGGGH is defined as mentclass2pt{minimx-direction, respectively , and x-direction (see red regions on the map), while negative values of this coefficient correspond to excitation of the plasmon travelling against the x-direction (blue regions on the map). Away the mode intercrossing regions, the incident electromagnetic wave excites rather weak plasmons in graphene. The transformation coefficient The coefficient of transformation of the electromagnetic wave into a propagating plasmon  by changing the gate voltages values. The plasmon propagation direction could be switched when the Fermi energy value under one of the sub-gratings remains fixed while the other Fermi energy value is changed  across the mode intercrossing  in dependence on the Fermi energy value x-direction. Positive x-direction to the sum of the absolute values of these power fluxes:P exceeds 6 in value). For the method of type I of plasmon direction switching, the unidirectional coefficients values after swapping the voltage values corresponding to mode intercrossing remain as high as 80 percent while the transformation coefficient P exceeds 6. This could be seen from Fig. We start from the method of type We have shown the possibility to realize electrically tunable terahertz active plasmonic directional coupler based on the periodical DGGGH. Proposed directional coupler allows for switching the plasma wave propagation direction between two opposite directions along the graphene layers. Important features of this coupler is high efficiency of transformation of incident THz wave into propagating plasmon and high directionality of the plasmon due to using the dual-grating-gate and the active graphene to excite undumped and/or amplified propagating plasmons.x-component of the electric field amplitude over the unit cell of the DGGGH in the passive graphene layer for the fixed Fermi energy values a little shifted from the values corresponding to the plasmon mode intercrossing  for frequencies exceeding 5 THz. The higher-frequency plasmon mode in this case has a lower radiative damping, therefore it becomes an amplified mode due to the gain provided by the active graphene, while the other mode remains absorbing39.An important feature to be discussed is the structure of the plasmon modes near the intercrossing regimes where the switching of the plasmon propagation direction occur. In Fig. Finally, the concept of the electrically controllable active plasmonic directional coupler of THz signal based on a periodical dual grating gate graphene structure is proposed. The advantage of effective energy transformation by using dual grating gate periodical graphene structure allows for excitation of unidirectionally propagating plasmon with great transformation efficiency and high directionality. One can control the plasmon propagation direction by applying different dc voltages to the subgratings of the grating gate. High transformation efficiency occurs due to amplification of plasmons in the structure by using pumped active graphene. Such electrically controllable directional coupler is of high importance for application in THz plasmonic integrated nanocircuits.Numerical calculations were performed using the electromagnetic approach based on the integral equation method. Basic steps of this approach are the following. (i) Maxwell equations, Ohm’s law and boundary conditions are written in the Fourier representation. (ii) The integral equations in respect of oscillating currents in the conducting areas of DGGGH are formed. (iii) The integral equations are numerically solved using the Galerkin procedure. The described method allows for calculation the field of plasma wave at any point of the structure. For more detail, see the Supplemental material.Supplementary Information.


In this paper, graphene-coated spherical nanoparticles are arranged around an infinite length dielectric cylinder to enhance its extinction cross-section. Initially, a single longitudinal one-dimensional periodic array is considered in different loci concerning the transverse electric (TE) incident plane wave. It is observed that regardless of the position of the particles, the extinction cross-section of the dielectric cylinder is considerably enhanced with respect to the bare one. Later, by increasing the number of longitudinal plasmonic arrays around the cylinder, each residing in a different azimuthal direction, the extinction cross-section is further manipulated to observe double pronounced Fano resonances. The origin of the Fano resonances is described by considering their planar counterparts constructed by the periodic assembly of plasmonic oligomers. Finally, the hexamer configuration is considered as the prototype, and the effect of various optical, geometrical, and material parameters on the optical response is investigated in detail. Interestingly, due to the spherical symmetry of the cells, the extinction cross-section is also enhanced for the transverse magnetic (TM) incident wave, which is unattainable using a continuous plasmonic cover made of metal or graphene. The potential application of our proposed structure is in the design of reconfigurable conformal optical absorbers and sensors. Also, core–shell cylindrical particles made of noble metals can be used to enhance luminescence, nonlinear optical activities, and for deep sub-wavelength-scale optical wave-guiding2. Moreover, a triple-layered tube consisting double metallic and single dielectric layers is proposed as a super-scatterer, designed using the dispersion engineering method3. By further increase of the stacked metal-dielectric layers, multiple super-scattering and invisible cloaking states can be observed for the possible application in electromagnetic tagging4. For the metal-based cylindrical scatterers, the plasmonic effects are mostly observed for TE polarized wave fields with respect to the cylinder axis5.Scattering analysis of cylindrical objects covered by patterned metallic elements or plasmonic metals has been the subject of many pieces of research because of the wide range of applications that can be provided by them. For instance, an infinite length dielectric cylinder coated with a sub-wavelength conformal array of slotted Jerusalem crosses can be used as a mantle cloak for both TE and TM polarizations of the incident waves6. Moreover, reconfigurable plasmonic waveguides and refractive index sensors are realized using localized surface plasmons of graphene shells around cylindrical objects8. As another application, double graphene shells around the dielectric cylinders are used to design single and double band super-scatters10. By attaining multiple peaks and dips in the scattering response using multiple plasmonic layers, an optical device for simultaneous super-scattering and super-cloaking is proposed11. Moreover, flatten conformal hyperbolic metamaterials are presented using graphene strips around a cylinder under magnetic bias12. The main limitation in all above-mentioned plasmonic devices is that they function only under TE polarized waves13. The goal of this paper is to introduce a dual-polarized graphene-based plasmonic cover for cylindrical wires.By the emergence of two-dimensional (2D) graphene material, the aforementioned applications are implemented with the graphene plasmons due to their exotic properties. For instance, graphene patches are wrapped around a cylindrical object to attain an invisible cloak14. By tailoring the corresponding intrinsic surface resistance, optical-resistive sheets and Salisbury absorbers have been implemented15. Moreover, by properly adjusting the homogenized surface reactance of the arrays made of silver particles wrapped around a dielectric cylinder, the dipole scattering mode has been canceled16. It should be noted that Clausius–Mosotti's effective medium approximation and bi-dimensional model have been used to theoretically demonstrate the functionality of the above meta-surfaces17. As further instances, different distributions and orientations of silver ellipsoidal particles have been considered around a dielectric sphere to implement a plasmonic cloak in the visible spectrum18. Another scheme for cloaking of the cylindrical objects has been proposed by core–shell nanoparticles around the cylinder and it is proved that perturbation of the periodic alignment does not significantly affect the performance of the structure19.Optical covers can be constructed by a periodic arrangement of plasmonic nanoparticles rather than using the metallic sheets with patterned elements which suffer from the practical realizably at high frequencies20. The surface plasmons of a periodic assembly of graphene-coated spherical particles are engineered to design different types of absorbers23. Following the design procedures of the particle coated optical curved structures, in the present research, a graphene-based optical cover is proposed using spherical nanoparticles to enhance the extinction cross-section of an infinite length dielectric cylinder. The potential of the structure for the double Fano resonance generation is also discussed. In particular, in the plasmonic oligomers, the nanoparticles with optimized sizes, shapes, and inter-particle distances are clustered in a way that the corresponding scattering cross-section contains two peaks (bright modes) having a dip (dark mode) between them, as a result of plasmonic hybridization24. Fano resonances can be possibly used for bio-sensing, surface-enhanced Raman scattering (SERS), molecular fluorescence, and solar energy conversion.Recently, the capability of the localized surface plasmons of graphene-wrapped spherical nanoparticles in the design of tunable optical meta-surfaces has been investigated25. In another approach, a wide dipole resonance is overlapped with narrower higher-order modes spatially and spectrally to give raise to plasmonic induced transparency (PIT)26. Moreover, the interface of electric and magnetic dipole modes can easily excite Fano resonances in all-dielectric oligomers27. Also, magnetic plasmon modes constructed by circulating currents in the rings of plasmonic nanoparticles lead to the Fano resonances with low radiation loss29. Another approach is anapole modes which are non-radiating configurations obtained by spectrally overlapped composition of electric and toroidal dipoles30. Symmetry breaking is another efficient approach for the Fano resonance generation that can be achieved in various ways including but not limited to the size, position, and incident angle engineering34. Our proposed device benefits from the incident angle symmetry breaking for the excitation of Fano resonances.In general, the generation of Fano modes can be related to the coupled excitation of various types of resonances. For instance, the destructive interaction between the narrowband and broadband dipolar plasmonic resonances in a gold-graphene sensor results in the plasmonic electromagnetic induced transparency (EIT) with a Fano line-shapeThe paper is organized as follows. In “35. This model is consistent with the well-known Kubo model of local surface conductivity σ in which graphene is characterized by the relaxation time τ and chemical potential μc36. Based on this model, graphene is considered as an infinitely thin spherical shell using the convert to sheet option in the software. Note that because of the large number of carbon atoms in the simulated shells, the curvature of the spherical nanoparticles does not affect the surface conductivity model available for the planar sheets37. To simulate an infinite length cylinder, image theory is used to set the top, and bottom faces of the solution domain with respect to the cylinder axis to perfect electric conductor (PEC) under TM waves and to perfect magnetic conductor (PMC) for TE waves. The details of image theory can be found in classic electromagnetic books38.In this section, various combinations of graphene-based plasmonic nanoparticles are considered around a cylindrical dielectric core. Later, the influence of the array position and configuration on the extinction cross-section of the structure is investigated in detail. All the simulations are carried out with the frequency domain solver of the CST 2017 commercial software using the built-in surface conductivity model of graphene materialrc = 500 nm and core permittivity εc = 2 under plane wave illumination, as shown in Fig. z-axis and the linearly polarized wave is transverse electric (TE) with respect to it. It is also assumed that a single one-dimensional periodic array with the periodicity along the cylinder axis covers the wire. The array is constructed by graphene-coated hollow spherical nanoparticles with the longitudinal periodicity of h = 250 nm. The possible locations of the array with respect to the plane wave are denoted by L (Left), R (Right), U (Up), or D (Down). The initial parameters are as follows: the radii of the spheres are rs = 100 nm, relaxation times of graphene shells are τ = 1.5 ps, and chemical potentials of graphene shells are μc = 0.5 eV.The structure under consideration is a dielectric cylinder with the radius 39 which are calculated using broadband far-field monitors in the simulations. It is observed that the presence of a one-dimensional periodic array of plasmonic nanoparticles enhances the extinction cross-section of the dielectric cylinder, considerably. This effect is due to the excitation of localized surface plasmons of the graphene shells and it depends on the position of the array with respect to the incident wave, which alters the incident angle of the illuminating wave. Therefore, as Fig. Initially, a one-dimensional periodic array is considered in R, L, U, or D locations concerning the incident wave, as illustrated in Fig. 40. These structures, mainly have 2D periodicity which is attained by pattering the oligomer on the planar surface44. The generation of the Fano resonances in our proposed structure benefits from the same strategy using a cylindrical substrate. Symmetry breaking provided by the different incident angles that each particle experience is the main mechanism for the Fano resonance generation. Note that for hexamer, heptamer, and octamer configurations, there are double Fano dips in the extinction cross-section. The meta-molecules can be engineered for the modification of the plasmon line-shape in double spectral positions, simultaneously, which have great potential in dual plasmonic sensor applications45. In this regard, a metallic double Fano-resonant nano-cluster is proposed as a four-wave mixing device46 and our proposed structure presents a novel double Fano resonant structure using graphene plasmons.The impact of increasing the number of one-dimensional periodic arrays around the cylinder on its extinction cross-section is investigated in Fig. 10. The main drawbacks of this method are its extreme sensitivity to the fabrication tolerance and material losses due to its highly resonant behavior. Therefore, very limited realizations of them are available in the low-frequency window11. Our proposed device performs more robustly considering these two aspects, as will be further clarified by the parametric analysis of the performance. Moreover, the invisible cloak design using plasmonic particle covers with different morphologies is a well-established topic19. Interestingly, the extinction cross-section enhancement can be considered as the opposite side of cloaking, where sensing is of interest rather than hiding. This purpose is attained using the same geometry as the previously published ones for the cloaking. The aforementioned cloaks can be fabricated by the self-assembly technique and have a robust performance against fabrication imperfections19. Since the graphene-coated hollow particles can be realized chemically47, the proposed cover can also be fabricated with the self-assembly technique.Note that a well-known approach for the extinction (scattering) cross-section enhancement of the cylindrical objects is exploiting the super-scattering phenomenon23. These resonances are the main mechanism of the performance.To further investigate the performance of the cylindrical wire with a cover constructed by graphene-coated particles, the hexamer configuration is considered as a prototype, and the impact of various material, optical, and geometrical parameters on the extinction cross-section of the wire is investigated. Before proceeding to the next sections, the excited localized surface plasmons on the three peaks of the extinction cross-section of the hexamer Fig. b are ill48. Based on Fig. 50. The proposed sub-wavelength plasmonic cover can be simply reconfigured by changing the bias voltage/doping density of the graphene covers. This fact is illustrated in Fig. c = 0.4–0.7 eV. As the figure shows, by increasing the chemical potential, the resonance frequency experiences a blue shift. This feature is unattainable with metal-based plasmonic oligomers and has only been implemented using a planar assembly of graphene-based disk-shaped particles51.This subsection studies the influence of the core material and graphene optical parameters on the extinction cross-section of the hexamer. As Fig. 52. In order to obviate the need for further modification of the geometry, the ion-gel method is proposed as another approach, which has been widely used for planar structures55. As Fig. ngel = 1.43 covers the particles54. The resulted extinction cross-section and its comparison with the initial design are provided in Fig. 7.Modulating the chemical potential of graphene shells is an important issue in practice. A simple approach for this purpose is to electrically connect the particles using cross-hair geometry or reside them on top of another simply biased structure37. Note that the longitudinal periodicity is fixed in all the previous simulations. Moreover, cylinders with larger core radii exhibit a remarkable Fano dip at high frequencies, as illustrated in Fig. The impact of the geometrical parameters on the modulation of the Fano dips is illustrated in Fig. 57 and this outstanding feature is attained thanks to using a spherically symmetric cell.An outstanding feature of our proposed cylindrical structure is its dual-polarized optical performance. To discuss this feature, in Fig. n = 1.333–1.363 to investigate the sensing performance of the generated Fano resonances. The extinction cross-section of the structure is illustrated in Fig. 58 are included in Fig. −159, our proposed structure performs better in all three plasmonic resonances due to the excitation of higher order modes  and scattering cross-section (RCS) of the hexamer are compared with the bare dielectric wire in Fig. One-dimensional and two-dimensional arrays of polarizable nanoparticles constructed by graphene-coated spherical particles have the capability of enhancing the extinction cross-section of a dielectric cylinder. By adjusting the number of particles, the amount of the extinction cross-section can be tuned. The structure has many degrees of freedom for optical response manipulation and it performs for both TE and TM polarizations. Our proposed structure can be possibly used is in the design of sub-wavelength reconfigurable absorbers for sensing applications.


The use of SERS for real-world bioanalytical applications represents a concrete opportunity, which, however, is being largely delayed by the inadequacy of existing substrates used to collect SERS spectra. In particular, the main bottleneck is their poor usability, as in the case of unsupported noble metal colloidal nanoparticles or because of the need for complex or highly specialized fabrication procedures, especially in view of a large-scale commercial diffusion. In this work, we introduce a graphene paper-supported plasmonic substrate for biodetection as obtained by a simple and rapid aerosol deposition patterning of silver nanowires. This substrate is compatible with the analysis of small (2 μL) analyte drops, providing stable SERS signals at sub-millimolar concentration and a detection limit down to the nanogram level in the case of hemoglobin. The presence of a graphene underlayer assures an even surface distribution of SERS hotspots with improved stability of the SERS signal, the collection of well-resolved and intense SERS spectra, and an ultra-flat and photostable SERS background in comparison with other popular disposable supports. Two-dimensional arrays of plasmonic nanoparticles are gaining consensus within the scientific community in surface-enhanced Raman scattering (SERS) detection ,2 of molIn this work, we introduce a highly responsive SERS substrate specifically designed for biodetection of small sample drops and aimed at overcoming some limitations of the previous system as a result of the use of a simplified fabrication procedure and the introduction of a graphene underlayer to improve the SERS response. On the one hand, the fabrication setup was implemented with a common nebulizer for aerosol therapy, assuring a facile deposition of AgNWs without the need for expensive, time-consuming, or highly specialized procedures. Several studies proposed the direct deposition of sprayed colloids on sample surfaces to probe the surface composition of atmospheric particles , inks, aw 40000), isopropanol (99.5%), myoglobin from horse skeletal muscle (Mb), and hemoglobin (Hb) were purchased from Sigma-Aldrich . Ethylene glycol  was purchased from Carlo Erba . Silver nitrate (AgNO3) and silver chloride (AgCl) were obtained from Cabro S.p.A. .Polyvinylpirrolidone  and UV-Vis spectroscopy . The concentration of the as-obtained AgNWs suspension was finally determined as 1 mg/L by gravimetric determination.AgNWs were synthesized by the polyol method. Briefly, 80 mL of EG was heated and thermally stabilized at 170 °C in a flask. Once the temperature had been stabilized, 112.5 mg of AgCl was added to the flask. Meanwhile, 495 mg of AgNO® 454 polyester film, thickness 125 µm, DuPont, Wilmington, DE, USA ) patterned with 1.5-mm in size round holes, obtained by mechanical punching. SERS substrates were fabricated by aerosol deposition of AgNWs on 50-μm thick graphene-based paper  as achieved by mechanical compression of small stacks of graphene, which in turn was obtained by exfoliation of expanded graphite. Different AgNWs dispersions, as obtained by sequential dilutions in isopropyl alcohol of the original solution, were aerosolized on varying the exposition time within the 1–15 min range by using a common compressor nebulizer emitting micron-sized AgNWs drops for aerosol therapy . A fixed spacing between the nebulizer output and the graphene paper was maintained by using a third-hand support clip. Before deposition, the graphene was wetted in ethanol and then adhered to a PET mask  . The morphology of AgNWs on graphene paper at the nanoscale was analyzed by tapping mode AFM by using a JPK NanoWizard III Sense  scanning probe microscope at a 250–300 kHz drive frequency and a 0.5 Hz scan rate and equipped with single-beam uncoated silicon cantilevers (μMash HQ:NSC15 Cr-Au BS).The as-fabricated substrates were analyzed using a micro-Raman spectrometer  working at 532 nm with 1200 grooves/mm grating, an integration time of 5 s, and laser power at the sample of 150 μW, unless otherwise specified. A 10× objective with 0.25 NA (7 μm waist) was used.The electric field distribution in the near proximity of the AgNWs had been evaluated using a commercial FEM package, the wave optical module of COMSOL multiphysics , and the MNPBEM MATLAB  toolbox for the simulation of metallic nanoparticles, using a boundary element method approach .0| values were averaged over 5 different polarization angles of the incident light. A further configuration including a single AgNW laying on graphene and surmounted by another crossed wire was presented in the We chose to depict the AgNW as a cylinder with hemispherical ends, a radius of 25 nm, a total length 5 μm, and a refractive index for silver taken from Rakić et al. . An eval2 piece of thin graphene paper (G-paper)  . The sys−6 M hemoglobin (Hb) once excited at 532 nm , while lower time values resulted in an uneven distribution a–c. Oncet 532 nm e. We wert 532 nm  down to t 532 nm e. The abWe point out that the as-fabricated substrates allow to support the analysis of minimal quantities (2 µL) of biomolecule solution, which were initially dropped onto an array spot. They were then confined by a high contact angle formed with the surrounding hydrophobic graphene barrier  and fina−1) and G (1584 cm−1) band signals (2), and flexibility , which makes it even more attractive. Our initial aim in choosing graphene as a low-cost support for nanoparticles was dictated by three main considerations: (1) superior hydrophobicity behavior with respect to other popular supports for disposable substrates as cellulose paper, thus enabling analyte confinement and concentration enrichment, as discussed above; (2) low Raman signals in the fingerprint region as compared to other candidate hydrophobic substrates as polytetrafluoroethylene (PTFE) and polydimethylsiloxane (PDMS) and limited to D :The amplification provided by the as-fabricated AgNWs@G-paper was evaluated by calculating the SERS enhancement factor (EF). EF is defined as the ratio between EF value of 4 × 106 was calculated, proving an enhancement ability of the same order of magnitude or higher than that from recently proposed low-cost disposable SERS substrates including those based on AgNWs assemblies [0|)graphene/AgNW/(|E|/|E0|)AgNW/AgNW = 1.2), could be explained by taking into account the higher refractive index of graphene in comparison to other popular supports, which boosts the near field within the gap between metal and graphene, as shown in Ex = 532 nm appears ∼2-fold larger (|E|/|E0| = 44) than that at a PTFE/AgNW (|E|/|E0| = 24) or cellulose/AgNWs (|E|/|E0| = 19) interfaces. As SERS enhancement is proportional to the fourth power of the E-field, an average EF = 3.7 × 106 is obtained in the former case. A large matching between simulated and calculated EF let us hypothesize that the electromagnetic mechanism (EM) largely prevailed in AgNWs@G-paper substrates over a chemical mechanism (CM), as previously reported, as a possible additional effect in the SERS response of graphene-based substrates [An semblies ,44,45. Wsemblies ,46,47. Tbstrates .−1 [Another significant aspect in the choice of a SERS substrate resides in its background contribution to the overall SERS signal and generated at the interface between plasmonic layer and underlying support, which becomes critical especially in the detection of species with reduced SERS response, such as biomolecules. In −1  by the n−1 , generat−5 M to 1 × 10−9 M (1.3 μg to 0.3 ng) (2 > 0.99) between the band area of the 1378 cm−1 mode and the amount of protein was observed , suggesting a high sensitivity of AgNWs@G-paper that overcomes previous detection systems based on unsupported colloidal particles for protein detection including hemoglobin [We finally evaluated the detection sensitivity of the AgNWs@G-paper substrate by decreasing Hb concentration from 1 × 10 0.3 ng) a. A sigmobserved b, which moglobin ,52.SERS substrates proposed so far are often unsuitable for the practical detection of biomolecules due to the lack of simultaneous presence of preferred characteristics, such as low manufacturing costs, disposable characteristics, and simplicity for routine use in turn limiting their use at or near point-of-need settings. In this work, we introduced a highly responsive SERS substrate relying on a simple aerosol deposition of AgNWs on graphene paper. The substrate was specifically designed for biodetection of small sample drops at submicromolar concentration. The proposed fabrication procedure relied on low-cost and facile steps that overcame a number of weak points frequently encountered in substrate preparation and patterning. These included the use of contaminating stabilizers to impart sufficient viscosity to nanoparticle inks as usually required in screen or ink-jet printing, as well as the need for dedicated instrumentation for top-down nanoparticle fabrication. Additionally, the presence of graphene improved the SERS response, conferring superior signal stability, low SERS background, and photostability. Overall, the proposed substrate exhibited high SERS efficiency, reliability, and sensitivity, as well as easy handling and usage aspects, meeting many of the requirements for effective and successful SERS detection of biomolecules. Future experiments on AgNWs@G-paper substrates will be aimed at exploring the SERS sensing of larger biological entities as cells for rapid and tag-free screening of tumor diseases  or to monitor light treatments  for therapeutic applications.