TDP-43 is a DNA and RNA binding protein involved in RNA processing and with structural resemblance to heterogeneous ribonucleoproteins (hnRNPs), whose depletion sensitizes neurons to double strand DNA breaks (DSBs). Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder, in which 97% of patients are familial and sporadic cases associated with TDP-43 proteinopathies and conditions clearing TDP-43 from the nucleus, but we know little about the molecular basis of the disease. After showing with the non-neuronal model of HeLa cells that TDP-43 depletion increases R loops and associated genome instability, we prove that mislocalization of mutated TDP-43 (A382T) in transfected neuronal SH-SY5Y and lymphoblastoid cell lines (LCLs) from an ALS patient cause R-loop accumulation, R loop-dependent increased DSBs and Fanconi Anemia repair centers. These results uncover a new role of TDP-43 in the control of co-transcriptional R loops and the maintenance of genome integrity by preventing harmful R-loop accumulation. Our findings thus link TDP-43 pathology to increased R loops and R loop-mediated DNA damage opening the possibility that R-loop modulation in TDP-43-defective cells might help develop ALS therapies. Amyotrophic Lateral Sclerosis (ALS) is an adult onset, progressive neurodegenerative disease, caused by the selective loss of upper and lower motor neurons in the cerebral cortex, brainstem and spinal cord. The nuclear TDP-43 RNA binding protein, is encoded by a major gene for ALS susceptibility whose mutations are found in 3% of familial and 2% of sporadic ALS cases. Thanks to its ability to recognize DNA and RNA, TDP-43 is involved in different steps of mRNA metabolism and in several mechanisms of genome integrity. This, together with the fact that R loops or DNA-RNA hybrids are a common source of genome instability, prompted us to investigate whether TDP-43 deficiency has any role in R loop homeostasis that could explain previously described DNA damage response defects of ALS cells. We show that TDP-43 plays a role in preventing R loop-accumulation and associated genome instability in neuronal and non-neuronal cells, as well as in patient cell lines. Thus, our study opens the possibility that R loop-modulation in TDP-43-defective cells might help develop ALS therapies. TDP-43 is a nuclear RNA binding protein (RBP) with a repressor role of HIV-1 transcription. It binds to the trans-active response element DNA sequence of the viral genome . Like otMutations in TDP-43 are associated with sporadic and familial cases of Amyotrophic Lateral Sclerosis (ALS), an adult onset, progressive neurodegenerative disease, caused by the selective loss of upper and lower motor neurons in the cerebral cortex, brainstem and spinal cord , 10. TARIn addition to transcriptional autoregulation, TDP-43 can be cleaved into smaller C-terminal fragments before being enzymatically degraded to maintain its physiological levels , 19 by aEvidence indicate that a major source of spontaneous DNA damage comes from the accumulation of R-loops, consisting in DNA-RNA hybrids and a displaced single strand DNA (ssDNA) . Non-phyAll this, together with the fact that a number of neurodegenerative diseases highlight a particular sensitivity of the nervous system and motor neurons are associated with deficiencies in RNA metabolism and DDR, prompted us to investigate whether TDP-43 deficiency, as found in ALS cells, have a role in R-loop homeostasis that could explain previously described DDR defects of ALS cells. We show that TDP-43 plays a role in preventing R-loop accumulation and R loop-mediated DNA breaks in neuronal and non-neuronal cells and in patient cell lines, thus opening the possibility that R-loop modulation in TDP-43-defective cells might help develop ALS therapies.A key regulatory role of TDP-43 in essential metabolic processes was previously suggested since silencing of TDP-43 in HeLa cells lead in dysmorphic nuclear shape, misregulation of the cell cycle, apoptosis, increase in cyclin-dependent kinase 6 (Cdk6) transcript and protein levels . As a maGenomic DNA-RNA hybrids in siTDP-43 HeLa cells were first assessed by immunofluorescence microscopy (IF) using the anti-DNA-RNA hybrid S9.6 antibody, and determining the levels of the S9.6 signal in the nucleoplasm after subtracting the nucleolar contribution , 34. As Next, we determined R-loop accumulation by the more accurate method of DNA-RNA immunoprecipitation (DRIP)-qPCR, based specifically on the purification of genomic DNA-RNA hybrids of different sizes. In this case the S9.6 signal was determined for genes expressed at different levels such as APOE, RPL13A, WDR90, EGR1 and MIB2, which have been previously validated for the detection of R loops , 33, 35,Then, we investigated the functional impact of nuclear DNA-RNA hybrid enrichment on DDR, given that hybrids have been shown to enhance transcription-replication conflicts . As can Our results show that TDP43 depletion causes R loop-accumulation, R loop-dependent DNA damage and accumulation of the transcription-replication collision-associated FANCD2 repair foci similar to depletion of other mRNP processing factors that function co-transcriptionally together with RNA polymerase II (RNAPII). Indeed, genome-wide ChIP-seq data obtained in K562 erythroblastoma cells  reveals that TDP-43 colocalizes with expressed genes defined by RNA-seq . 6011 ouIn ALS patients harboring TDP-43 mutations, TDP-43 mislocalizes from the nucleus to the cytoplasm in detergent-resistant aggregated forms either full-length (43 KDa) and fragmented forms , which can be ubiquitinated and hyperphosphorylated . Protein-containing supernatants were collected and stored at −80°C. Total soluble protein lysates were loaded onto 8–16% SDS-PAGE gel (Bio-Rad). Samples were transferred to nitrocellulose membrane with Trans-Blot Turbo Transfer System (Bio-Rad). After 5% non-fat dry milk blocking, nitrocellulose membranes were incubated ON at 4°C with the following antibodies: anti-TDP-43 antibody , anti-GAPDH antibody . Densitometric analysis of the bands was performed using ImageJ software from three independent experiments.g). Cytoplasmic proteins were collected and stored at −80°C until use. The pellet was re-suspended in an ice-cold hypertonic nuclear extraction buffer  and incubated on ice with agitation. The nuclear extracts were centrifuged at the maximum speed and the nuclear proteins were collected. GFP antibody , rabbit polyclonal anti-H1  and mouse monoclonal anti-lactate dehydrogenase  were used as nuclear and cytoplasm loading controls.SH-SY5Y subcellular fractionation was performed according to Pansarasa et al. 2018. Cells were first re-suspended in an ice-cold hypotonic lysis buffer  and incubated on ice. 10% Nonidet NP-40 was added and samples were centrifuged at the maximum speed  ENCSR033VAZ entry, while DRIPc-seq, DRIP-seq and RNA-seq was gathered from Gene Expression Omnibus under accession number GSE127979 We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.Yours sincerely,Dmitry A. GordeninAssociate EditorPLOS GeneticsGregory BarshEditor-in-ChiefPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: In the manuscript “TDP-43 deficiency links Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated damage,” Giannini and co-authors explore the role of TDP-43 in the maintenance of genome integrity and try to dissect the mechanism of the neurodegenerative pathogenesis caused by the mutated version of the protein. They test the hypothesis that TDP-43 prevents the accumulation of DSB-inducing, deleterious R-loops and claim that an R-loop-dependent increase in DSB foci and Fanconi Anemia repair centers is caused by mis-localization of the mutated protein in transfected neuronal cells and in cell lines from ALS patients. Without a doubt, the findings reported in the manuscript are significant and worth further investigation; however, the conclusions drawn from the data are not sufficiently substantiated.Major concerns.1. The depletion of TDP-43 in HeLa cells by siRNA leads to a minor increase in S9.6 signal intensity . Even though the increase is statistically significant, the biological relevance of such an increase is unclear without comparison to the changes in intensity after depletion of the known protein involved in R-loop processing. This concern is exacerbated by the absence of an increase in DRIPqPCR product for the APOE gene in siTDP-43 treated cells . Taking into account this marginal effect on R-loop formation on the one hand and the multiple functions of TDP-43 in mRNA processing and its interaction with DNA on the other, the increase in gammaH2AX and FANCD2 foci  could reflect consequences of TDP-43 depletion that are unrelated to R-loop processing. The decrease in foci in cells expressing RNaseH could also be an indirect consequence of the protein depletion.2. The statement from the abstract, “we prove that mislocalization of mutated TDP-43 (A382T) in transfected neuronal SH-SY5Y and lymphoblastoid cell lines (LCLs) from an ALS patient cause R-loop accumulation, and R loop-dependent increased DSBs and Fanconi Anemia repair centers” is misleading. Mis-localization of both proteins - mutant and wild type - leads to an increase in S9.6 intensity , and the difference in S9.6 signal intensity between the cells with mutant and wild type proteins is not significant. It is well documented that overexpression of proteins can cause their mis-localization and misfunction. So, “significantly decreased” nuclear localization of the mutant protein in SHSY-TDP382 cells (decrease of less than 15%) compared to wild type SHSY-TDP+ cells  can result from (slightly) different expression levels of each of the proteins in the corresponding cell lines. In other words, the somewhat increased deleterious consequences of expression of the mutant allele could be caused by a higher level of overexpression of the mutant protein, compared to the wild type allele, rather than the presence of a missense mutation.Unless the levels of TDP+ and TDP382 proteins are measured and found similar, the “mutation” effects could be attributed to the differential expression levels of TDP-43 alleles in the cells.3. The same critique applies to the experiments with the lymphoblastoid cells from the patients.4. The data in Figure 6 are neither quantified nor normalized. The quantity of the loading control proteins varies from lane to lane. Also, it is unclear how many times the experiment has been performed, so the discussion of the results of this experiment is mere speculation.5. In Figure 3A, the increase in S9.6 signal in the cells with overexpression of TDP+ cannot be attributed to R-loop formation because overexpression of RNaseH does not decrease the signal. Nevertheless, the authors make the following conclusion: “Therefore, we conclude that mislocalisation of TDP-43 also causes R-loop accumulation “(page 8). This statement contradicts the data. It is unclear whether the levels of overexpression of RNaseH were monitored during the experiments in different cell lines. One explanation of these data could be that the expression of RNaseH in the specific cell line was different (lower) from the others.6. Throughout the study, the only assay utilized for providing evidence for DNA damage or activation of DNA damage response is the detection of DNA repair foci. Additional, independent tests  are necessary to address physiological consequences of TDP43 malfunctioning.Minor concerns.1. There is no description of how overexpression of RNaseH is achieved in Figures 1-5. Reference #33, used for an explanation , does not provide relevant information.2. Since in Figures 3A and Supplemental Figure 1, the data presented as “signal per nucleus” and the differences in these signals are minor, it seems to be important to make sure that the area (size) of the nuclei are similar among cells transfected with different constructs.3. Flow cytometry plots in Figures 5B and C do not include the plot of S9.6 signal for the LCL-SALS cells (cells from sporadic ALS patient), making the data representation skewed.Reviewer #2: In this manuscript, Gianini et al. have investigated in detail the role played by TDP-43 in the generation of R-loops and RNA:DNA hybrids in neuronal and non-neuronal cell lines. In general, the manuscript contains interesting data for the field and several novel observations that contribute to a better understanding of the protein in disease. A few required additions and clarifications are as follows:-The title “TDP-43 deficiency links Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage” does not seem appropriate since the manuscript principally focuses on a disease-associated mutation, A382T. Therefore, it might be better to change it in “TDP-43 mutation links Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage”.- The order followed in the abstract is not the one respected in the results  in transfected neuronal SH-SY5Y and lymphoblastoid cell lines (LCLs) from an ALS patient cause R-loop accumulation and then a non-neuronal model of HeLa cells depleted of TDP-43. However, in the results they present first the depletion of TDP-43 in Hela cells and then the transfection of mutated TDP-43 in SHSY-5Y. Please be consistent in the order of the text.- In the work the authors used silenced TDP-43 in Hela cells but transfected mutated TDP-43 in SHSY-5Y. Please explain better the choice of the two cell lines and how they are connected.- In the LCLs mutated in TDP-43 the authors observe an increase of R-loops compared to CTL and SALS LCLs. RNAse H could revert partially the S9.6 increase signal. How can this partial decrease be explained?- Experiments were run in SHSY5Y and in LCL carrying only one mutation A382T. It might be interesting to carry out at least part of the reported experiments in SHSY-5Y cells overexpressing TDP-43 with another mutation. These data would confirm whether the readouts observed with this mutation are specific or not.Reviewer #3: The paper by Gianini et al focuses on understanding how deficiency of TDP-43 observed in ALS is connected with R-loop homeostasis and R-loop mediated DNA damage. Previous work from Livinsgton lab has demonstrated that TDP43 is involved in prevention/repair of transcription-associated DNA damage through R-loop-related mechanisms in neuronal cells , therefore the novelty of this paper is partially compromised by this publication. One interesting aspect which the authors were trying to pursue is to uncover if TDP-43-disease-associated mutations which result in re-localisation of usually nuclear TDP43 to the cytoplasm has any effect on R-loop-mediated DNA damage. This is certainly an interesting question worth addressing. The authors pursued these questions in HeLa cells , neuroblastoma cells  and lyphoblastoid cells . Using these model systems, the authors have shown that TDP43 deficiency results in accumulation of R-loops (by IF and DRIP-qPCR), DNA damage (γH2AX) and Fanconi Anemia repair centers. Unfortunately, the authors have not managed to take these initial findings, some of which were already reported in Hill et al PNAS 2016. (i.e. R-loop and DNA damage increase), into subsequent mechanistic investigations. For the reasons of the quality and amount of data presented in this paper, at this current stage the paper is not suitable for publication in PLoS Genetics.Major comments:1. The amount of data presented in this paper is minimal and not of a good quality which overall does not give me the confidence of the general paper conclusions. In particular the conclusions drawn from S9.6 IF analysis are rather ‘pushed’ and not visible from the images presented. For example, Fig 1A does not have a clear nuclear signal in any of the images shown . The differences between quantified signals are very small and represent only a background signal, in my opinion). The RNase H over-expression does not result in any visible change of the signal on the images. The positive controls (known factor which up-regulates R-loop level) are missing. The authors have not quantified nucleolar signal (are there any changes there?), though they have chosen to study gene encoding ribosomal protein in their DRIP analysis (see below).2. It is surprising to me that the only gene which shows statistically significant accumulation of R-loops is RPL13A, which is a ribosomal protein. I would like to see more comprehensive analysis of R-loop increase by DRIP-qPCR. Other multiple genes of different categories should be tested in different model systems.3. Experiments in Fig 3-4 are carried out with neuroblastoma cells, where TDP-43 Wt or mutant (TDP382) are expressed on the endogenous background. The authors need to characterise this system a bit better, since the imaging in Fig S1 is not sufficient. They need to present Westen blot showing the level of expression of endogenous and over-expressed proteins in different cellular fractions  in all three cell lines. What is the level of endogenous protein contribution to all results observed? The authors have not really explained why do we observe a significant decrease of nuclear signal for TDP43 WT cells compared to control cells? These data are important for further interpretation of IF and DRIP results. All these experiments should have been performed on the background of depleted endogenous TDP-43 protein to have a clear interpretation.4. The authors have carried out IF in Lymphoblast cells from patients with TDP43 and ALS mutations. Similar to my comments for Figures 1A and 3A, the IF data are not convincing and show extremely small changes between cell lines studied. These cells derived from different people and are subject to patient –to patient genetic variation. Since the data are showing rather small changes, the authors should use multiple cell lines representing each category. Possibly using alternative approaches such as slot blot may provide more quantitative data than IF.5. Figure 6 contains results of S9.6 IP experiments in lymphoblast cells. The authors propose that they observe less TDP43 interaction with R-loops when TDP43 is mutated. This is not visible from the protein gel presented, and as far as I am concern represents a wishful thinking of the authors and not the result of a given experiment. There is absolutely minimal pull-down observed with TDP-43 in the nucleus, and mainly there is a pull down of TDP-35 which should be really cytoplasmic, and not in the chromatin fraction? Clearly these TDP-35 fragments are also IPed in the WT conditions – why is this happening? This experiment lacks RNase H control to show that interaction between TDP43 and R-loops is specific. Comments regarding stronger pull-down of TDP35 in TDP43 mutant in cytoplasm are not really substantiated since it is clear that this sample is over-loaded on the gel as can be seen from H3 and GAPDH westerns.Minor comments:1. On page 9 first paragraph, the authors say that ‘TDP-43 mutation causes an increase in DNA breaks derived from R-loop accumulation that promotes transcription-replication collisions..’ . I think that this is an over-statement since it has not really been tested if accumulation of R-loops following TDP-43 depletion results in transcription-replication collisions.2. The authors need to explain why SNRPN gene does not have DRIP signal – it is not expressed?3. All the data in this paper can be grouped in 2-3 figures max (one figure for each cell model used).4. The paper would benefit from additional English editing (i.e. - ‘significant’ on pages 6 and 11 should be spelled correctly..etc)5. Concluding model demonstrating TDP43 role in disease and R-loop biology would be helpful.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: No: The numerical data underlying graphs are not provided in spreadsheet form.Reviewer #2: YesReviewer #3: Yes**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  and your responses to reviewer comments. 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Gordenin, Ph.D.Associate EditorPLOS GeneticsGregory BarshEditor-in-ChiefPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: The revised manuscript by Gianini et al., “TDP-43 deficiency links Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated damage,” is much improved compared to the original version.Most of the issues that caused concerns in the paper’s previous version regarding data interpretation and appropriate controls are addressed and resolved in the updated manuscript.The added comparison of the consequences of TDP-43 depletion on R-loop formation (reported as changes in S9.6 fluorescence intensity) to the measurements already published in the studies of the contribution of proteins directly involved in R-loop processing , as well as DRIP-seq of additional R-loop - prone targets, allows appreciation of the biological relevance of relatively minor changes in fluorescence levels.The data from the new experiments involving treatment of the cells with RNAse III for the removal of dsRNA  eliminate the ambiguity of the results reported in the first version of the manuscript and validate the statement, “we prove that mislocalization of mutated TDP-43 (A382T) in transfected neuronal SH-SY5Y and lymphoblastoid cell lines (LCLs) from an ALS patient cause R-loop accumulation…”Measurements of the expression levels of the wild type and mutant constructs  strengthen the conclusion that indeed mislocalization, rather than an increased level of the mutant protein, causes accumulation of R-loops.Several points still require the authors’ attention.1. Even though the new genome-wide analysis of TDP-43 localization  does not provide direct evidence of the contribution of TDP-43 to R-loop processing but instead suggests that TDP-43 binds to highly expressed loci in DNA, these data offer new insights into TDP-43 functions. Nevertheless, the statement “…TDP-43 RBP controls transcription at expressed genes that are prone to accumulate R loops...” is not substantiated by the data and should be toned down.2. Since statistically significant “reduction in nuclear fraction” of endogenous TDP-43 was NOT observed either following expression of SH-TDP+, or SH-TDP382, it is formally incorrect to attribute an absence of a reduction in SH-TDP294 to any phenotype . .3. The model in Figure 6C must be adjusted to accommodate the data showing that a decrease (compared to control) in nuclear localization of overexpressed wild type TDP-43  does not cause an increase in S9.6 intensity .4. The spreadsheets with the raw data reflected in graphs and figures are not provided.Reviewer #2: The authors have provided several new data and have addressed in detail most of the queries raised by reviewers.Reviewer #3: The authors have made an effort to improve this manuscript including additional genes for DRIP-qPCR analysis (as requested in my comment 2) and some modest visual improvement for S9.6 IF.Related to previous comment 1:To address my concern related to small changes in IF signal in the nucleus associated with Pol II genes, the authors introduced genomic data from publicly available Encode and previously published data, showing a correlation between TDP-43 binding and R-loops in expressed genes under physiological conditions in K562 cells (fig 3). Based on these datasets TDP-43 is mainly enriched at the 5’end /TSS of the expressed genes, while R-loops can be found in multiple locations . However, these new genomic data in Fig 3 does not clarify what happens with this correlation when TDP-43 is depleted? Do R-loops accumulate only at the locations where TDP-43 is bound which would support the direct effects of TDP-43 on the regulation of these R-loops? The authors have not demonstrated if they see a specific enrichment of TDP-43 binding at R-loops of their tested genes by DRIP-qPCR supporting the specificity and direct effects of TDP-43 on these R-loops.Related to previous comment 4:Unfortunately the authors have failed to introduce additional patient cell lines to verify the effects of R-loop accumulation in patients with different genetic backgrounds. Therefore, in my opinion, providing a direct evidence that TDP-43 binds at the regions of R-loops affected following its depletion (as discussed above) is crucial.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the No: The data underlying the figures are not providedReviewer #1: Reviewer #2: YesReviewer #3: Yes**********what does this mean?). 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PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via  28 Nov 2020PGENETICS-D-20-00749R2 TDP-43 mutations link Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage Dear Dr Aguilera, We are pleased to inform you that your manuscript entitled "TDP-43 mutations link Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. 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Spanish cañón “canyon” /kaɲon/ [ka.'ɲon]). With these crosslinguistic differences in mind, we ask: (1a) Do L1 English learners of L2 Spanish produce acoustically distinct Spanish /n/ and /ɲ/ and (1b) Does the distinction of /n/ and /ɲ/ vary by proficiency? In the case that learners distinguish /n/ and /ɲ/, the second question investigates the acoustic quality of /ɲ/ to determine (2a) if learners' L2 representation patterns with that of an L1 Spanish representation or if learners rely on an L1 representation  and (2b) if the acoustic quality of L2 Spanish /ɲ/ varies as a function of proficiency. Beginner (n = 9) and advanced (n = 8) L1 English/L2 Spanish speakers and a comparison group of 10 L1 Spanish/L2 English speakers completed delayed repetition tasks in which disyllabic nonce words were produced in a carrier phrase. English critical items contained an intervocalic heterosyllabic /nj/ sequence ; Spanish critical items consisted of items with either intervocalic onset /ɲ/  or /n/ ['xa.na]. We measured duration and formant contours of the following vocalic portion as acoustic indices of the /n/~/ɲ/ and /ɲ/ ~/nj/ distinctions. Results show that, while L2 Spanish learners produce an acoustically distinct /n/ ~ /ɲ/ contrast even at a low level of proficiency, the beginners produce an intermediate /ɲ/ that falls acoustically between their English /nj/ and the L1 Spanish /ɲ/ while the advanced learners' Spanish /ɲ/ and English /nj/ appear to be in the process of equivalence classification. We discuss these outcomes as they relate to the robustness of L1 phonological constraints in late L2 acquisition coupled with the role of perceptual cues, functional load, and questions of intelligibility.This study examines whether L1 English/L2 Spanish learners at different proficiency levels acquire a novel L2 phoneme, the Spanish palatal nasal /ɲ/. While alveolar /n/ is part of the Spanish and English inventories, /ɲ/, which consists of a tautosyllabic palatal nasal+glide element, is not. This crosslinguistic disparity presents potential difficulty for L1 English speakers due to L1 segmental and phonotactic constraints; the closest English approximation is the heterosyllabic sequence /nj/ (e.g., “canyon” /kænjn/ ['k A lasting question that has occupied a central role in the study of second language (L2) phonology across several decades asks which factors modulate the acquisition of L2 contrastive sounds that are not part of the first language (L1) grammar. A look at the collective body of research reveals that, while there is robust evidence that novel L2 sounds are acquirable , which is restricted to heterosyllabic position. With these crosslinguistic differences in mind, to converge on the L2 Spanish target, the learner's grammar must come to allow a single alveopalatal nasal segment. The question, then, that follows, is whether these constraints can be overcome in the L2. While there are no L2 Spanish /ɲ/ acoustic or perception data to inform our predictions for the current studyIn the present study, we examine L1 American English speakers' acquisition of the alveopalatal nasal /ɲ/ in L2 Spanish. This sound is a challenge for L1 American English speakers for a number of reasons. First, this is a scenario in which the L2 sound does not exist in the L1. Second, it is the least frequent phoneme in the Spanish inventory  includes an alveopalatal nasal that contrasts with the other nasals in word-medial onset positionju:zik]) . Together, these L1 constraints yield a learning task in which the grammar must come to allow a single nasal segment alveolar and palatal places of articulation in syllable onset position.According to Martínez Celdrán and Fernández Planas , the alvTo determine whether L2 Spanish learners produce a single segment (/ɲ/) or a sequence (nj), we follow Bongiovanni  by acousj/~/C/ contrasts , with Diehm also reporting production data of within-language and between-language contrasts.As mentioned, although this is the first study to our knowledge to examine L1 English speakers' acquisition of the L2 Spanish palatal nasal, we can look to a small body of research that has examined L1 English speakers' acquisition of L2 Russian palatalized consonants to inform predictions for the current study. Similarly to Spanish /ɲ/, and unlike the English /nj/ sequence, Russian palatalized consonants are single complex segments with dual places of articulation that contrast phonemically with non-palatalized counterparts. Therefore, the L2 learning task for L1 English speakers is similar. In onset position/. Diehm  and Luky/. Diehm  examinedj/ ~ /Cjj/ contrast, Lukyanchenko and Gor's data from an ABX task reflected above-chance accuracy in low-proficiency (~73%)j/ and /Cjj/ more accurately than low-experience learners when followed by a low vowel  and that the increase in /Cj/ accuracy corresponded with a decrease in inaccurate identification of /Cj/ as /Cj/. While the learners in these two studies are neither at ceiling nor in line with L1 Russian accuracy, the data suggest that learners can develop perceptual acuity that at least partially circumvents the L1 phonological constraints that yield /Cj/ perception. Production data from Diehm, the only study to our knowledge to report a within-subjects comparison of L1 English and L2 Russian production data, points to partial acquisition in the oral modality. She found that even advanced L2 Russian learners' productions of /CjV/ and /CjjV/ syllables did not differ in F2 trajectory or duration from the point of consonantal release to the offset of the palatal element. This result lends support to the observation that L1 English learners decompose L2 /Cj/ into an L1-like /Cj/ sequence. Interestingly, a crosslinguistic comparison of a subgroup (n = 4) of advanced learners' L1 English and L2 Russian productions revealed that, while one learner produced L1 /Cj/ and L2 /Cj/ as (L1-like) /Cj/, the other three produced a distinct sequence in Russian that fell between their L1 English /Cj/ and the L1 Russian /Cj/ comparison. That is, although the L2 Russian /CjV/ F2 trajectories did not approximate those of the L1 Russian comparison group, they were shorter and had a more negative slope  than their English /CjV/. Diehm posited that this intermediate representation was indicative of partial L2 acquisition.Regarding perception of the /CjV/ ~ /CV/ contrast is higher than the functional load of the Spanish /ɲ/~/n/ contrast. Russian, which has 42 consonantal phonemes, has 15 pairs of consonants that are phonemically distinguished by palatalization; palatalized consonant phonemes range in frequency ranking from 14 to 42  overcoming the relevant L1 English constraints is possible. However, we recognize that the learning scenario is different in L2 Russian vs. L2 Spanish. Specifically, the functional load  of the Russian /CThere are two research questions that drive the current study. The first regards whether learners acquire the relevant contrast in the L2, independent of how their /ɲ/ productions compare with the L1 Spanish /ɲ/.(1a) Do L1 English learners of L2 Spanish produce acoustically distinct Spanish /n/ and /ɲ/, as measured by duration and formant trajectories of the following vocalic portion?(1b) Does the distinction of /n/ and /ɲ/ vary by proficiency?Following the acoustic description of the nasal segments in section Nasal consonants in English and Spanish, distinct segments are predicted to take the form of a longer vocalic portion following /ɲ/ than following /n/; /ɲ/ is expected to present a higher F2 and a lower F1 than /n/, with an overall flatter shape for /n/ vs. /ɲ/.In the case that learners distinguish /n/ and /ɲ/, the second question concerns the acoustic quality of /ɲ/.(2a) If learners distinguish /n/ and /ɲ/, (i) do they rely on an L1 representation to produce /ɲ/ or (ii) have they overcome L1 constraints to establish a novel L2 representation? In this latter case, does the acoustic quality of the L2 representation pattern with that of an L1 Spanish representation?(2b) Does the acoustic quality of the L2 Spanish /ɲ/ vary as a function of proficiency?There are two logical outcomes. The first is that we will encounter evidence that learners have mapped Spanish /ɲ/ in the input onto their representation of English /nj/. In the Speech Learning Model . The Spanish/English bilinguals were divided into three groups based on order of acquisition and level of proficiency: (1) L2 Beginner (n = 9), 2) L2 Advanced (n = 8) and (3) L1 Spanish (n = 10). The L2 Beginner and L2 Advanced groups are comprised of L1 English speakers who learned Spanish as an L2. The L1 Spanish baseline comparison group mirror the L2 groups and are L1 Spanish speakers who learned English as an L2. The use of a Spanish baseline from a mirror-image bilingual group avoids the problematic comparison of bilinguals to monolinguals and acknowledges that a bilingual's systems do not act in isolation  and Modern Language Association (MLA) that was first used in Slabakova and Montrul CV1.ɲV2 and (C)CV1.nV2 structures, respectively; critical and control items in English consisted of (C)CV1n.jV2 and (C)CV1.nV2 structures, respectively. Across conditions, V1 was a mid or low vowel  and V2 was /a/ in Spanish and /ɲ/ in English. Distractors followed the same general (C)CV.CV structure as the control and critical stimuli. English and Spanish stimuli were recorded by phonetically trained female native speakers of Midwest American English and Northern Peninsular Spanish, respectively. The experiment consisted of Delayed Repetitions Tasks ; audio stimuli were presented over Sennheiser HD-280 PRO headphones through a MOTU Ultralite mk3 interface. Recordings were made in a sound-attenuated booth using a head-mounted Shure SM 10A dynamic microphone and a Marantz PMD 661 solid-state recorder at a 44.1 kHz sampling rate.The experiment was conducted in a single session divided into Spanish mode and English mode segments, with the language mode order counterbalanced across participants. After providing informed consent, participants started the first segment with a 10-min interview in order to establish the first language mode, followed by the delayed repetition task in that language. The English mode segment ended with completion of the BLP and the Spanish mode segment ended with the Spanish written proficiency assessment. The L1 Spanish comparison group only participated in the Spanish mode segment and completed the interview, repetition task, and English written proficiency assessment, in that order.Following the research questions presented in section Research questions and predictions, this study examines the duration and formant trajectories of the following vocalic portion as acoustic indices to differentiate between nasal segments. To that end, we used Praat [6.1.16]  the visual presence of an abrupt change in formant structure and frequencies (onset) and 2) a breaking up of the formant structure and a loss of energy and periodicity in the waveform (offset). Following Bongiovanni  perceptual mapping of /ɲ/ to English /nj/ or (ii) development of a novel L2 representation. We first compared the advanced learners' /ɲ/ to their English /nj/ and to the L1 English baseline  and a between-groups comparison (L2 Spanish /ɲ/ vs. L1 Spanish /ɲ/). Recall that, in addition to non-overlapping confidence intervals, differences between English /nj/ and Spanish /ɲ/ are expected to present in the form of a higher F2 peak and lower F1 valley for English /nj/ vs. Spanish /ɲ/. Keeping that in mind, For the Beginner L2 group, the within-group comparison (Beginner /ɲ/ vs. Beginner /nj/) revealed no overlap in confidence intervals for F1 (maximum difference =0.47 Bark), with the exception of the point where the two curves cross at 35–45%. The same pattern presents for F2, with no overlap in confidence intervals between 0–35 and 70–100% (maximum difference =0.51 Bark) and an overlap between 35 and 70% where the formant curves cross. The formant trajectories reflect the expected differences between /nj/ and /ɲ/, i.e., a lower F1 valley and a higher F2 peak for /nj/ vs. /ɲ/. The between-groups comparison (Beginner /ɲ/ vs. L1 Spanish /ɲ/) revealed no overlap in F1 confidence intervals between 0–55% and 85–100% (maximum difference =0.32 Bark), with an overlap between 55 and 85% where the curves cross. Further, there was no overlap in F2 confidence intervals between 0 and 65%, with a maximum difference of 0.51 Bark.For the advanced L2 group, the within-groups comparison (Advanced /ɲ/ vs. Advanced /nj/) revealed that the F1 confidence intervals overlap between 0 and 50% and then run adjacent to one another from 50 to 100%. The F2 confidence intervals overlap at the beginning of the formant trajectory (0–20%) and then again when the curves cross around 65%. There is no overlap between 20 and 55% nor between 70 and 100% (maximum difference =0.21 Bark). Visually, the formant trajectories illustrate a higher F2 peak for English /nj/ vs. Spanish /ɲ/ but a lower F1 valley for Spanish /ɲ/ vs. English /nj/. However, the confidence intervals are overlapping at both of these points, thus rendering this distinction non-significant. The between-groups comparison (Advanced /ɲ/ vs. L1 Spanish /ɲ/) revealed zero overlap in the F1 confidence intervals with a difference of 0.64 Bark units at their most different. The F2 confidence intervals do not overlap at the beginning and the end of the trajectory (between 0 and 20% and between 75 and 100%) with a maximum difference of 0.22 Bark units). Visually, the Advanced /ɲ/ demonstrates a higher F2 peak  when compared with the L1 control /ɲ/.To summarize the results for RQ 2: As with RQ1, there were no differences in duration within learner groups or between learner groups and the L1 Spanish group. In terms of the beginners' formant trajectories, there was a significant three-way distinction between the beginners' Spanish /ɲ/, their English /nj/, and the L1 Spanish /ɲ/. In contrast, the advanced data's considerable overlap between /nj/ and /ɲ/ formant contours suggests a lack of difference. However, comparably limited overlap between the advanced /ɲ/ and L1 /ɲ/ contours indicate a significant difference in formant trajectories .This study examined the speech production of beginner and advanced groups of L1 English/L2 Spanish learners and a comparison group of L1 Spanish/L2 English speakers to determine whether their production evidences a phonemic distinction between Spanish /n/ and /ɲ/, and, if so, whether learners establish the contrast via L1 English /nj/ or creation of a novel L2 representation. Between-segment patterns were established via two acoustic indices: Z-score transformed durations of the vocalic portion that follows the nasal segment and formant trajectories of the same vocalic portion. Durational differences were predicted to take the form of longer duration for /nj/ than /ɲ/ and for /ɲ/ than /n/. The formant trajectory for /nj/ was expected to consist of an F1 with a lower valley and an F2 with a higher peak compared with /ɲ/; /n/was predicted to evidence an earlier and higher F1 peak and an overall lower F2 contour.Before we turn to the discussion of the results, there are two notes regarding our analysis: First, because this is the first study to measure these sounds in L1 English/L2 Spanish bilinguals and there are no data to inform relative cue strength for these contrasts, we avoid the arbitrary assignment of relative weights to the indices of duration, vowel height (F1), and vowel frontedness (F2). Instead, we treat them as three separate strategies that speakers may use to distinguish between these nasal segments. Second, we eschew an arbitrary quantification of how little overlap in the confidence intervals of the spline curves constitutes a meaningful difference between the nasal segments and focus our qualitative interpretation on the first half of the formant trajectories (0–50%). In doing so, we home in on the nature of the transitions in /nj/ vs. /ɲ/ rather than differences in the vocalic portion, which is expected to differ due to cross-linguistic differences in the following vowel ([ə] in English vs. [a] in Spanish).Our first research question asked whether L1 English learners' L2 Spanish production reflects a distinction between /n/ and /ɲ/ in Spanish and if that distinction is subject to differences in proficiency. Beginning with the duration data, our beginner group did not evidence a significant difference in duration between /n/ and /ɲ/ but the advanced group did, producing longer vocalic portions following /ɲ/ than /n/ with a medium effect size. This difference between the learner groups suggests that the durational difference increases as a function of L2 proficiency. In comparison to the advanced group, however, the L1 Spanish group distinguishes via duration to a greater degree. Thus, while the advanced L2 Spanish and L1 Spanish both distinguish the segments via duration, the degree to which the L2 group does so does not approximate the L1 Spanish comparison.If we were to use duration as the only acoustic index of the /n/~/ɲ/ distinction, we would conclude that beginner learners have not acquired the /ɲ/ phoneme in Spanish since there is no durational difference between Spanish /n/ and /ɲ/. However, the formant trajectory data indicate that the beginners and advanced learners alike utilize height (F1) and frontedness (F2) of the vocalic portion to distinguish /n/ and /ɲ/. Given that the Spanish /n/~/ɲ/ contrast varies by proficiency, the question that follows is: Why do beginner learners use vowel height and frontedness to make a distinction, but not duration? There are two points to consider. First, it could be the case that duration is not the primary cue that learners attend to in the input to distinguish the /n/~/ɲ/ contrast, but rather that it is a later-acquired cue that learners have available to them at advanced proficiencies . Second, we remind the reader of the large standard deviation values for the duration results , which ir(17) = 0.08, p = 0.760]. Thus, the individual data are suggestive of individual differences in cue weighting, which have been documented in L2 acquisition  = −0.30, p =0.237], the weakness is likely due to the variation in the beginners' duration differences (proficiency scores < 30).Our second research question concerned the quality of the learners' Spanish /ɲ/. That is, (a) do they rely on their L1 English /nj/ to approximate the novel Spanish contrast, or (b) have they overcome L1 constraints and established a single segment? We begin with the duration results, which are limited here to the advanced group since the beginner group did not use duration to contrast Spanish /n/ vs. /ɲ/. Since the learners' Spanish /ɲ/ was not different from their English /nj/, we posit that they do not use duration to differentiate them. Solely based on this outcome, we might conclude that the learners rely on English /nj/ to approximate the L2 Spanish /ɲ/ target. However, neither of these was different from the L1 Spanish /ɲ/. What might explain a scenario in which a learner's L1 and L2 sounds do not differ from each other and also do not differ from the L2 target? One possibility is that the learners' L2 Spanish /ɲ/ has affected their L1 English /nj/. L2 influence on the L1 aligns with a scenario of equivalence classification in which /ɲ/ is initially mapped onto /nj/ and, over time, the representation shifts in the direction of the L2 sound. Nevertheless, the advanced learners' English /nj/ did not differ from the English baseline . These inconclusive findings cast doubt on the reliability of duration as an acoustic correlate in this case, at least at the group level. A look at the individual-level duration difference between the advanced learners' English /nj/ and Spanish /ɲ/  and frontedness (F2) as illustrated by non-overlapping formant contours in the first half of the vocalic portion that follows the nasal segment see , 8. The Based on these comparisons of duration and formant contours, our tentative response to RQ 2a is that (i) the advanced group relies on their L1 /nj/ representation when producing Spanish /ɲ/ while the beginner group does not, and (ii) neither group approximates the L2 Spanish target as measured by an L1 Spanish baselineThe finding that beginner and advanced L2 learners differ from one another as well as from the L1 Spanish baseline is not unexpected; intermediate representations have been commonly documented in L2 production research Zampini, . In factWhat we find is that the F1 contour is indeed different, but in the opposite direction of what would be predicted , and there is no difference in F2. Thus, there is no evidence of a shift toward Spanish. Going forward, longitudinal data will best inform the following outstanding questions regarding the relationship between /ɲ/ and /nj/ via within-group developmental observations: (1) Do learners who first establish a three-way crosslinguistic distinction (/n/~/ɲ/~/nj/) eventually develop a single representation that is used for perception of English /nj/ and Spanish /ɲ/? (2) In the case of a shared representation, does the representation trend in the direction of the L1 or L2? And finally, as a complement to the variables we have examined here: (3) Is the representation that learners use to produce /ɲ/ tautosyllabic or heterosyllabic? That is, is there evidence that learners can overcome the L1 English constraint that militates against /nj/ in onset position? To address this question, future analyses will (a) examine production data for syllabic breaks via identification of glottalization  consonants that have been shown to influence outcomes in L2 phonology—the functional load of a contrast (e.g., Best and Tyler, j/ representation that differed from their English /Cj/ representation, thus outperforming the advanced L2 learners in our study. We could reasonably hypothesize that the difference between the L2 Russian and L2 Spanish learners can be attributed to the comparatively higher functional load of the contrast in Russian. However, the L2 Russian learners' new representation did not converge on the L2 target, which suggests that high functional load might be necessary but not sufficient for L2 convergence.First, recall Diehm's  finding Instead, a lack of L2 convergence might be at least partially attributed to cue robustness. Archibald , 2009 prcañon” (Diversity Style Guide, Returning to the question of intelligibility, if an L2 speaker's message is not at risk of being lost, what are the practical implications of these findings? Pronunciation pedagogy objectives have shifted away from adherence to native-speaker norms and toward intelligibility (see e.g., Levis, The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by University of Illinois Office for Protection of Research Subjects. The patients/participants provided their written informed consent to participate in this study.SS and JC contributed equally toward conceptualization, methodology, and validation of the project, as well as preparation of the manuscript. SS was responsible for data collection and data analysis. JC was responsible for the theoretical framework. 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.


All these devices have one point in common: they have been built using graphene-based materials like graphene, graphene oxide, reduced graphene oxide, inkject printing graphene, graphene-based composite sponges, graphene screen-printed electrodes or graphene quantum dots.This Special Issue compiles a set of innovative developments on the use of graphene-based materials in the fabrication of sensors. In particular, these contributions report original studies on a wide variety of sensors, such as gas sensors for NO Therefore, sensing is one of the many fields that can benefit from the use of this exciting material.Graphene has become the most explored material since Novoselov and Geim (Nobel Prize winners for Physics in 2010) achieved its isolation in 2004. The exceptional properties of graphene have attracted the attention of the scientific community from different research fields. Graphene is a monolayer of hexagonally arrayed spHowever, the development of a method for the production of high-quality graphene in large quantities at a competitive cost is essential to further exploit its full potential. For that reason, the use of graphene-based materials as graphene oxide (GO) and reduced graphene oxide (rGO), among others, has gained widespread consideration, as a compromise between the interesting properties of graphene and the synthesis price and complexity. Consequently, not only graphene but also other graphene-based materials are considered very good substitutes of graphene in many applications. In particular, graphene-based materials have been widely used for sensing applications in the last few years due to their high specific surface area, high electronic mobility and low electrical noise. A wide range of chemical sensors, biosensors and gas sensors have been developed using graphene materials.This Special Issue includes seven works focused on sensors based on diverse technologies for different applications with the common particularity that all of them use graphene materials.2-Decorated rGO as NO2 Gas Sensor” [2 commercial powders into mono-to-few-layer flakes of WS2, and dispersed them with rGO flakes to obtain a WS2-decorated rGO. They deposited it on Si3N4 substrates with platinum finger-type electrodes to build a chemo-resistive NO2 sensor that operates at near room temperature conditions and achieves a detection limit of 400 ppb NO2 and reproducible gas responses. Cross-sensitivity tests with humid air showed a very low influence of water vapor on the NO2 response.In “Near Room Temperature Light-Activated WS Sensor” , Paolucc2 and NH3 Sensing Characteristics of Inkjet Printing Graphene Gas Sensors” [2 and NO3. Here, the sensitivity of graphene-based chemiresistor gas sensors, fabricated through inkjet printing, is investigated using different concentrations of graphene in the inks. The response of these sensors towards humidity, nitrogen dioxide and ammonia has been characterized, showing that when the sensing layer is not homogeneous and does not cover all the electrode area, the reaction of the sensor towards gases is slower. Another important parameter is the thickness of the graphene film. This work shows that when the layer is too thick, the current flows through independent parallel paths, which brings it to a lower sensitivity. Conversely, if the film is too thin, thermal noise degrades the signal to noise ratio at the output. Moreover, thinner graphene films show a higher sensitivity and faster recovery time.In “NO−22 g. The benefit of stress immunity indicates the great robustness of the proposed sensor against external disturbances in real conditions.In “Stress-Insensitive Resonant Graphene Mass Sensing via Frequency Ratio” , the appThe addition of nanomaterials such as nanoparticles, carbon nanotubes or graphene-based materials may enhance the sensitivity of biosensors. In particular, “Graphene-Based Glycan Biosensor for Electrochemical Label-Free Detection of a Tumor-Associated Antibody”  describeAlthough graphene has been widely used as a nano-filler to enhance the conductivity of porous materials, it is still a requirement to prepare graphene-based sponge porous materials by simple and low-cost methods to enhance their mechanical properties and make them have good sensing and capacitive properties. In “Multifunctional Graphene-Based Composite Sponge” , a multi“Mechanical, Electrical, and Piezoresistive Sensing Characteristics of Epoxy-Based Composites Incorporating Hybridized Networks of Carbon Nanotubes, Graphene, Carbon Nanofibers, or Graphite Nanoplatelets”  comparesTo finish, “Development of Graphene Quantum Dots-Based Optical Sensor for Toxic Metal Ion Detection”  exhausti


The ability to change the secondary electron emission properties of nitrogen-doped graphene (N-graphene) has been demonstrated. To this end, a novel microwave plasma-enabled scalable route for continuous and controllable fabrication of free-standing N-graphene sheets was developed. High-quality N-graphene with prescribed structural qualities was produced at a rate of 0.5 mg/min by tailoring the high energy density plasma environment. Up to 8% of nitrogen doping levels were achieved while keeping the oxygen content at residual amounts (~ 1%). The synthesis is accomplished via a single step, at atmospheric conditions, using ethanol/methane and ammonia/methylamine as carbon and nitrogen precursors. The type and level of doping is affected by the position where the N-precursor is injected in the plasma environment and by the type of precursors used. Importantly, N atoms incorporated predominantly in pyridinic/pyrrolic functional groups alter the performance of the collective electronic oscillations, i.e. plasmons, of graphene. For the first time it has been demonstrated that the synergistic effect between the electronic structure changes and the reduction of graphene π-plasmons caused by N doping, along with the peculiar “crumpled” morphology, leads to sub-unitary (< 1) secondary electron yields. N-graphene can be considered as a prospective low secondary electron emission and plasmonic material. This phenomenon is responsible for the formation of electron clouds (e-clouds) in particle accelerators, which affect particle beam trajectories and introduce beam instabilities. In addition, electron bombardment of the tube walls introduces pressure rise and additional heat loads of these cryogenic systems. All this makes e-cloud effect the major limitation of beam luminosity of modern particle accelerators. Similarly, e-cloud formed in microwave generators of satellite communication devices affects electron trajectories and introduces noise in the communication systems.The development of low Secondary Electron Emission (SEE) materials is of great importance for modern technologies, overarching from space applications (e.g. telecommunication satellites) to particle accelerators. For instance, a significant potential difference between the dielectric and conductive part of satellites can arise due to a difference in their secondary electron yields (SEYs) induced by cosmic rays, igniting discharges that can disrupt normal operation or even destroy the equipment. Additionally, in the presence of an alternating electric field and under resonant conditions, secondary electrons can accelerate to high enough velocities to strike the material and free additional electrons, which can then be accelerated and so forth, leading to an exponential increase of the number of free electrons, a process known as multipacting15. SEE is a surface process that often is not well determined because it depends on the type of the material but also on surface finish, i.e., surface contaminants and morphology. Equally important to have surfaces with low SEY is assuring that these are chemically stable, i.e., inert, as well as conductive. In principle, the potential for electron avalanches formation is avoided by using materials with SEY < 1, but in some applications the threshold can be higher.Since SEE occurs in the topmost 5 nm layer of the material being struck by the primary electrons, the techniques usually employed to mitigate multipacting rely on surface modification by deposition a thin film of a material with lower SEY16. Recent investigations have demonstrated that SEY can be further reduced using a graphene coating of the surface18. Potential advantage of this material in respect to amorphous carbon is its superior chemical inertness. Sian et al. have reported a maximum SEY of 1.4 in Stainless Steel samples coated with graphene using electrophoretic deposition (EPD), down from the 2.4 for the uncoated substrate19. Jie Wang et al. have shown that depositing 6–8 layers of graphene in a copper substrate by CVD lowers the maximum SEY to 1.2520. However, considering practical applications, depositing previously synthesized graphene powder is highly relevant and advantageous as compared of direct growing graphene on the substrates. Sub-unitary values of SEY were reported by Montero et al., in aluminium surfaces coated with graphene nanoplatelets, with significant improvements being obtained by simple change of substrate roughness21. Although, the lack of data regarding essential characteristics of the used graphene  as well as surface finish makes difficult to assess contribution of graphene to the low SEY values reported in their work.Altering the surface composition in order to reduce the SEY has been extensively studied. Carbon-based thin films, in particular of the amorphous kind, are routinely used for this purpose in particle accelerators due to their relatively low SEY (0.95–1.05). By applying an amorphous carbon thin film to a Stainless-Steel surface using DC Magnetron Sputtering, the SEY can be reduced from 2.4 to about 1 while leaving the bulk of the material and its properties virtually unchanged22 and different means of its coating on metallic surfaces. However, in spite of significant development of the graphene market (there are about 250 companies worldwide producing graphene and its derivatives in different forms) massive application of this material is not yet achieved in any area. It has been recently argued by Kauling and co-authors that the reason why graphene did not yet experience a takeoff in any technology is in a low quality of the commercially available graphene and in the lack of its derivatives with tailored properties23. The results of the systematic analysis of commercial graphene purchased from 60 different companies ended up with disappointing conclusions: material produced by the majority of companies contain no more than 10% of graphene; not a single company offers a material with more than 50% of pure graphene; not a single product contains more than 60% of sp2 carbon. To this end, a highly competitive plasma-based alternative that circumvents the drawbacks of conventional chemical technologies has been developed24.The search for low SEY materials to mitigate e-cloud effect has become an important technological demand. New materials with better performance should, ideally, be produced via large-scale, cost-effective, and environmentally clean technologies. In this respect, graphene could become a prime low SEY material candidate due to its electronic structure, chemical inertness31. The advantages of this approach are in its reproducibility and ability to control morphological, structural and functional properties of the synthesized material. The end-result is production of graphene sheets, highly doped by nitrogen (up to 8%at.), collected with ~ 40% in the form of single atomic layers, with very low-content of oxygen (< 1%) and high-ratio of sp2/sp3 carbons (~ 15)31. Coating of stainless steel and copper by this material has recently been performed using electrophoretic deposition, which reduced SEY of samples from about 2.4 to 1.0. Moreover, air exposure of such sample and/or high vacuum baking at about 150 °C for 64 h changes the SEY for just few percent32.It has been reported, for the first time, application of microwave-driven plasmas in atmospheric pressure for the single-step production of N-doped high quality free-standing graphene sheets33. Both processes contribute to the generation of internal secondary electrons by their direct excitation from the valence band or via Landau damping, respectively34. During the transport of internal secondary electrons through the material, which is the second step of SEE, energy loss processes again take place, so that only minority of electrons reaches the surface with sufficient energy to be emitted. The energy loss efficiency of primary and internal secondary electrons depends on the electronic structure, e.g. density of states in the valence band and above the vacuum level, or the ability for plasmon excitation. Doping of graphene by foreign atoms, such as nitrogen, modifies its electronic properties, and significantly affects graphene plasmon performance22. In addition to the usual for pristine graphene, π and π + σ plasmons, low-energy 2D-plasmons appear in doped graphene22. In this regard, the properties of N-graphene are conferred by N atoms within the carbon lattice, where they typically form pyridinic, pyrrolic, graphitic and oxidized nitrogen functional groups. For instance, it was reported that incorporation of nitrogen in the graphene scaffold affects the material work function, depending on the type of functional groups formed: graphitic nitrogen decreases work function, whilst presence of pyridinic and pyrrolic groups affects its increase35. N-doping shifts the Fermi level and opens a band gap near the Dirac point. Each graphitic N can contribute ~ 0.5 electron to π network of the graphene lattice, resulting in an n-doping effect. However, pyridinic and pyrrolic N are formed at the edges or defect sites. These defects impose the p-doping effect accompanied with a downshift of the Fermi level towards the Dirac point and withdrawing electrons from the graphene sheet, i.e., inducing electron-deficient nature of the valence band38.Secondary electron yield of graphene can be further improved by tailoring its electronic structure. Following the three-step model commonly considered describing the SEE, in the first step primary electrons impinging surface lose their energy inside the material mainly by excitation of valence electrons and the creation of plasmons42. Other direct synthesis methods, such as ball milling, solvothermal synthesis, segregation growth, arc plasma, etc. have the potential to provide homogeneous doping, contrasting with post-treatment methods that provide surface doping only. Solvothermal approach typically involves mixing of lithium nitride with tetrachloromethane under moderate to high pressure and temperature that facilitate the interaction of precursors during the synthesis43. The method provides high yield and high doping levels but with very high oxygen content (larger than nitrogen one). Graphene oxide (GO) and nitrogen precursors  are the initial materials for wet chemical methods and thermal treatment. The high temperature required for GO thermal treatment makes the process costly and inefficient. Although the wet chemical approaches can provide cost-efficient large-scale production, they rely on the use of harsh chemistry (e.g. strong acids)48. Moreover, these methods are based on lengthy/complex batch procedures that do not provide control over the assembly process. Plasma post-treatment methods offer short reaction times, low power consumption, but also low yield, inhomogeneous doping, and high level of oxygen content47. To foster N-graphene as a cost-effective material with low SEY several challenges need to be addressed. In the first place is development of a method for large-scale production of N-graphene relying on cost-effective and environmentally friendly synthesis processes, capable of providing controllable and reproducible nitrogen doping.Despite of the numerous approaches available for N-graphene synthesis, either by direct or post-synthesis treatment, many issues remain unsolved, such as the ability to control the type and level of doping. The direct CVD method, for instance, requires high temperature, vacuum systems and suffers from metal interference, low yields and high costs2 radicals, and C atoms, in the high energy–density plasma environment, which translates in an effective control over the energy and material fluxes towards growing nanostructures in the assembly “mild” zone of the reactor, constitutes these methods’ most crucial advantage. Raman spectroscopy, scanning electron microscopy, high resolution transmission electron microscopy (HRTEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and Near Edge X-ray-absorption fine-structure spectroscopy (NEXAFS) were used to probe the morphological, chemical and microstructural features of the produced material. The secondary electron emission properties of synthesized free-standing N-graphene sheets with different level of doping and dominant pyridinic/pyrrolic N bonding configuration have been examined. The morphology of N-graphene sheets, characterized by wrinkles and lateral corrugations, contributes to suppression of secondary electron emission due to the efficient recapturing of emitted electrons. Furthermore, the changes in the electronic structure of graphene induced by well-controlled level and type of nitrogen doping, with the correspondent changes in the performance of graphene plasmons, have demonstrated the potential to translate in sub unitary (< 1) SEY values. Since the procedure for electrophoretic deposition of free-standing graphene that preserves its overall properties was already developed32, properly customized free-standing N-graphene with low SEY is a promising low secondary electron yield material of interest in accelerators and space technologies.To this end, the synthesis of N-doped graphene through plasma treatment presents significant advantages relatively to other technologies because plasma systems simultaneously comprise thermal and chemical reactor functions along with catalytic properties. Moreover, it provides the ability to control the amount and localization of energy and matter delivered from plasma bulk to developing nanostructures at atomic scale level, the key to achieve the desired morphological, electronic and functional properties of targeted materials. In this work we intent to provide substantial evidence that microwave plasma technologies can be used as a competitive and disruptive alternative route to the chemical methods for the production of self-standing N-graphene sheets with controllable level and type of N doping, i.e. pirydinic, pyrrolic, graphitic, etc. A novel plasma approach is used to achieve in situ doping of the graphene sheets during their assembling and growth in the “mild” plasma zone of the reactor. The control on the type and level of doping has been achieved by changing of N-precursor injection position in the plasma environment, flux of the precursors, i.e. number density of N atoms as well as type of the N bonds configuration in the precursor utilized. Moreover, the possibility to tune the density and energy of the carbon building units, i.e. C24  induced microwave plasma at atmospheric pressure conditions24. The Carbon precursor (e.g. ethanol) mixed with background argon gas is injected in the "hot" high energy density zone where decomposition processes of the injected carbonaceous molecules take place as a result of particles collisions and intensified chemistry. The created radicals become precursors of chain reactions leading to creation of the main building blocks, i.e. carbon atoms and C2 radicals. The next "mild" zone in axial (z) direction includes the "near" plasma afterglow where the gas temperature drops from about 3,000 to 500 K. It is important to note that the gas temperature changes also radially due to the radial thermal losses. The radial profile of the temperature is approximately parabolic. The transport of gas-phase carbon atoms/molecules into colder zones through the so-called vaporization surface27 results in transformation into solid carbon nuclei. An isothermal plasma surface of about 2,500 K is considered as a “boundary of vaporization”. The transport of plasma generated carbon atoms/molecules into colder zones (outside of the vaporization boundary) of the reactor results in formation of solid carbon nuclei that are gradually withdrawn in the outlet plasma stream where kinetic processes of assembly and growth of “flowing” carbon nanostructures take place. Given the fact that the nucleation and growth processes are determined by the interplay of kinetic and thermodynamic factors, the engineering of structural qualities of targeted nanostructures was achieved via synergistic tailoring of the "hot" plasma environment and thermodynamic conditions in the “mild" zone of the plasma reactor. Therefore, by tailoring the temperature gradients, the density of carbon molecules and atoms as well as their residence time, selective synthesis of graphene sheets in a narrow range of operational parameters can be achieved. The processes of assembly and growth of graphene sheets take place in the “mild” plasma zone, during their flight with background gas flow.The reactor can be virtually separated in three different zones as can be seen from the scheme presented in Fig. 3NH2 → 2·CN + 5H2) in the “hot” discharge zone. Furthermore, the formation of HCN as “building blocks” for N-graphene, is due to reactions such as, ·CN + H2 → HCN + H· and ·CN + H· → HCN, occurring in the “mild” plasma zone. Considering down-to-top injection, a gas jet containing the N precursor is sprayed in the “mild” zone  with sufficiently high energy towards "mild" plasma zone. Here, in the larger volume, the departure from the supersaturation conditions of the environment prone to foster nanosheets assembly is deliberately preserved28. Therefore, in the larger volume, the density of carbon nuclei decreases, and, as a result, the conditions favor the creation of planar nanostructures. Moreover, the assembling process requires very fast delivering and stacking of building units and equally fast supply of enough energy to overcome all the actual potential barriers. Thus, the fluxes were adjusted to satisfy conditions for well-tuned residence time of the flowing nanostructures in the “assembly” zone of plasma reactor.Using the reactor with an expanding radius allows large power densities inside the sections with smaller radius and to achieve larger fluxes of building units .Free-standing N-graphene sheets have successfully been fabricated at a relatively high yield (~ 0.5 mg/min) using ethanol and methane as carbon precursors and ammonia and methylamine as nitrogen precursors. It should be noted that the selective synthesis of N-graphene sheets can be obtained in a very narrow range of discharge operational conditions as previously reportedIn the down-to-top scheme the nitrogen precursor injection point is in the “mild” plasma zone at a distance z = 12 cm from the launcher  across different locations, thus indicating nearly homogeneous nitrogen doping of graphene structure. The 2D to G peak intensities ratio (~ 1.2) and the full width at half maximum of the 2D-band (~ 52 cm−1) illustrate that the sample contains graphene sheets composed by few monolayers52. Synthesized free-standing graphene sheets contain few atomic layers, as estimated from the Raman and TEM results. Having in mind that the interlayer distance in the sheets is about 3.46 Å (as shown by XRD) the thickness of these structures is about 1–2 nm.Furthermore, considering a top-to-down injection scheme, carbon/nitrogen precursors have been injected into “hot” plasma region. Typical graphene morphology and corresponding Raman spectra measured at three randomly chosen sample regions are shown in Fig. 2 carbons, while the peak fitted at ~ 286 eV is mainly attributed to carbon bound to nitrogen53 , is quantified. Therefore, N-graphene prepared with methane/methylamine, holds a high nitrogen doping level (Atomic Concentration = 8%). The obtained results reinforce the advantages of the novel plasma-based method as compared with the chemical ones. The graphene lattice structure containing more than 60% sp2 carbons is well preserved after doping and the oxygen level is kept at residual amount, by contrast with the materials synthesized via chemical methods.The use of methane/methylamine as carbon/nitrogen precursors significantly increases the graphene doping level. The survey XPS spectrum Fig. a shows t58 and the σ* resonance at ~ 292 eV59 are detected. Additionally, a peak at ~ 287 eV is observed, which is attributed to C=N bond60. A pronounced peak is detected in N K-edge spectrum  and 402–410 eV (C) are attributed to pyridine-like bonding that arises from transitions from the K-shell to the unoccupied π* orbital64 and to N–C/N–H bonding65, respectively.Furthermore, the Near Edge X-ray-absorption fine-structure (NEXAFS) spectra, presented in Fig. X-ray diffraction analysis made shows that interlayer spacing is about 3.46 Å and exhibits small variations with level of doping . The measured levels of interlayer spacing are larger than those of graphite (3.35 Å) is to be noted.66. The measurement geometry utilizes a four-contact scheme, where pinching point contacts are located on the periphery of the pellet. This design provides measurement of the longitudinal conductivity as far as the current lines are parallel to the pellet surface. Due to the compressing, the individual sheets have parallel orientation, as seen in Fig. In order to assess the electrical conductivity of the graphene sheets and its temperature dependence, 0.1 g of the N-graphene powder was pressed into disc pellets of 8 mm diameter and about 3 mm of thickness. The electrical conductivity was measured applying the Van der Pauw method3 S m−1 are observed at temperature of 300 K. However, the real electrical conductivity should be even higher, considering that the measurements were performed on compressed disks made from individual N-graphene sheets, where a conductive network is formed through the matrix of highly conductive sheets having multiple interfaces between them.Figure 67. A non-monotonic temperature dependence in the measured electrical conductivity and “insulating” behavior at low temperatures is reported for CVD grown monolayer graphene68 and bilayer graphene69. The anomalous transport dependence on temperature in graphene is theoretically studied as arising entirely from disorder effects70. It should be noted that the graphene samples studied consist of compacted graphene sheets and therefore, the temperature dependencies σ(T) could be analyzed in the frame of disordered systems not being the aim of this investigation. We should note that the room temperature values σ (300 T) reveal explicit influence of the N doping content, with the higher atomic percentage showing higher conductivity.The temperature characteristics show thermally activated transport and non-metallic behavior. As resistance increases, conductivity decreases accordingly at decreasing temperature (i.e. dσ/dT > 0), as opposed to typical metal behavior. The measured σ(T) values increase by 65% as temperature T increases from 10 to 300 K. Typical insulators (high work function) or localized systems have dσ/dT > 0. Graphene is a rather unusual electronic material that often exhibits dσ/dT > 0 at certain carrier densities and temperature range while showing the expected "metallic" behavior dσ/dT < 0 in other density and temperature rangesConsidering the morphology and the high electrical conductivity, the properties of synthesized N-graphene flakes as a low secondary electron emission material have been investigated. Moreover, the typical graphene crumpled nature, yielding in highly corrugated surface morphology, can also contribute to reduce SEY. The SEY values of Highly Oriented Pyrolytic Graphite (HOPG) were used as a reference, since secondary electron emission properties of HOPG are well known.71. The setup consists of an electron gun and a large Faraday cup, connected to an electrometer. The sample is mounted on a sample holder placed inside the Faraday cup, in front of the entrance aperture. All measurements have been performed at normal incidence. The sample holder is galvanically decoupled from the Faraday cup. On the entrance of the Faraday cup there is a suppression electrode in the form of an aperture, kept on the potential of − 20 V, in order to reflect the electrons that would otherwise escape. When the sample is in shortcut with the Faraday cup, primary electron beam current Ip is measured. When the sample is biased negatively to − 15 V, the current of all electrons leaving the sample surface Itot is measured. Therefore, the ratio between the two currents is the total electron yield although its major part is secondary electron yield, and it is calculated by the following ratio:SEY measurements in the 40–1,000 eV have been performed on a home-made experimental setup similar to the one described inIt should be noted that SEY measured in this way encompasses not only so-called true secondary electrons (with kinetic energies bellow 50 eV), but also those that are elastically and inelastically backscattered.To restrict the study only to the graphene/N-graphene properties as low SEE materials, the graphene samples for SEY measurements were prepared by powering small quantity of graphene/N-graphene powder on an indium plate, which is then placed in-between two pieces of aluminium foil. Applying pressure of ~ 105 Pa on the plate provides enough adhesion between graphene and indium, while the surface morphology of the graphene sample is not affected. The estimated thickness of such graphene patch is few hundred micrometres, so that primary electrons cannot reach the substrate.32. The pristine graphene morphology is similar to that of N-graphene with low level of doping as evidenced by SEM images  excitation of inner shell electrons in electron-atom collisions , (b) excitation of valence electrons and (c) via excitation of bulk and surface plasmons, i.e., collective oscillations of valence electrons excited by primary electrons during their penetration into the material. The plasmon decay results in the excitation of single valence electrons, i.e. creation of electron–hole pairs in the process of Landau damping53. As seen, this peak is strongly decreased for N-graphene, which in turn results in lower production of internal secondary electrons via plasmons decay. In fact, the π–π* intensity decreases for higher doping levels as seen in Fig. 29, remain nearly the same regardless of the relative amount of N. Furthermore, a very small shift of π electron delocalization towards lower energies is observed for N doped graphene  and (14.5–27 eV), respectively. Both plasmon types have sufficient energy to generate internal secondary electrons in the process of Landau damping. A detailed comparison of the energy loss features in C 1s XPS spectra of pristine and N-graphene is shown in Fig.  eV and  and required doping configurations is about 160 €, much less than the prices of high-quality N-graphene available on the market , while exhibiting much lower SEY values than those of commercial carbons from reference suppliers. The synthesis method shows potential for controllable, large-scale fabrication of other graphene derivatives, while promoting microwave plasmas as a competitive, green, and cost-effective alternative to presently used chemical methods.Furthermore, the possibility to tune or suppress SEE is of fundamental importance in fostering N-graphene’s potential as a key player in improving the performance of low SEE coatings while preserving the properties of the substrates. The synergistic effect of the electronic structure modification and changes in the graphene plasmons behavior that result from the introduction of N atoms in the graphene scaffold, along with the peculiar “crumpled” morphology of the material lead to a decrease in the SEE.These results indicate that plasma-synthesized N-graphene can be considered as a prospective low secondary electron emission material. The demonstrated possibility to tune graphene plasmons behavior is particularly important in improving the performance of plasmonic materials and in inspiring innovative solutions.Supplementary information.


This approach eliminates the generation of toxic gases, simplifies the washing steps, and reduces the time required to prepare rGO. To validate the proposed method, we present spectroscopical and morphological studies, using energy-dispersive X-ray spectroscopy (EDS), UV-visible spectroscopy, infrared spectroscopy, Raman spectroscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Thermal gravimetric analysis (TGA) is used to analyze the thermal properties of GO and rGO. This eco-friendly method proposes a complete guideline protocol toward large-scale production of oxidized graphene, with potential applications in supercapacitors, fuel cells, composites, batteries, and biosensors.The oxidative exfoliation of graphite is a promising approach to the large-scale production of graphene. Conventional oxidation of graphite essentially facilitates the exfoliation process; however, the oxidation procedure releases toxic gases and requires extensive, time-consuming steps of washing and reduction to convert exfoliated graphene oxide (GO) into reduced graphene oxide (rGO). Although toxic gases can be controlled by modifying chemical reactions, filtration, dialysis, and extensive sonication are unfavorable for large-scale production. Here, we report a complete, scalable, and green synthesis of GO, without NaNO Althougon atoms . Keepingon atoms ,10 or oxon atoms ,12. The on atoms , whereason atoms ,15,16. Son atoms ,18,19 toon atoms ,21.2−sp3 hybridization as a direct consequence of the degree of oxidation acquired during the oxidation process [4 and NaNO3 in concentrated H2SO4. This procedure involves the generation of toxic gases, such as NO2 and N2O4, and the residual Na+ and NO3− ions are difficult to remove during the processes of synthesizing and purifying GO [Typically, the oxidation–reduction of graphite uses strong oxidizing agents to yield graphene oxide (GO), an insulating carbon material . To date process  . Hummerfying GO ,30,31. W3. The authors successfully demonstrated that this modification does not decrease the yield of the product, but reduces toxic gas evolution and makes the disposal of wastewater more straightforward due to the absence of Na+ and NO3− ions [To circumvent these environmental and technical issues, Chen et al.  have impO3− ions . NeverthO3− ions . Hence, To support this direction, Abdolhosseinzadeh et al.  reported3 [To the best of our knowledge, a reliable method with the capability to supply the large demand for pristine GO and (undoped) rGO by using a green approach, a short-sonication time, and viable washing steps is still lacking. In this communication, such a method is presented, considering the environmental problem and technical limitations. We successfully demonstrate, through several spectroscopy and morphological studies, an eco-friendly and complete guideline protocol for large-scale production of GO by excluding NaNO3  and addi3 .2SO4, ACS reagent, 95.0–98.0%), potassium permanganate , hydrochloric acid , and citric acid  were obtained from Sigma Aldrich. All chemicals were used as received, without further purification. Graphite powder , sulfuric acid  was added, and the solution was stirred for 20 min at 2O2, 30%, Merk) and stirred up to turn the color of the solution from dark brown to yellowish. The resulting graphite oxide suspension was washed with 1:10 HCl solution and distilled water several times, each for 10 min by means of centrifugation at 3000 rpm until the pH was at ~6. The precipitated material was dried at 80 °C for 24 h.It is important to stress once more that the synthesis of GO is based on the approach of Ref. . HoweverInitially, 50 mg of resulting solid was dispersed in 500 mL of distilled water by sonication for 0.5 h, employing an ultrasonic bath  in continuous operation. Bath sonication is preferred to tip sonication because the direct positioning of the tip in the medium results in strong material fragmentation . The obtThe characteristic absorption spectra of GO and rGO were recorded using UV-vis spectroscopy . Infrared spectra were collected using a Fourier transform infrared spectrometer Jasco FT/IR-4000 . The thermal stability of GO and rGO was investigated using thermogravimetric analysis . The structure and surface morphology of the obtained materials were taken out on a transmission electron microscope  operating at 80 kV, and a scanning electron microscope  equipped with a JEOL-made dispersive X-ray spectrometer (EDS) with the accelerating voltage of 15 kV. Raman spectra were obtained using a Jasco NRS-500  spectrometer with a 532 nm laser wavelength . SEM samples were prepared by drop casting on aluminum substrates and dried at 80 °C for 2 h. Similarly, TEM and Raman samples were prepared by drop casting onto formvar-coated copper grids and glass substrates, respectively.The main intent of this work is to propose a general eco-friendly approach to large-scale production of oxidized graphene. In this sense, we have selected CA because it is a green and low-cost alternative for conventional reducing agents of GO  , C−1), C−1), and C−1). The broad peak observed at 3426 cm−1 is due to the hydroxyl groups  and more stable oxygenated functional groups, respectively [ectively . On the ectively . TGA cur−1 and the G peak at −1. Other less intense peaks also have been detected: the 2D peak at −1 and the D+G peak at −1. The D and D+G (the combination of D and G bands) peaks are ascribed to structural imperfections of GO and rGO  wD/IGI) is a common index for the density of defects on GO and rGO. As shown in earlier studies [The peak ratio between the intensity of D and G peaks  of rGO is shifted with respect to the 2D peak (−1) of GO. The latter was impossible to be fitted, and the maximum intensity value was removed from the data (−1. Furthermore, the 2D peak became more symmetrical in rGO, indicating the presence of few-layer-reduced graphene oxide.Unlike the 2D peak of monolayer graphene, which has high intensity, the 2D peak of GO and rGO is complex in shape and evolves with the number of layers . The prethe data c. The reSEM and TEM micrographs of GO and rGO are shown in 3 [.This asseveration is supported by TEM analysis. 3 ,30.Figu3 in the oxidation and adding CA in the reduction, which demonstrated to be an effective and inexpensive reducing agent for GO. EDS measurements showed a reduction effectivity from 0.5 to 1.5 h, while GO is in contact with CA under vigorous agitation. UV-visible spectra confirmed the reduction of GO into rGO. FTIR analysis demonstrated the presence of oxygen functional groups, which decreased in intensity after reduction. The thermal stability of rGO was found to be higher compared to GO. Raman spectra showed a high intensity of the D peak in GO, indicating the presence of basal/edge defects; however, the characteristic 2D peak of graphene arose in rGO. SEM measurements showed that the semitransparent layers were relatively free of impurities. TEM analysis presented similar features. However, rGO exhibited a more regular, transparent, and thin nanosheet. The proposed method prevents toxic gas evolution, prolonged sonication, and complicated washing steps, making it effective at preparing oxidized graphene on a large scale.In summary, we have established a complete and green protocol for the preparation of GO and rGO by excluding NaNO