TARDBP confer a baseline increase in cytoplasmic TDP-43 thus suggesting that small changes in the subcellular localization of TDP-43 could in fact drive early pathology. In this review, we bring forth the theme of protein mislocalization as a key mechanism underlying ALS, by highlighting the importance of maintaining subcellular proteostasis along with the gain- and loss-of-functional consequences when TDP-43 localization is dysregulated. Additional research, focusing on early events in TDP-43 pathogenesis  will provide insight into disease mechanisms, therapeutic targets, and novel biomarkers for ALS.Since its discovery as a primary component in cytoplasmic aggregates in post-mortem tissue of patients with Amyotrophic Lateral Sclerosis (ALS), TAR DNA Binding Protein 43 kDa (TDP-43) has remained a central focus to understand the disease. TDP-43 links both familial and sporadic forms of ALS as mutations are causative for disease and cytoplasmic aggregates are a hallmark of nearly all cases, regardless of TDP-43 mutational status. Research has focused on the formation and consequences of cytosolic protein aggregates as drivers of ALS pathology through both gain- and loss-of-function mechanisms. Not only does aggregation sequester the normal function of TDP-43, but these aggregates also actively block normal cellular processes inevitably leading to cellular demise in a short time span. Although there may be some benefit to therapeutically targeting TDP-43 aggregation, this step may be too late in disease development to have substantial therapeutic benefit. However, TDP-43 pathology appears to be tightly linked with its mislocalization from the nucleus to the cytoplasm, making it difficult to decouple the consequences of nuclear-to-cytoplasmic mislocalization from protein aggregation. Studies focusing on the effects of TDP-43 mislocalization have demonstrated both gain- and loss-of-function consequences including altered splicing regulation, over responsiveness to cellular stressors, increases in DNA damage, and transcriptome-wide changes. Additionally, mutations in  TAR DNA Binding Protein 43 kDa  have provided crucial insight into common pathogenic themes in ALS [Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective loss of motor neurons resulting in mortality within an average of 2-5 years . Though s in ALS .Zn/Cu Superoxide Dismutase 1 (SOD1) and Fused in Sarcoma (FUS) [TARDBP have been linked to ALS, further supporting TDP-43 dysfunction as a critical component in ALS [TARDBP can also cause frontotemporal lobar dementia (FTLD), which itself shares some clinical parallels with ALS and displays TDP-43 pathology in ~ 45% of cases [TDP-43 bridges the divide between sporadic and familial ALS and remains a dominant protein of interest to understand disease pathogenesis. TDP-43 was identified as a primary component of ubiquitinated and hyper-phosphorylated cytosolic aggregates observed from post-mortem tissue of patients with ALS , 9. Thisma (FUS) –15. Furtt in ALS , 16–18. of cases –21. HereTARDBP mRNA [TDP-43 is a highly conserved and essential DNA/RNA binding protein belonging to the heterogenous ribonucleoprotein family that preferentially recognizes UG-rich and TG-rich motifs of RNA and DNA, respectively –26. TDP-DBP mRNA –32. DeleDBP mRNA –48. StruDBP mRNA , 28, 29.DBP mRNA , 49–51. DBP mRNA , 52, 53.DBP mRNA . Not onlDBP mRNA .In ALS, truncated forms of TDP-43 are found in ALS aggregates, more predominantly in the cortex but also to a lesser extent in the spinal cord –59. The There are several features that commonly define aggregates of TDP-43 in ALS. These include the accumulation of post translational modifications such as ubiquitination, poly-ubiquitination, and aberrant phosphorylation (sometimes referred to as hyperphosphorylation) of full length TDP-43; specifically phosphorylation of TDP-43 at serine 409 and 410 (S409/410) is widely used as an indicator of aggregated TDP-43 , 58, 61.The exact mechanisms mediating the formation of TDP-43 aggregates remain elusive. In ALS aggregates, TDP-43 was found to colocalize with important markers of stress granules (SGs) –95. SGs TDP-43 plays an important role in regulating the dynamics of SG formation and disassembly where loss of TDP-43 reduces SG formation , 105. TrUbiquilin-2 (UBQLN2), and is important for degrading full-length TDP-43 in addition to CTF-35 and CTF-25 [CYLD Lysine 63 Deubiquitinase (CYLD) was identified to cause ALS and FTLD [SQSTM1, TANK Binding Kinase 1 (TBK1) and Optineurin (OPTN) [Clearance of TDP-43 remains an important biological process tightly coupled with cytotoxicity. The ubiquitin-proteasome system is disrupted by ALS-linked mutations in d CTF-25 –120. Inhd CTF-25 , 122. Reand FTLD . The autn (OPTN) , 124–128n (OPTN) , 130.KIF5A or DCTN1, cause ALS [The reduced clearance of aggregates can lead to another toxic gain-of-function mechanism: blocking intracellular transport. Aggregates are observed throughout the cytoplasm, often in the soma, but are also observed in the axons and dendrites , 132. Inause ALS –139. Thiause ALS –142.drive cell-death. Clearly, TDP-43 aggregation is not the only feature at play.Studies have suggested that not all aggregates are equal in their ability to cause toxicity. Similar to other neurodegenerative diseases, large protein aggregates such as amyloid-like structures may not be as toxic as smaller ones that preceded them such as oligomers –147. HowΔNLS resulted in depletion of endogenous TDP-43WT from the nucleus and promoted the formation of insoluble inclusions in the cytoplasm [ΔNLS under a neurofilament heavy chain promoter for brain and spinal cord expression, mice displayed a rapidly progressive motor phenotype, loss of body mass, neuromuscular denervation, and spinal motor neuron loss [In the presence of ALS-causing mutations, TDP-43 often demonstrates an altered nucleocytoplasmic distribution  in comparison to its wild-type counterpart , 163. Thytoplasm . In a trron loss . These mArabidopsis thaliana-derived Cryptochrome 2 (CRY2) protein to allow for optogenetic instigation of LLPS [ΔNLS or mildly stressed cells inducing a mild mislocalization resulted in cytoplasmic inclusions positive for ALS-like hallmarks. Additionally this model demonstrated in vitro that there is a relatively short time course between initial induction of LLPS to aggregate formation (within hours) and inevitable cell death in less than 6 weeks [The cellular stress model, particularly oxidative stress (e.g. via sodium arsenite treatment), to induce SGs and TDP-43 inclusions is a widely used model to study TDP-43 mislocalization and aggregation , 164. Ye of LLPS , 165. In of LLPS . However 6 weeks , 111. ThSTMN2) transcript [Increasing evidence suggests that nuclear-to-cytoplasmic mislocalization of TDP-43 induces toxicity through both loss- and gain-of-function mechanisms. Classic roles for TDP-43 pertain to mRNA maturation in the nucleus, specifically acting as a repressor of alternate splicing, cryptic exon splicing, and alternate polyadenylation , 166–173anscript , 174. TDanscript , 175. Dianscript , 176–178anscript , 180. Inanscript –183. Addanscript , 184–189anscript , 189. MiVacuolar Protein Sorting 4B (VPS4B) to repress its transcription [VPS4B repression leading to an increased interaction with the ALS-linked protein Charged Multivesicular Body Protein 2B (CHMP2B) thereby disrupting dendritic recycling-endosome trafficking and reducing ALS-linked ERB-B2 Receptor Tyrosine Kinase 4 (ERBB4) surface expression [The nuclear functions of TDP-43 are not limited to its RNA binding functions; TDP-43 also binds DNA at TG-rich regions to regulate gene expression and exon skipping , 190. Focription . Loss ofpression –193. Anopression , 194–200pression , 202. Lopression , 204.Not all consequences of TDP-43 mislocalization are attributed to nuclear loss-of-function as TDP-43 has defined roles in the cytoplasm including stress granule regulation, mRNA stability, translational regulation, local synaptic RNA regulation, mRNA trafficking, microRNA regulation, and regulation of autophagy . The exSOD1 rarely present with TDP-43 pathology [Although we have focused strictly on nuclear and cytoplasmic TDP-43, it is important to highlight a role for TDP-43 at mitochondria. TDP-43 misregulation through genetic manipulation of the NLS, presence of an ALS-causing mutation, or overexpression result in an increased localization to mitochondria . Within athology .Together, these data suggest that both loss- and gain-of-TDP-43 function mediated by nuclear-to-cytoplasmic mislocalization cause systemic cellular dysfunction in ALS. This recognition calls for a better understanding of the native subcellular functions of TDP-43 and the consequences of mislocalization independent of aggregation.It is apparent that TDP-43 mislocalization on its own is toxic and can contribute to many of the cellular characteristics observed in ALS. However, the mechanisms governing TDP-43 localization remain largely elusive. Identifying the mechanisms driving mislocalization will be crucial to identify key mechanisms that are misregulated early in disease and can be therapeutically targeted to prevent TDP-43 pathology all together Fig. . These mTARDBP mutations, suggesting extrinsic factors from TDP-43 govern its subcellular localization [TARDBP or C9ORF72 [C9ORF72 repeat expansion, dipeptide repeats (DPRs) generated through repeat-associated non-AUG (RAN) translation of the expanded hexanucleotide (CCCCGG) repeat blocks and disrupts the nuclear pores leading to TDP-43 pathology [C9ORF72 DPRs and could contribute to nuclear pore defects in conjunction or independently from DPRs [C9ORF72 DPRs can disrupt nuclear pore function. Thus, nuclear pore complex disruption is an important part of TDP-43 pathogenesis, however, this mechanism may not always precede TDP-43 mislocalization. These studies warrant further investigation into mechanisms that may hinder TDP-43 translocation into the nucleus as potential aggravators of disease.As previously described, TDP-43 contains several subcellular-regulatory sequences including an NLS, the controversial NES, and mitochondrial localization sequences M1, M3, and M5. ALS-causing mutations however rarely reside within these motifs , and TDP-43 mislocalization exists outside of lization , 211–222lization . Furtherlization . Mutagenlization –51. Neve C9ORF72 –228. Agg C9ORF72 . In caseathology –235. TDPIt is clear that regulation of TDP-43 is crucial for proper function, yet relatively little is known about how TDP-43 is regulated. Post translational modifications play an important role in regulating protein function (Reviewed by Buratti ). Along The role of TDP-43 cleavage into CTF35 and CTF25 is gaining traction as potential contributors of normal and toxic TDP-43 function. CTF35 for example assembles into SGs and plays roles in RNA processing, however, CTF25 does not localize to SGs and remains diffuse throughout the cell . CytoplaAs with TDP-43 aggregates, interpreting the extent of TDP-43 mislocalization in patient tissue remains challenging as observations are made post-mortem at the late stage of disease. It may be implied that mislocalization of TDP-43 has occurred where there are cytoplasmic TDP-43 aggregates, however the mechanism(s) of biogenesis of this pathogenic hallmark remain(s) elusive. Future studies should systematically analyze the extent of TDP-43 mislocalization in addition to aggregation in human tissue to gain a better understanding of TDP-43 pathogenesis. Relying on key late-stage hallmarks such as phosphorylation of TDP-43 or SQSTM1 sequestration may limit the understanding of early pathogenesis and may lead to the dismissal of models that do not recapitulate late-stage pathology. Though studies have not systematically analyzed the extent of TDP-43 mislocalization throughout the central nervous system, data suggest that TDP-43 mislocalization correlates with aging in the vulnerable motor neurons of mouse spinal cord tissue . FurtherSenataxin (SETX), a poorly understood protein thought to act as a DNA/RNA helicase which cause a rare juvenile-onset form of fALS and sALS [SETX recapitulate TDP-43 mislocalization, aggregation, and ALS-like phenotypes observed in patients [TARDBP models, many models that display TDP-43 proteinopathy rely on overexpression approaches. With the advent of genome engineering (e.g. via CRISPR/Cas9) in animals and iPSCs, models have pushed towards studying endogenous TDP-43, moving away from the heavy reliance on transgenic and toxic overexpression models. Several knock-in mouse models expressing mutant TDP-43 display a range of phenotypes depending on the mutation, but most behavioural phenotypes are quite subtle and occur at a very-late stage [Q331K knock-in mouse model does not display significant motor phenotype whereas the TDP-43Q331K transgenic mouse display some, but not robust, motor deficits [A90V patient iPSCs display TDP-43 mislocalization (likely due to disruption of the NLS), TDP-43M337V results in slight cytoplasmic granular staining of TDP-43 in iPSC-derived motor neurons, and mislocalization of TDP-43 is observed in TDP-43M337V patient iPSC-derived astrocytes [There is a need for developing better models that recapitulate important aspects of ALS, including behavioural, pathological, and molecular phenotypes to further understanding of disease. Although most cases of ALS display TDP-43 proteinopathy, only some transgenic mouse models, and fewer endogenous mouse models, recapitulate TDP-43 pathology and ALS-like phenotypes . An intand sALS –260. Botpatients . Within te stage , 262. Fodeficits . This dideficits ) howevertrocytes –267. TheAlthough protein aggregation has gained the most attention to understand ALS and identify novel therapeutic targets, studying the earlier components of TDP-43 mislocalization may provide an important level of insight into the disease onset and progression. However, studying subcellular localization comes with its own challenges ranging from limitations in technology to convoluted interpretations. TDP-43 mislocalization is primarily studied using cellular fractionation methods or microscopy-based methods. Developing and optimizing more reliable methods to quantitatively analyze TDP-43 subcellular localization will enhance our understanding of critical regulators of TDP-43. For example, identifying nuclear-, mitochondrial-, and cytosol-specific post translational modifications will allow for the generation of antibodies facilitating more rapid and quantifiable detection of mislocalized TDP-43. These would parallel the antibodies raised against phospho-S409/410 TDP-43 which function as a gold-standard for detection of TDP-43 aggregates through microscopy . The genVasolin Containing Protein (VCP) in addition to spinal cords from sALS patients [SFPQ gene. The authors further suggest FUS mislocalization may be a more common hallmark of ALS than previously recognized however more evidence is required. Proteomic approaches such as immunoprecipitation-mass spectrometry or proximity-labeling mass spectrometry (e.g. APEX Proteomics or BioID) comparing wild-type to mislocalized TDP-43 will provide insight into locale-specific interactors [Integrative “omic” approaches will be important to identify robust biomarkers capable of diagnosis and providing early insight into disease progression. Determining the direct consequences of TDP-43 mislocalization as they pertain to ALS remains a challenge due to the incomplete understanding of TDP-43 function and general dysfunction associated with disease. Therefore unbiased, systems-based approaches will be important to understand TDP-43 biology surrounding mislocalization. Bulk RNA sequencing has provided great insight into transcriptomic changes in ALS mediated by TDP-43. This technology, however, has limited abilities to detect subtle biologically significant changes that may be cell-type specific. Incorporating single cell RNA sequencing or methods of enriching populations of interest  will help target cell-type specific changes affected by TDP-43 mislocalization in animal and cell models that can translate to human disease , 275. Aspatients . FUS miseractors , 280. Aderactors –283. Enheractors . Integramost ALS cases, and more informative biomarkers for diagnosis and analysis of progression for ALS. Lastly, given that TDP-43 pathology can also coexist with other aggregate-prone proteins, such as C9ORF72 DPRs, Tau, α-Synuclein, and poly-Q expanded Huntingtin, insight into the role of TDP-43 mislocalization in its pathogenic function will serve to better understand pathology and modes of degeneration across a spectrum of neurodegenerative diseases [Increasing evidence suggests that TDP-43 aggregation is not a single driver of pathology in ALS. TDP-43 mislocalization plays significant roles in cellular dysfunction independently and in parallel to aggregation. Increasingly the field has begun to focus on understanding the regulatory mechanisms of TDP-43 mislocalization. To this end, as protein mislocalization is likely more readily reversible than protein aggregation, understanding the mechanisms regulating TDP-43 subcellular localization will be critical for therapy development. Specifically, a better understanding of TDP-43 localization regulators will surely shed light on novel therapeutics that have the potential to be more effective earlier in disease, more generalizable to diseases , 285–288


The effect of Mn intercalation on the atomic, electronic and magnetic structure of the graphene/Cu(111) interface is studied using state-of-the-art density functional theory calculations. Different structural models of the graphene–Mn–Cu(111) interface are investigated. While a Mn monolayer placed between graphene and Cu(111) (an unfavorable configuration) yields massive rearrangement of the graphene-derived  As an ideal 2D material, graphene has many possible attractive practical applications, such as protective coatings10, touch screens12, gas sensors13 and many others. With respect to the graphene–metal systems, the synthesis of graphene on metals is considered as one of the most promising technological approach11, with further processing of the obtained graphene layers. Moreover, the graphene–metal interfaces, particularly graphene–ferromagnet, are proposed for the realization of the spintronics applications based on the transport of electrical charge or/and spin, such as the spin filters14 and stable spin emitters16. Therefore, the understanding of the crystallographic structure and electronic properties of such graphene-metal interfaces18 is an initial prerequisite for the further studies of these systems.Since the discovery of the unique properties of graphene19: (1) in the weakly bonded case, graphene is always doped (n-type or p-type), while the electronic structure characteristic for freestanding graphene remains almost intact  and (2) in the strongly bonded case, graphene overlayer demonstrates the significant distortion of the linear dispersion relation for the graphene k-vector overlapping), that leads to the complete disappearance of the Dirac point in graphene .From the point of view of electronic structure, graphene adsorbed on metal surfaces shows two distinct cases28, graphene/Au/Ni(111)29 and graphene/Cu/Ni(111)30; (2) Such intercalated layers lead to the change the carrier concentration in graphene, and even change the carrier type (from holes to electrons) such as in the case of Cu intercalation in graphene/Ir(111)31; (3) intercalated metals may also enhance the magnetic coupling between a ferromagnetic substrate and graphene, such as graphene/Fe/Ni(111)32 and graphene/33, which improves the possible spin-filtering properties of graphene; (4) the intercalated layer in itself may bring new properties to graphene, such as in the case of intercalated lithium, where superconductivity in graphene has been predicted to occur34.The electronic structure of graphene on metals can be altered in different ways, like intercalation of different species in graphene/metal interfaces, adsorption of atoms or molecules on top of graphene, graphene’s edge engineering, creation of the graphene nanostructures, etc. It is well known that the intercalation between graphene and metallic substrates can strongly affects the properties of the graphene–metal interface. There are several possible results of this process: (1) The intercalated species may decouple graphene from strongly interacting substrates, such as in the case of graphene/Al/Ni(111)weakly-bonded graphene on Cu(111) has been well studied, the intercalation of Mn atoms , top–hcp (TH), and fcc–hcp (FH) are usually considered19  as a reference system. When studying the interface between graphene and close-packed metal surface, three “high-symmetry” structures, which preserve 311 as a rL-gap surface state around the 35. Using the graphene’s lattice constant, the calculated binding energy of this state at the 35)]. In our calculations for graphene/Cu(111) we detect the upward energy shift of the metal surface state by 274 meV compared to clean Cu(111), that is in agreement with the experimental observations37. This effect, observed earlier also for the adsorption of graphene on Ag(111) and Au(111) surfaces40, can be explained by the stronger localization of the metal surface state wave-function upon physisorption of graphene on Cu(111) compared to the clean Cu surface. There is also a charge transfer from the Cu(111) surface to graphene  allows to detect hybridization between the Cu 3d and graphene 41.For the clean metal surface, Cu111), the well-known 11, the wentclass1pt{minimafcc hollow or hcp hollow sites above Cu(111). Together with already mentioned three high-symmetry structures for graphene on Cu(111), this results in 6 structural possibilities  , several scenarios are possible. Let us start with an assumption that Mn atoms form a 2D hexagonal layer commensurated with the  surface was previously investigated by means of DFT in the studies on the grain boundaries suppression during the graphene growth45. In this work the energetically most stable arrangement corresponds to the structure shown in Fig. 42. On the second step, the annealing of the predeposited Mn leads to intercalation of Mn with a formation of system, which is supposed to be an ordered graphene/45 shown in Fig. According to available experimental and theoretical data, epitaxial Mn films grown on Cu111) form a surface alloy 11 form an-doped due to the charge transfer from the metal to graphene valence band states , the calculated interaction energy between graphene and mentclass2pt{minimmentclass2pt{minimK point for the most energetically stable configuration of the graphene/K point on the C-atoms projected weights corresponding to different carbon atoms in the unit cell of the graphene/K point in to three band’s branches. For the first one, associated with the carbon atoms above the Cu-hcp atoms of the Cu(111) slab  are shown in Fig. lab Fig. a, the liyer Fig. b, the liyer Fig. c. The siIn Fig. n-doped and in case of the sharp graphene/Mn/Cu(111) interface the Dirac cone is fully destroyed. For the most realistic case of the graphene–Mn–Cu(111) system, the formation of the ferromagnetic K point. Here, graphene-derived Using state-of-the-art DFT calculations the electronic properties of different systems obtained via intercalation of Mn in graphene/Cu(111) were studied. Three models were considered—sharp graphene/Mn/Cu(111) interface and two configurations for the graphene/ab initio simulation package (VASP)49. The Perdew–Burke–Ernzerhof (PBE) exchange-correlation functional50 was employed. The electron-ion interaction was described within the projector augmented wave (PAW) method51 with C , Mn , and Cu  states treated as valence states. The Brillouin-zone integration was performed on 52 was employed. The k mesh for sampling the supercell Brillouin zone are chosen to be as dense as at least 53. During structure optimization, the convergence criteria for energy was set equal to 55 with the code BandUP. The STM images are calculated using the Tersoff-Hamann formalism56.Spin-polarized DFT calculations based on plane-wave basis sets of 500 eV cutoff energy were performed with the Vienna x, y, z-coordinates of carbon atoms as well as z-coordinates of the top three layers of metal atoms are relaxed until forces became smaller than The studied interfaces are modeled by a slab consisting of 10 Cu-layers, a graphene adsorbed on the top side of the slab, and a vacuum gap of at least 20 Å. The bottom layer of the slab is protected by H-atoms. In the case of the graphene–Mn–Cu(111) system, a Mn layer is added between graphene and Cu(111). In order to describe graphene as realistically as possible, the lattice constant of copper is set to be compatible with the optimized graphene lattice constant (Supplementary Informtaion.


In this paper we present a facile method for the synthesis of aminated graphene derivative through simultaneous reduction and amination of graphene oxide via two-step liquid phase treatment with hydrobromic acid and ammonia solution in mild conditions. The amination degree of the obtained aminated reduced graphene oxide is of about 4 at.%, whereas C/O ratio is up to 8.8 as determined by means of X-ray photoelectron spectroscopy. The chemical reactivity of the introduced amine groups is further verified by successful test covalent bonding of the obtained aminated graphene with 3-Chlorobenzoyl chloride. The morphological features and electronic properties, namely conductivity, valence band structure and work function are studied as well, illustrating the influence of amine groups on graphene structure and physical properties. Particularly, the increase of the electrical conductivity, reduction of the work function value and tendency to form wrinkled and corrugated graphene layers are observed in the aminated graphene derivative compared to the pristine reduced graphene oxide. As obtained aminated graphene could be used for photovoltaic, biosensing and catalysis application as well as a starting material for further chemical modifications. Significant efforts are being made to obtain graphene derivatives covalently modified by various functional groups, containing oxygen, nitrogen, sulfur, halogens and other elements4. As a result, the family of functionalized graphenes has grown dramatically during recent years5. Such excitement for the graphene functionalization is a result of wide opportunities in tailoring its physical and chemical properties which are being opened by adding certain type and number of organic moieties onto either graphene basal plane or its edges. Graphene derivatization allows to tune material electrical resistivity, luminescence properties, and optical transmittance, open and vary band gap what is of high interest for electronic, optoelectronic and electrochemical applications8. The addition of chemically reactive moieties, such as carboxyls, amides or amines10 modifies graphene reactivity, surface energy and surface chemistry, substantially improving the performance of graphene-based catalysts, gas sensors, and biosensors13. And last but not least, functionalization supports the successful dispersion of graphene in organic solvents, which the main issue in processing the graphene-based materials14.In the past years derivatization of graphene has become one of the central topics in the field of nanocarbon materials studies18, as well as fluorographene and graphane20, amino-functionalized graphene is another derivative also being extensively studied nowadays. Primary amines represent attractive functionalities that enable an easy graphene grafting through amide coupling or so-called “click” reactions. Such an approach makes possible to covalently functionalize graphene with a large variety of biomolecules, particularly DNA strands and aptamers, as well as with carboxylated forms of carbon nanotubes or fullerenes21. Moreover, as amine is proved to be an electron-withdrawing group the functionalization of graphene with amines modify its electronic structure, in particular, enhance conductivity and provide controllable work function engineering22. As a net result, aminated graphene is regarded as a promising material for various applications in photovoltaic, gas sensing and biosensing, drug delivery and composite formation25.Besides the most known graphene derivatives, graphene oxide (GO) and reduced graphene oxide (rGO)et al.11 have demonstrated successful graphene grafting with about 9 at.% of primary amines using electron beam produced Ar/NH3 plasma. Zhang et al.24 have also reported the formation of amino-functionalized graphene via Hoffman rearrangement using graphene oxide as a starting material with amine content of around 4 at.%. However, the applied procedure involves several stages, requires hazardous reagents and works only on the edges of graphene flakes. The hydrothermal approach is widely used for graphene functionalization and, particularly, for nitrogen doping of graphene oxide via reaction with ammonia, melamine, etc28. However, all these reactions entail the use of autoclave operating at rather high temperatures (up to 195 °C) and, therefore, mostly provide incorporation of such nitrogen-containing heterocycles as pyrroles and pyridines, than the formation of amines. Recently, one-pot graphene oxide amination and reduction via Leuckart reaction, which involves the conversion of a carbonyl group of an aldehyde or a ketone into the amine group, was reported by Aguilar-Bolados et al.29. Although the proposed method is simple and easy operational its efficiency is noticeably limited by localization of the formed amines on the edges of GO platelets. Additionally, the as-synthesized GO commonly contains a rather small amount of carbonyls (around 2–3 at.%) and the increase of their content requires additional GO processing, for instance, via liquid-phase partial reduction30.Various strategies for amine functionalization of graphene are currently used. For instance, Baraket 2-conjugated graphene network is the most attractive and effective way to obtain amino-functionalized graphene. This cannot be done straightforwardly in mild conditions; however, one solution is to use an additional step of GO reductive bromination. Earlier it was demonstrated that GO treatment with bromine solutions or hydrobromic acid results in graphene oxide reduction and functionalization by bromine with as high as ~5 at.% bromine concentration32 Considering the high reactivity of bromine moieties, especially for substitution reactions, one can further easily obtain aminated graphene by treating prepared brominated graphene with ammonia.Apparently, the direct substitution of GO basal plane groups (hydroxyl and epoxide ones) by amine groups with the simultaneous restoration of the spThis paper reports for the first time a scalable and facile approach for the formation of aminated graphene (rGO-Am) through two-step GO treatment with hydrobromic acid and ammonia solution in mild conditions. The effect of bromine and amine functionalization on morphology and electronic characteristics of graphene is discussed as well, providing further insights into the tuning of graphene physical and chemical properties via its derivatization.5/2) and 68.8 eV (Br3d3/2) is related to free Br− 32, two analogous doublets at 70.0 and 71.0 eV, and at 72.7 and 73.8 eV, respectively, correspond to bromine atoms covalently bonded to carbon33 and to oxygen34. The relative areas of these doublets are easily determined and are, ~23.8% for free Br−, 74.9% for C-Br, and 1.3% for O-Br. Accordingly, the concentration of C-Br species appears to be about ~3.96 at.%, what is comparable and even higher than the values obtained by other researchers33.X-ray photoelectron spectroscopy (XPS) was used to determine the chemical composition of the initial graphene oxide (GO), brominated graphene (rGO-Br) and aminated graphene (rGO-Am). Figure 36. The curve fitting of the obtained high-resolution N1s spectrum  and carbon atoms with the bonds distorted due to attachment of functional groups at a neighboring atom (C-C)43. The C=C peak exhibits asymmetric shape due to excitonic screening in sp2-conjugated graphene network of aromatic domains observed not only in graphite or graphene C1s XPS spectra but as well in the same spectra of highly reduced graphene oxide obtained by its high-temperature annealing (rGO-HT) , carbonyl (C=O), and carboxyl (COOH) groups, respectively45. The last resolved peak at ~290.2 eV corresponds to π − π* shakeup satellite of the peak C=C. Quantitative analysis of the deconvoluted C1s XPS spectra ). These oxygen species are known to be highly stable to elimination via various reduction techniques30 retaining even after GO thermal reduction with the presence of the peaks located at 286.1–286.3 eV and 533.4–533.6 eV in the C 1 s and O 1 s XPS spectra, respectively.After the amination step, this ratio demonstrates further growth reaching for the rGO-Am the value of 8.85, due to the additional elimination of oxygen-containing groups and by their substitution with amines. The noted value of the C/O ratio is close to that obtained for rGOs prepared by chemical reduction using common reducing agents, namely, hydrazine, benzylamine, various alcohols and sodium borohydrideThe structural features of the obtained brominated and aminated graphenes were further studied by the means of different techniques such as atomic force microscopy (AFM), X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and Raman spectroscopy. Representative bright field low magnification TEM images Fig.  demonstret al.48 based on the analysis of electron diffraction tilt series to determine the graphene nanorelief. The slope of the diffraction spot intensity dependence on the reciprocal space applicate square variation measured as g2 corresponds to the average square of the graphene sheet corrugation amplitude (more details are in Supporting Information and at the reference). Applying this method, we have found that functionalization leads to some increase of the corrugation amplitude , D + D′ band (2935 cm−1) and 2D′ band (3202 cm−1). The appearance of these bands is related to the interaction of the incorporated bromine and amine moieties in the double resonant processes that involve two phonons and was observed earlier for the aminated graphene24.Figure The morphology of the obtained aminated graphenes was also studied by scanning electron microscopy (SEM) at various scales. Regardless of the solvent type used during the deposition process, which was varied from the polar ones  to non-polar solvents (trichloromethane and tetrachloromethane), rGO-Am platelets display a wrinkled and twisted structure Fig. . This re2 adsorption experiments has been measured to be ~265 m2g−1. This value is lower than those achieved in formation of the structured GO and rGO aerogels54, but still sufficiently high for rGO-Am applications in the catalysis and adsorption of metallic or dye pollutants55.To further facilitate the porous network arrangement lyophilization of rGO-Am dioxane suspension was carried out, resulting in the formation of aminated graphene aerogel Fig. . The BruIt is worth noting that despite the rGO-Am films contain many voids, cracks, and ripples due to the corrugation of the graphene layer, SEM studies of the arrays of individual rGO-Am platelets reveal that the applied modification procedure does not lead to tearing and reduction of the lateral size of GO platelets. The obtained SEM images demonstrate , which can be transformed to acyl chloride moieties by the treatment with thionyl chloride (SOCl2), phosphorus trichloride (PCl3), or phosphorus pentachloride (PCl5)58. Thus, the chosen test reaction allows to demonstrate the feasibility of rGO-Am covalent grafting with various biomolecules, dyes, functionalized nanocarbon materials, etc.The formation of primary amines via the applied procedure and their chemical reactivity were further analyzed by a chemical test based on the reaction between rGO-Am and 3-Chlorobenzoyl chloride. This organic compound carries acyl chloride functional group, -COCl, known to react readily with primary amines forming covalent bond: so-called amide coupling3/2) and 201.9 eV (2p1/2) related to the C-Cl bonds59 are observed. The chlorine concentration calculated from the XPS survey spectrum is ~2.8 at.%. Since as it was earlier estimated the concentration of amine groups is ~4 at.%, the efficiency of the performed amide coupling appears to be about 74% determined as a relation between the concentration of bonded chlorine and the presented amines. Besides the XPS results, rGO-Am covalent grafting by the 3-Chlorobenzoyl chloride is also indicated by the obtained FTIR spectrum , green (high concentration of the Cl− ions) or blue (low concentration of Cl- ions). Amines from the rGO-Am are weak bases, which should interact with the Cl− ions and, thus, reduce their content in the solution, leading to the blue color of the resulting solution. Indeed, this effect we observed in the case of the mixture with the addition of rGO-Am  is observed between Fermi level and ~2.2 eV in the case of GO, indicating the presence of band gap of ~4.5 eV which is in a good agreement with the published data64. On the contrary, both rGO-HT and rGO-Am demonstrate non-zero DOS in the region from 3 to 0 eV as a result of the increase of C2p-π electron content due to enlargement of the π -conjugated polyaromatic sp2-domains during the GO reduction. In rGO-HT VB spectrum peak at ~3.2 eV corresponding to C 2p-π states is more distinguishable compared to the one in rGO-Am suggesting the more complete restoration of the delocalized π-conjugated system. Note, that this difference can be also attributed to the corrugated nature of rGO-Am lattice which distortion also influences the number of 2p-π electronic states. Despite this similarity between rGO-Am and rGO-HT valence band spectra, several bands related to the introduction of the nitrogen species appear in the former one. Particularly, the bands at ~5.0 eV and ~7.3 eV, corresponding to electronic states of N lone pair and the delocalized C–N π bonds can be distinguished in the rGO-Am valence band spectra66. Moreover, slight changes in the density of states near 2.3 eV arise due to the states related to C–C and C–N π bonds, existing in a planar sp2-graphene structure67. Finally, a broad peak centered between 18 and 20 eV can be observed after amination. While studying N-doped HOPG Favaro et al.68 have discovered the appearance of such a feature in the VB spectra of HOPG upon its N implantation and assumed that it originates from the presence of the –CN groups. Considering the absence of this peak in the spectrum of the rGO-HT sample and following Favaro et al. we assume that it is related to the electronic states arising due to the incorporated amines.To explore the effect of the functionalization with amine groups on the electronic structure of graphene, valence band spectra of the initial GO, rGO-HT, and rGO-Am using a photon at 130 eV were measured Fig. . All them, can be calculated using the following equation: eΦm = hν – (EF – ESEC), where hν = 130 eV is the photons energy, EF and ESEC are the positions of Fermi level and cut-off threshold both represented in the kinetic energy scale69. The obtained in such a way eΦm values are represented in the Fig. 71. On the contrary, the value of the work function of initial GO (~5.1 eV) appears to be somewhat higher the typical values (~4.9 eV) obtained by other groups72, probably due to the higher degree of graphene oxidation in our case.The effect of the amine functionalization on the graphene work function (WF) has also been studied Fig. . The wor73. Hence, the observed decrease in rGO-Am work function may be attributed to the contribution of the amines as well as pyridines, which both form a noticeable amount of rGO domains with the lower value of eФm.Interestingly, the SE spectrum of rGO-Am demonstrates the presence of two slopes indicating that the surface of this sample consists of two types of domains. The work function of first type domains has the value of ~4.2 eV, while for second type domains it becomes very close to the work function of the rGO-HT sample (~4.65 eV). This fact points out the substantial local decrease of the rGO-Am work function reaching the value of ~0.5 eV see Fig. . The priIn summary, the approach for scalable production of the aminated graphene from graphene oxide is developed. The proposed method leads to the reduction of graphene and subsequent incorporation of up to 4 at.% of amines with low content of other nitrogen species (pyridines and graphitic N) as indicated by XPS and XAS data. Note, that the amination efficiency can be further enhanced by increasing Br concentration via modification of the bromination method. Both TEM and SEM studies revealed that due to the presence of amines and elimination of oxygen functional groups, the aminated graphene exhibits complicated morphology with a tendency to form wrinkled and corrugated structure. This facilitates the formation of the porous films and aerogels from the obtained aminated graphene, making it perspective for electrocatalyst and sensing applications. The use of the aminated graphene in biosensing applications, particularly aptasensors manufacturing, is further enhanced by its chemical reactivity through amide coupling what is emphasized by the successful covalent linking of 3-Chlorobenzoyl chloride to the obtained material. In addition, the amination increases the conductivity of graphene layers and alters its valence band structure and work function that is of interest not only for optoelectronic and photovoltaic applications but also for the study properties of variously functionalized graphenes.74. Graphite powder (4 g) was oxidized by using concentrated H2SO4, KMnO4 and 30% H2O2 solution. No nitrates were used to prevent nitrogen doping of graphene oxide during the synthesis. The rest of the GO preparation procedure is analogous to that described in our previous work30 and its main steps are as follows. The resulting mixture was centrifuged at 3500 rpm for 1 hour, and the supernatant was decanted away. The remaining material was additionally centrifuged at 1500 rpm for 10 min to obtain aqueous GO suspension as a supernatant. Sonication was excluded throughout the whole process to prevent damaging of graphene oxide flakes and obtain suspensions with GO flakes lateral size up to 100 μm.Graphene oxide was synthesized by the Hummers methodGraphene oxide bromination was performed as follows. The GO aqueous suspension (0.05 wt.%) was centrifugated , the supernatant was decanted away. After that, HBr acid  was added to the sediment and suspension was intensively shacked for 60 seconds. The described procedure was repeated three times and the finally obtained suspension was stirred using magnetic stirrer during 20 hours in closed flask. The obtained brominated graphene was copiously washed with centrifugation  and rinsing the obtained sediment with organic solvent by mechanical stirrer (5 minutes stirring). This procedure was repeated 5 times. During the first 2 cycles chloroform was used as a solvent and in the last 3 cycles it was changed to the isopropyl alcohol.Amination was carried out by centrifugation  of rGO-Br isopropyl alcohol suspension (0.05 wt.%), decanting the supernatant away and rinsing the sediment by a saturated solution of ammonia in isopropyl alcohol. The described procedure was repeated 3 times. The washing procedure is the same as in the case of rGO-Br synthesis.Covalent modification of rGO-Am by 3-Chlorobenzoyl chloride was performed as follows. rGO-Am was filtered using a glass filter and 3 times washed with acetonitrile. Afterwards, 50 mg of rGO-Am was dispersed in 10 ml of acetonitrile and 100 μl of triethanolamine and 50 μl of 3-Chlorobenzoyl chloride were added while stirring. The obtained suspension was further stirred for one hour in a closed flask. The as-prepared suspension was filtered using glass filter with subsequent washing using acetonitrile, deionized water, ethyl alcohol, and chloroform.30 with some extensions concerning the study of the aminated and brominated materials as follows. Measurements using X-ray photoelectron spectroscopy (XPS) were made using Thermo Fisher ESCALAB 250Xi XPS system equipped with an Al Kα X-ray source providing 1486.6 eV line. Calibration of the spectra was performed using the Au 4f7/2 line at 84.0 eV as a reference. Effect of surface charging of low-conducting GO surface was treated by aligning of XPS spectra according to the C1s line at 284.6 eV of a conductive rGO-HT  mode realized by sweeping the incident photon energy and simultaneously recording the sample drain current. The thus-obtained TEY XAS spectra were then subjected to appropriate normalization and smoothing.N-K edge X-ray absorption spectra (XAS), Valence band spectra and Work function spectra were recorded at the Russian-German beamline of electron storage ring BESSY-II (Helmholtz-Zentrum Berlin) using the beamline ultrahigh vacuum experimental station30 with some differences as can be seen in the following. X-ray diffraction (XRD) technique was implemented using Bruker Smart Apex Duo set-up equipped with a CuKα source and an Apex 2D detector. Sample for these studies was prepared by the fixation of a material portion on a cactus needle end by a nitrocellulose lacquer. Then a series of diffraction patterns were acquired at various incidence angles (of the X-ray beam incidence on the detector), and the obtained 2D data was recalculated to the means of 2θ scan. DIFFRAC.EVA (Bruker Cor.) software was used for the obtained diffraction curves analysis based on the data from Powder Diffraction File ICCD PDF-2 release [JCPDS-International Centre for Diffraction Data (http://www.icdd.com)]. The transmission electron microscopy including electron diffraction (ED) studies were performed on a JEM ARM200F cold FEG probe and image aberration corrected electron microscope equipped with a large solid-angle CENTURIO EDX detector, Gatan GIF QUANTUM, and ORIUS CCD camera and Jeol JEM-2100F microscope. TEM sample was prepared by wetting a TEM-grid with carbon lacey film in a diluted water dispersion of the studied material. Jeol JSM-7001F microscope was used for SEM studies. Langmuir−Blodgett method was exploited for monolayer films formation in a way described elsewhere76. AFM images were obtained using Veeco Dimension 3100 atomic force. Operation in tapping mode using RTESP probes was used for surface morphology and thickness of the rGO-Am films determination.Structural characterization was mainly performed analogously to that presented in our previous workSpecific surface area was measured by Brunauer-Emmett-Teller (BET) method. Immediately prior to the measurements, the sample of rGO-Am was preheated to 180 °C and kept at this temperature for an hour in a vacuum to remove any products that could be adsorbed on the highly developed surface of the sample.Supplementary information.


Termination of pregnancy (TOP) is a common cause of maternal morbidity and mortality in low- and middle-income countries. Population-based surveys are the major data source for TOP data in LMICs but are known to have shortcomings that require improving. The EN-INDEPTH multi-country survey employed a full pregnancy history approach with roster and new questions on TOP and Menstrual Restoration. This mixed methods paper assesses the completeness of responses to questions eliciting TOP information from respondents and reports on practices, barriers, and facilitators to TOP reporting.The EN-INDEPTH study was a population-based cross-sectional study. The Full Pregnancy History arm of the study surveyed 34,371 women of reproductive age between 2017 and 2018 in five Health and Demographic Surveillance System (HDSS) sites of the INDEPTH network: Bandim, Guinea-Bissau; Dabat, Ethiopia; IgangaMayuge, Uganda; Kintampo, Ghana; and Matlab, Bangladesh. Completeness and time spent in answering TOP questions were evaluated using simple tabulations and summary statistics. Exact binomial 95% confidence intervals were computed for TOP rates and ratios. Twenty-eight (28) focus group discussions were undertaken and analysed thematically.Completeness of responses regarding TOP was between 90.3 and 100.0% for all question types. The new questions elicited between 2.0% (1.0–3.4), 15.5% (13.9–17.3), and 11.5% (8.8–14.7) lifetime TOP cases over the roster questions from Dabat, Ethiopia; Matlab, Bangladesh; and Kintampo, Ghana, respectively. The median response time on the roster TOP questions was below 1.3 minutes in all sites. Qualitative results revealed that TOP was frequently stigmatised and perceived as immoral, inhumane, and shameful. Hence, it was kept secret rendering it difficult and uncomfortable to report. Miscarriages were perceived to be natural, being easier to report than TOP. Interviewer techniques, which were perceived to facilitate TOP disclosure, included cultural competence, knowledge of contextually appropriate terms for TOP, adaptation to interviewee’s individual circumstances, being non-judgmental, speaking a common language, and providing detailed informed consent.Survey roster questions may under-represent true TOP rates, since the new questions elicited responses from women who had not disclosed TOP in the roster questions. Further research is recommended particularly into standardised training and approaches to improving interview context and techniques to facilitate TOP reporting in surveys. Accurate termination of pregnancy (TOP) rates are difficult to obtain, particularly in low- and middle-income countries (LMICs) with restrictive laws, or where incomplete coverage of routine data collection systems requires reliance on household surveys where under-reporting is common. Globally, some reduction has been seen in TOP rates from an estimated 40 per 1000 females aged 15–49 years in 1990–1994, to 35 per 1000 in 2010–2014 . HoweverData regarding TOP in LMICs is predominantly generated from health facility data and household surveys, notably Demographic and Health Surveys (DHS), plus special Maternal Health Surveys . Other sDHS surveys have, until now, generally employed standard questionnaires with a Full Birth History approach (FBH) to collect information on pregnancy losses, not distinguishing between induced (TOP) and spontaneous abortions (miscarriages) –17. The Termination of pregnancy rates are known to be related to fertility levels of populations and access to TOP. The total fertility rates for the sites included in the EN-INDEPTH study were Matlab, Bangladesh—2.6; Dabat, Ethiopia—3.8; Kintampo, Ghana—4.1; IgangaMayuge, Uganda—4.3; and Bandim, Guinea-Bissau—4.2 (urban) and 5.1  . All couFew studies have assessed the performance of survey questions by virtue of their completeness of responses in the capture of TOP data in LMICs. Sedgh et al. observed the challenge in generating valid and reliable abortion statistics, recommending additional research to improve monitoring of trends . The extQuantitative analysis of performance of TOP survey questions: To evaluate modified and new questions’ influence on TOP reporting by women in a population-based survey, including completeness, time spent responding to questions, and plausibility of reported TOP rates.Qualitative assessment regarding TOP reporting: To describe community perceptions, practices, barriers, and facilitators to reporting TOP from the perspective of interviewers and mothers, and how these influenced TOP capture in the EN-INDEPTH survey.This paper is part of a series of papers from the Every Newborn-International Network for the Demographic Evaluation of Populations and their Health (EN-INDEPTH) study in five Health and Demographic Surveillance System (HDSS) sites in Africa and Asia, to improve the measurement of pregnancy outcomes in population-based household surveys. This paper addresses the following objectives:The EN-INDEPTH study was a population-based cross-sectional study undertaken between July 2017 and August 2018. Five HDSS sites belonging to the INDEPTH Network were involved in the study. The sites are located in Dabat, Ethiopia; Bandim, Guinea-Bissau; IgangaMayuge, Uganda; Kintampo, Ghana; and Matlab, Bangladesh. The study’s primary objective was to compare two methods of retrospective recording of pregnancy outcomes used in the DHS, i.e. a Full Birth History with additional questions on pregnancy losses (FBH+), as per the DHS7 standard and the 2016 Nepal DHS Full Pregnancy History (FPH). Whilst some sites were familiar with FBH+, no sites had previous experience of FPH. To contribute to the understanding of the measurement of TOP in population-based surveys, focus group discussions (FGDs) with women survey respondents and interviewers  standard TOP questions from the FPH roster were used to generate TOP rates/ratios for all five HDSS sites in the 5 years preceding the survey —100.0%; on methods employed—100.0%; and place where it was restored—90.3% Table . The lifCompleteness of responses was 99.8% to the question on actions being successfully taken to end unwanted pregnancies, and 100.0% for questions on steps taken to terminate unwanted pregnancies, terminations that occurred in the past 5 years, main reasons for the termination, how the termination was done, person who suggested the termination, who performed the termination, where the termination was done and the cost of the termination process Table . The newThe median response time on the roster TOP questions was below 1.3 minutes in all sites. Mean times of responses to roster questions was relatively low in Dabat 1.1 ± 0.8 min) and high in Matlab (3.7 ± 4.7 min) Perceived shame associated with TOP was mentioned by women and interviewers as contributing to TOPs being kept secret, and hence rarely reported, especially in young females.It is shameful to tell people that you have aborted a pregnancy, so it is not possible to easily disclose it Women observed that grieving openly for a TOP was virtually non-existent as such pain was considered self-inflicted. Interviewers noted that livebirths were easier to report than TOP and some reported them as miscarriages instead.Interviewers felt unable to ask questions on TOP apprehensive of legal, traditional, and religious implications—women also confirmed this notion. According to interviewers, they were perceived at times as disrespectful when they attempted probing women about TOP. Interviewers and women observed that stigmatisation of TOP was very commonly reported in three out the five sites, to the extent that those who had engaged in it were, in many cases, referred to as murderers.I think the perception the community has about abortion…how they stigmatise those who have done abortions... Sometimes you are seen as a murderer, so someone who has had an abortion would not like to open up This could contribute to TOP being less reported as compared to miscarriages, which are perceived as natural. Younger females out of shyness and perceived stigma were also not willing to disclose TOP as per observations of interviewers.There was a perception amongst interviewers and women that TOP reporting was not comfortable. Furthermore, unlike information on livebirths that is recorded in antenatal care records, documentation on TOP is hard to come by. Interviewers therefore could only depend on reports from persons who have experienced TOPs.Interviewers perceived good interviewer communication techniques to facilitate TOP reporting. These included speaking the same language  as the interviewees and the use of “softer” contextually appropriate expressions other than a direct reference to TOP.What makes it easier for us is instead of asking the woman how many times she has had an abortion, you ask her how many times she has ‘taken out her stomach’ With the diversity of reasons for engaging in TOP and its stigmatised nature, interviewers described needing to develop the tact of adapting to each interviewee’s individual needs whilst not being judgmental. The informed consent process was viewed by interviewers as important in enhancing TOP disclosure by assuring respondents of confidentiality and that information gathered would be used to the benefit of the community.It was observed by interviewers that when interviewees perceived they are speaking to a healthcare professional, they were much more open in disclosing TOP and other adverse pregnancy outcomes, potentially due to a level of trust for the health professional that might not be available for non-health professionals.Interviewers observed that individual-level factors perceived to facilitate reporting by the women included personality of the respondent, with some outspoken persons much more likely to report TOP; when the woman felt justified in having a TOP, for example when conceiving immediately after a successful pregnancy; or conversely when a woman regretted an event after she was left with no children.Measuring TOP is methodologically challenging, and the structure and format of the interview itself is believed to be relevant in eliciting disclosure. Based on data from five countries and 34,371 women of reproductive age, we found that responses to the new questions on TOP were high and facilitated TOP disclosure from women who had not reported them with the roster questions. The new questions could therefore help improve TOP measurement. Termination of pregnancy was perceived as stigmatising, and uncomfortable to disclose, being particularly shameful amongst young women. As such, survey teams should prepare the members to eschew judgmental and stigmatising behaviours and be accommodating of interviewees’ specific circumstances.Completeness of responses to roster TOP questions, and new Menstrual Restoration and TOP questions were generally high, implying that respondents understood them. A key observation was that once women acknowledged a TOP or delayed menstruation, they reported on most of the other questions related to that event.With the exception of Guinea-Bissau where no prior rates were identified, significant differences were observed between TOP rates reported in the current study and those from previous studies in countries representing the other study sites , 24–27. Shi and colleagues describe the relevance of response times in determining the characteristics of questions used in surveys, their influence on interviewee’s comprehension, and interviewer performance . Our finRespondents in the current study brought to light certain community perceptions and practices that influence TOP reporting. Cultural and religious perceptions of TOP as inhuman and immoral influenced its reporting. Associated societal stigma, moral condemnation, and shaming observed in Cameroon, Kenya, and elsewhere , 35–37 rSome perceived barriers to reporting TOP emerged in the current study. Perceived and internalised stigma influence reporting or openly discussing TOP , 26, 38.Perceived facilitators of TOP reporting as per the current study were good interviewer skills such as knowledge of contextually appropriate TOP expressions, adapting to each woman’s individual needs and using a common language, similar to findings of a Cameroonian study . InterviInterviewee trust in healthcare professionals and well-performed informed consent procedures were perceived to enhance TOP reporting in the current study. Consenting for the roster and new questions on TOP were the same; hence, differences in responses could be attributed to the type of questions asked. Question framing and sequencing were perceived to facilitate TOP reporting in the current and Huntington and colleagues’ studies , possiblPotential next steps for improving direct TOP measurement in women’s questionnaires in population-based surveys premised on findings of this study include (1) enhanced pre-survey interviewer training sessions, to include training in TOP specific terminology used in the communities and training to address stigma and judgmental attitudes, and the need to accommodate interviewee’s specific circumstances; and (2) improved survey processes, including attention to accurate translation of TOP specific questions and interviewer prompts to include commonly understood lay-terms and ensuring the informed consent process is appropriately executed, describing the benefits or otherwise of the study to participants and the wider society.“It is not uncommon for a woman to get pregnant at a time when circumstance would make it difficult to have a child. Have you ever gotten pregnant at a time when it would have been difficult for you to have a child, or when you did not want to have one?” was asked prior to the standard question on whether the woman successfully did anything to end that pregnancy. It is possible that this lead-in question may have made women feel more at ease in reporting these events, and the potential for including a similar question in the roster could be considered. A notable observation was that respondents who reported a pregnancy ending in a TOP or Menstrual Restoration went on to respond to further questions regarding that event.Specific implications for DHS and Multiple Indicator Cluster Surveys (MICS): The mean and median times for TOP interviews point to the fact that TOP questions would not overly burden respondents or the interview team. Some respondents with a past TOP did not report the event with the standard roster questions, but only with the additional new questions where an additional lead-in question Strengths of the study include a large and diverse group of study participants from HDSS sites in five countries on the continents of Africa and Asia. A lot of effort went into developing standardised and uniform data collection tools that were tested ahead of the study; this ensured that data generated were comparable across the five study sites and made pooled analysis possible. The Survey Solutions software allowed the assessment of the time women spent in responding to TOP questions, which was a very important aspect of this study. The new questions on Menstrual Restoration and TOP exhibited their potential for eliciting information on women’s management of delayed menstruation and extra TOP events, respectively. Perspectives on factors influencing TOP reporting were enriched by the views of survey interviewers and women who had first-hand experiences related to TOP reporting. We also note that varying legal, religious and cultural differences in the study contexts may have influenced TOP reporting differentially.It would be prudent to design and undertake experimental studies powered to ascertain the capacity of the new questions on TOP in eliciting information on TOP in comparison to other questions in population-based surveys. Further exploration of paradata on response time could help determine the characteristics of TOP and menstrual restoration questions that influence interviewee and interviewer performances in such surveys. Except for Bangladesh, Menstrual Regulation/Restoration is rarely reported from the other countries involved in this study; further studies exploring this phenomenon will bridge that knowledge gap.All TOP question types were well responded to by the women. The roster TOP questions are likely to under-represent the true population TOP rates. Findings from the current study support further exploration of the new questions using experimental designs in estimating TOP and Menstrual Restoration reporting. During preparations for future population-based surveys, cultural competencies of research teams should be enhanced and the need to adhere to appropriate informed consenting procedures should be emphasised to improve TOP reporting.Additional file 1: Details of qualitative methods (FGDs), EN-INDEPTH studyAdditional file 2: TOP rates per 1000 women 15–49 years over the five years preceding EN-INDEPTH survey from roster TOP questions.Additional file 3: TOP ratio per 1000 livebirths over the five years preceding EN-INDEPTH survey from roster TOP questions.Additional file 4: Table of EN-INDEPTH survey TOP questions.Additional file 5: STROBE guidelines checklist.Additional file 6: Table of socio-demographic characteristics of focus group discussion participants.Additional file 7: Percentage of women with lifetime and five-year termination of pregnancies from FPH new questions on TOP (three HDSS sites).Additional file 8: Time taken in minutes to complete TOP section by interviewers using paradata.Additional file 9: Ethical approval of local Institutional Review Boards.


Since the production of ferromagnetic graphene as an extremely important matter in spintronics has made a revolution in future technology, a great deal of efforts has recently been done to reach a simple and cost-effective method. Up to now, controlling the magnetic properties at extremely low temperature have been investigated only by adding and removing atoms in graphene lattice. In this regard, the effect of strain on the magnetic and electronic properties of graphene has been probed. Here, the ferromagnetic properties are what have been created by strain, magnetic field, and temperature along with observation of the parallel magnetic domains in ferromagnetic graphene for the first time as a great achievement. In this way, we have represented the following: First, introducing three novel methods based on temperature, magnetic field, and strain for producing ferromagnetic graphene; Second, obtaining ferromagnetic graphene at room temperature by significant magnetization saturation in mass-scale; Third, probing the electronic systems and vibrational modes by Raman and IR spectroscopy; Fourth, introducing stacking and aggregation as two types of gathering process for graphene sheets; Fifth, comparing the results with leidenfrost effect-based method which the temperature, magnetic fields, and strain are simultaneously applied to graphene flakes (our previous work). In the honeycomb structure of graphene, the presence of one free electron per atom is responsible for high electron mobility7and the carriers transport is described by Dirac equation2. Moreover, other electrical properties such as room-temperature quantum Hall effects8, unique band structure, and ambipolar electric field effects2, covers the electronic application.Graphene, as a 2D material9. Indeed, graphene is suitable for spin logic devices due to its properties such as room-temperature spin transport with long spin-diffusion lengths10. Although the magnetic properties do not naturally exist in graphene, it is valuable in spintronics. Recently, the magnetic property is created in graphene by manipulating electronic systems. Interestingly, magnetic domains structure directly effects on magnetic properties. Generally, ferromagnetic materials are composed of one magnetic domain, with all dipoles aligned in the same direction which is a consequence of a strong driving force for parallel alignment caused by exchange energy11 . This energy is a quantum mechanical effect which tends to align electron spins, and in consequence their magnetic dipole moments, simultaneously. Noticeably, magnetic domains formation would certainly minimize the exchange energy; hence, it reduces total magnetic energy of ferromagnetic materials formation and raises their stability. By the same token, production of these domains in graphene during magnetization can thus make a revolution in future technology. Essentially, magnetism in graphene can be created by many methods such as functionalization, doping, and adding atoms. In fact, the symmetry of the electronic structure is locally broken around the new bonds and the magnetic moments are created in this area30. Additionally, vacancy and edge defects, Introduced as another method in magnetization. By removing one carbon atom and rearrangement of others in vacancy defect, the magnetic moment is created due to remaining a dangling bond which is theoretically estimated 1 μB. Experimentally, vacancy defects are created by ionic bombarded and reduction of graphene oxide41. Among these methods, the highest magnetization is occurred in the doping methods by sulfur27 and nitrogen24 but at extremely low temperatures.Apart from electronics, graphene is also attractive material in spintronics, where in addition to charge, the spin of electrons is considered44. Indeed, the creation of the pseudo-magnetic field in highly strained nanobubbles of graphene is one of the extreme approaches in graphene magnetism42. In our previous study, Leidenfrost effect-based (LFE) method for preparation ferromagnetic graphene are introduced45. Evaporating graphene droplets under high temperature and external magnetic field leads to magnetism in graphene. In this method, temperature, magnetic field, and strain is simultaneously applied to graphene flakes and magnetization occurred. Here, all these parameters are considered separately and determined their contributions.Observation of the pseudo-magnetic field in strained graphene without damaging the lattice structure is the huge gap in the magnetization process47. After first isolation of graphene by Scotch tape1, many methods are proposed to produce graphene, from exfoliation50 of graphite to growing graphene on the substrate by nucleation of carbon atoms51. On the other hand, most mechanical exfoliation methods49 such as the Liquid-phase exfoliation53 produce high-quality graphene flakes in mass-scale. Therefore, in this study, the graphene flakes are obtained by the Liquid-phase exfoliation method by using of ethanol solution54.Graphene properties are extremely influenced by the preparation methods54. To follow the instruction, pristine graphite was added to the ethanol solution in which the graphite concentration was 5 mg/ml and the ethanol/water ratio was 20:80. Then, sonication was performed for 30 min at high power and was subsequently centrifuged at 3000 and 4000 rpm (just for LFE) for 10 min, to eliminate larger flakes  and after drying the ferromagnetic graphene particles were collected. Here, the droplets were rotated and evaporated base on the Leidenfrost effect. In this method, the source of the magnetic field and temperature were Heater-stirrer and the source of pressure was the pressure inside the droplets. In fact, all parameters were simultaneously applied to graphene flakes55. Apart from strain, temperature and the magnetic field are significant parameters to manipulating the electronic and magnetic properties of graphene which is studied here. In all the methods, ferromagnetic graphene powders (FGPs) was created  and negligible impurities such as Ni, Co, Fe, and Mn, the weak ferromagnetic behavior is observed in pristine graphite  and the results are shown in Fig. 45. Because the temperature and pressure are applied in all directions, all flakes can be felt the same conditions in Oven- and Autoclave-method. The details of the hysteresis loop were summarized in Supplementary Table Similar to LFE-method, the slope created in the VSM diagram of Heater-method in high magnetic fields, related to the non-uniformity of particles. In Heater-method, the non-uniform conditions are felt by flakes because of gradual removal of the solution, non-uniform of the magnetic field, and non-uniform heat flow. In LFE-method, the slope created is related to the different behavior of droplets56. In Fig. Magnetic force microscopy (MFM) is used to observe the magnetic domains. This technique is similar to atomic force microscopy (AFM), based on cantilever oscillations. The surface of materials is probed by the magnetic tip in different distances. Each line is scanned twice, in one of them, the tip moves near the surface to collect the topography data and in another one the tip moves far from the surface to collect magnetic data60.Raman and IR spectrum complement each other and show an excellent map of active vibrational modes. By means of these techniques, a lot of information is obtained about the electronic systems and the lattice vibrational modes of graphene at Brillouin Zone (BZ). Raman spectroscopy is widely considered as a key diagnostic tool for symmetric vibrational modes, whereas IR is used for recognizing the asymmetric modes. Although most vibrational modes are active, some of the modes are silent. Generally, in Raman spectroscopy, the laser light is used to excitation and Raman scattering is detected. In addition, the electronic band structures can involve in the resonance process such as double resonance (DR) and triple resonance (TR) Raman process. For example, the phonon modes far from the center of BZ are activated by the DR Raman process. Whereas, at Γ point the phonon is usually activated by first-order Raman process57. Raman spectra of all FGPs and graphite are shown in Fig. Lattice structure, the lattice vibrations, the electronic systems, and their changes can be investigated by Raman spectroscopy. Therefore, each parameter that affects the electronic systems and phonon modes of graphene can be detected and probed by Raman spectroscopy6h, which is belonged to one-layer graphene and graphite, at the Γ point. The irreducible representation of them are ΓGraphene = A2u + B2g + E1u + E2g and ΓGraphite = 2(A2u + B2g + E1u + E2g). Three optical modes in graphene are E2g (Raman active) and B2g (silent). In graphite, nine optical modes exist containing three IR active, five Raman active, and one silent (B2g) modes. IR active modes are a doubly degenerate E1u appear in ~ 1588 cm−1, generated from asymmetric in-plane vibrational mode and one asymmetric out-of-plane mode, A2u, located in ~ 868 cm−161. In Supplementary Fig. 1u and A2u modes are observed in most samples. Also, these two peaks are too weak in graphite and LFE-sample. For graphite and LFE-sample, E1u peak is located at ~ 1525 cm−1 and in the other samples broadening and redshift of E1u peak is observed.Interpretation of the vibrational mode is based on the structural symmetry and group theory. The highest symmetry is D3d and D3h at Γ point, respectively. Also, the symmetry information along the Γ–K direction is different and their phonons are activated by DR Raman process. For example, the symmetry of monolayer and NLG (even) at M point is D2h and C2h, respectively62. Generally, changes in the electronic system and the lattice structure can create new modes, and even the combination and splitting of some modes can appear the complex modes in the Raman spectrum.By increasing the number of layers, the symmetry is reduced and subsequently, the modes are changed. For example, N-layer graphene (NLG) symmetry with the even and odd layers is D−1. These doubly degenerate, E2g, is called G mode and its position is sensitive to external perturbations, such as defects, doping, strain, and temperature. The two other peaks appear in ~ 2670 and 1200 cm−1, called 2D and D, respectively57.Basically, three characteristic peaks exist in graphene Raman spectra. One of the main peaks in Raman spectra is generated from symmetric in-plane vibrational mode, located in ~ 1580 cm2g (~ 128 cm−1) and E2g (~ 43.5 cm−1) modes, respectively. As another example in AB-2LG, C and LB modes are located at ~ 31 cm−1 (Eg) and ~ 90 cm−1 (A1g), respectively. In addition, C mode in AB-3LG and ABC-3LG are located at ~ 33 cm−1 and ~ 19 cm−1, respectively. Generally, interlayer vibrations depend on twisting, stacking order, and the number of layer68. In Fig. −1, however, many modes are observed in Fig. Ultralow-frequency modes are directly related to interlayer vibrations. In graphite, the out-of-plane and in-plane interlayer vibrations, layer-breathing (LB) and shear (C) modes, are related with Bnd E2g ~ 3.5 cm−1 nd E2g ~ 3.5 cm−1 D/IG is used to estimate the amount of defects in graphene flakes57. Only edge defects can be created in the samples, related to the sonication process in the dispersion of graphene. Because of using the same dispersion method in all samples, it is expected that the edge defects are the same and ID/IG are equal  can interact. Therefore, the position and the shape of the G peak are changed and led to broadening and splitting72. Broadening of G mode in Heater-method can be related to the new modes such as E2  is observed under stress. In addition, the G mode are sensitive to temperature80.Changing the interatomic distance in crystal lattice under stain and stress lead to manipulating electronic systems. Recently, the strain effect on electronic systems is considered and remarkable results such as shifting the Dirac cones, opening band gap, shifting and splitting Raman modes, and inducing strong pseudo-magnetic field is reported. As an example, in graphene, shifting the vibrational frequency and splitting the G mode (G−1) is activated by the resonance process. The component of the 2D peak dependent on electronic bands and laser wavelengths. Here, the laser wavelength is the same in all measurements. Due to several electronic bands are in multilayer graphene, the many 2D components can be expected. Because of these features, the 2D peak is used to determine the number of layers. In addition, the stacking order also can be strongly effected on electronic bands and change the spectral profile of the 2D peak. Therefore, the complexity of 2D LFE-sample and Autoclave-sample compared to other samples can be related to more manipulation of electronic systems . Ethanol solution is prepared by pure ethanol (Merck) and deionized water. After dispersion, elimination of larger flakes is occurred by centrifuge (Hettich EBA20). To prepared ferromagnetic graphene Heater-stirrer  and oven (Memmert) are used. For investigating magnetization of graphene, Vibrating Sample Magnetometer  is used. Natural defects of graphite and manipulation of electronic structure are investigated by Raman spectroscopy (HORRIBA). UV–Vis (mini 1240) and FTIR (8400S) spectrophotometer analyzes are performed by SHIMADZU instruments for investigation graphene concentration and vibrational modes, respectively. Magnetic field sensor  is used to measure magnetic fields of Heater-stirrer.Supplementary Information.


The activity of this new material was evaluated in situ and in vivo. The performance of the synthesized material was measured in different saline solutions  as an electrolyte. The results showed that the new material exhibits very good performance (157 F/g at 5 mV/s and 1.0 M NaCl compared to 19.5 F/g for pure graphene oxide). In the desalination test, which was performed in batch mode, the salt adsorption capacity and the efficiency of salt removal were 9.2 mg/g and 98%, respectively. To check the stability, the desalination test was repeated several times, and no change in the performance was observed. The results provide evidence that the newly synthesized material is a potential electrode material for CDI water desalination with satisfactory salt removal ability.In this study, N-doped and TiO Capacitive deionization (CDI) is a promising new technology compared to other traditional desalination methodologies, such as thermal or nonthermal distillation, due to its low energy consumption, low capital and processing costs, easy operation and maintenance processes, and environmentally friendly characteristics, with no hazardous chemicals used for electrode regeneration. Furthermore, CDI does not require high pressures, has low equipment and operational costs, employs low voltages and low power (low energy cost), is safe and involves no expendables. Small CDI units can be used in remote locations and run by solar panels, and some of the energy can be recovered by utilizing stored energy. CDI can achieve arsenic removal to below drinking water standards. CDI is strongly recommended as an environmentally friendly and economical technique for removing salt ions from saline water. The main concept of CDI is to use the electrosorption/desorption processes of an electric double layer capacitor as an electrochemical water purification method. By applying an external electrical potential , salt ions in the feed water will be attracted by the oppositely charged electrodes. Reversing the potential leads to ejection of the desorbed ions and regeneration of the electrodes for a continuous desalination process3.Most existing desalination technologies require a great deal of energy. Even nuclear power has proven too expensive for desalination and is the most expensive water supply option. Thus, energy conservation has prompted researchers to develop new water desalination technologies5. The most suitable materials that can be used as electrodes are carbon-based materials, including aerogels7, activated carbon (AC)8, mesoporous carbon (OMC)10, carbon nanofibers (CFs)11, carbon nanotubes (CNTs)12, and graphene13, which are the best candidates for CDI electrodes. Moreover, other high-performance carbon electrodes have been developed15.Researchers are in a race to develop CDI technology, and at the heart of this research are the electrodes. Effective electrodes should have high electrical conductivity, a high specific surface area with adjusted and well-controlled pore sizes, and excellent electrolyte wettability. The electrode materials should have a potential at zero charge, which is the potential at which anions and cations are adsorbed onto a surface . Many innovative electrode materials have been registered in patents16. Other electrode materials have also been investigated. Ryoo et al. used modified activated carbon cloth as an electrode to enhance the capacitive deionization performance. However, this was only very fundamental research17.The ability of an electrode to absorb ions basically depends on the surface properties of the electrode material, such as the electrical conductivity, surface area, and pore size and distribution. Therefore, carbonaceous mixtures, such as carbon nanotube and nanofiber (CNT–CNF) composite films, have also been proposed as electrosorption electrode materials21. Unfortunately, the known high hydrophobicity of graphene harms its performance in aqueous media due to the low contact angle, which adds additional resistance. However, the oxygenated groups on the surface of graphene oxide (GrO) make this material more hydrophilic than graphene. On the other hand, the electrical conductivity of GrO, compared to that of graphene, is negatively affected by these active groups. In fact, GrO’s electrical conductivity depends on the carbon/oxygen ratio in the sample. During the oxidation step, the sp2 carbons are replaced by sp3 carbons with oxygen functionalities, and this process will create a band gap by pulling the bands apart. Consequently, graphene oxide can behave as an insulator when graphene is fully oxidized. However, the partially oxidized GrO behaves as a semiconductor due to the appearance of sp3 and sp2 regions22, and the electrons can pass through the sp2 regions via Klein tunneling22. Insertion of a transition metal oxide and nonmetals within the GrO layers could play a crucial role in enhancing electron transfer23.Among the reported nanostructure carbonaceous materials, graphene has drawn the most attention due to its very large surface area and excellent electrical conductivity2 and N-doped graphene oxides have been developed as efficient nonprecious electrodes for capacitive deionization. It is expected that this fabrication process will meet many CDI electrode requirements.In this work, a fast and green synthesis route for controlling the morphological structure is presented. TiO4 from Cica-reagent, Japan) and urea hydrazine were used without any further purification.Commercial graphite powder (<20 μm) purchased from Sigma Aldrich was utilized as a graphene precursor. Titanium chloride solution , 0.1 g urea (as a source of nitrogen) and 150 micro liter hydrazine were added. All these mixtures were subjected to closed reflux system at 150 °C for 5 h. Then filtration and drying at 60 °C under vacuum for 1 day.The graphene oxide (GrO) was synthesized via a modified Hummer’s methodX-ray diffraction was conducted to analyse the phase and crystallinity using Rigaku X-ray diffractometer  with Cu Kα (λ = 1.54056 Å). 2θ angles from 10° to 80° has been used. A spectrometer (JY H800UV) equipped with an optical microscope was used to collect Raman spectra at room temperature. Scanning electron microscope , field-emission scanning electron microscope  and Transmission electron microscope  operated at 200 kV equipped with EDX analysis has been used to investigate the surface morphology of the produced samples.27. Three-electrode system were used: counter electrode from platinum wire, Ag/AgCl, reference electrode and the prepared materials as the working electrode. VersaStat4 potentiostat device was used to control the whole system. The specific capacity was simply calculated by integrating the area under the CV curve to determine the average value according to the following equation29.CDI is a solt seperation process that can be simply described as adsorption/desorption processes occurring on the electrode surface by means of electrostatic force. The salt ions are adsorbed on the charged electrode and form an electric double layer (EDL). As polarity reversed, the regeneration process take place and the ions are desorbed from the electrode surface and released back to the solution. The electrode is the main part of CDI unit. the salt adsorption capacity (SAC) of the CDI electrode material strongly depends on the properties including high electrical conductivity, large capacitance, high wettability and electrochemical stability. The salt adsorption capacity was analyzed by cyclic voltammetry cell simulate the CDI flow cell using the proposed materials. Cyclic voltammetry measurement was done by using different NaCl concentrations  after adjusting the sweep potential range from −0.4 to 0.6 V vs Ag/AgCl in the electrochemical cell. This range was selected according to that reported for graphene and graphene doped materials2 is one of the most widely used semiconductors with supercapacitor electrodes that exhibit excellent performance and a wide band gap and are placed in contact with a redox electrolyte. The purpose of using TiO2 and N-doped GrO is to improve the electrochemical characteristics, as TiO2 intercalates between the graphene oxide nanosheets.The Hummers approach was adopted to synthesize single- or few-layered chemically exfoliated graphene oxide (GrO), followed by postprocessing with thermal treatment or chemical reduction to achieve a better reductive degree of graphene, also known as reduced graphene oxide (rGO). Moreover, GrO with enough oxygen functional groups, small particles, and massive exposed edge sites offers a promising matrix for surface modification, especially for heteroatom doping. TiO2 and N-doped graphene oxide nanostructure can be easily controlled, as shown in Fig. 2 and N-doped GrO semisphere with a diameter of nearly 10 µm. This SEM image confirms that the sample contains spherical TiO2 nanoparticles. The right image shows a magnified part of the image on the left. TiO2 and N-doped GrO is in the form of layers with a thickness on the nanoscale. These introduced forms of TiO2 and N-doped GrO separating layers are very effective for use in capacitive deionization with highly improved salt adsorption capacity; consequently, an optimum CDI electrode can be obtained.The morphology of the TiO2 and N-doped GrO material. Ultrathin wrinkled graphene oxide NPs with systematic distribution can also be observed. The image indicates that TiO2 intercalated into the flexible and ultrathin wrinkled graphene oxide sheets with a few layers to form a needle-like structure. Decoration of the prepared graphene oxide by small particles of TiO2 could be proved by TEM images , (004), (200) and (204) crystal planes, respectively, indicate the formation of the anatase phase of TiO2.Figure 30. This technique can efficiently and easily prove the formation of graphene oxide, as it can distinguish GrO from the utilized graphite precursor. For instance, in the case of pristine graphite, the obtained Raman spectrum shows a prominent G peak at 1581 cm−1, which corresponds to the first-order scattering of the E2g mode31. However, the G band is broadened and shifted to 1594 cm−1 in the case of graphene oxide. Moreover, because of extensive oxidation, the D band, which appears at 1363 cm−1, becomes dominant, indicating that the in-plane sp2 domains decrease. Finally, the D/G intensity ratio in the case of graphene oxide approaches unity, while that of graphite is very small31. Figure 2 nanoparticles and doping with nitrogen. It can be easily concluded from these spectrum corresponding to pristine GO that the chemical procedure utilized was successfully applied to prepare graphene oxide. Raman spectroscopy can also easily distinguish the two compounds. For the reduced graphene oxide, the intensity of the D band is higher than that of the G band. Furthermore, the D/G ratio is very high, especially in the case of single (or few)-layered graphene. However, in our case, as shown in the figure, no change in the ratio can be observed after decoration and doping. Actually, the presence of functional groups on the surface of graphene oxide makes it preferable in capacitive deionization applications due to the enhanced ability for ion adsorption compared to graphene.The rotational, vibrational and other low-frequency modes of compounds can be easily investigated by Raman spectroscopy32. This result confirms the doping of the prepared composite by nitrogen. Decoration of the prepared graphene oxide by small particles of TiO2 could be proven by XRD analysis  analysis is a powerful technique to investigate surface elements and their electronic structure. Moreover, some elements cannot be detected by XRD, such as nonmetals and even noncrystalline metals. However, XPS is a powerful analytical technique that can detect all elements except hydrogen and helium. Figure sis Fig. . As showCyclic voltammetry (CV) is a good electrochemical technique to investigate the surface activity of functional materials.2 and N-doped GrO electrode at different scan rates. The analyses were conducted using different NaCl solution concentrations  at 25 °C in an applied voltage window of −0.4~0.6 V (vs. Ag/AgCl reference electrode). As shown in the figure, smooth ellipse-shaped cycles were obtained at all the assigned scan rates for all concentrations. This finding indicates, due to the absence of any redox peaks, that the surface is very stable and free of any functional groups. Moreover, an increase in the cycle width indicates an increase in the amount of adsorbed ions, which can be understood as an improvement in the formation of the electric double layer. Actually, the high surface area due to the exfoliated GrO sheets increased the number of absorbed ions. Additionally, no chemical reactions are involved, allowing the ultracapacitor to be rapidly charged and discharged  thousands of times, which is highly beneficial in CDI technology.Figure 2 only as a dopant did not distinctly enhance the electrochemical absorption of the ions, especially at a content close to that used in this study25. To show the advantage of the utilized codoping strategy (TiO2 and nitrogen), Fig. 2 and N-doped GrO in the presence of 1.0 M NaCl solution at a scan rate of 5 mV/s. As shown in the figure, a very considerable enhancement in ion adsorption for the introduced material was obtained compared to that of pristine graphene oxide. Moreover, the observed tail  in the case of GrO can be assigned to the oxygen evolution reaction. In other words, pristine GrO possesses electrocatalytic activity toward the water reduction reaction to produce oxygen gas. Even though this activity is very small, it is undesirable in CDI technology. In addition to the improvement in ion adsorption, the proposed doping process annihilates the electrocatalytic activity.Notably, our previous study concluded that utilizing TiO2 and N-doped GrO and pristine GrO, respectively, which translates to an 8-fold increase in the specific capacitance upon doping of graphene oxide.Numerically, the observed cycles in the above figures can be translated into the specific capacitance values by applying Eq. 37:, V (L) is the total volume of the solution, and M (g) represents the mass of the electrodes. Figure Practically, the real performance of any proposed electrode for the CDI unit is estimated by investigation of its ion adsorption and durability. Figure 2 nanoparticles and doping by nitrogen atoms. Incorporation of the metal oxide and doping by nitrogen distinctly improve the specific capacitance of the graphene oxide, resulting in an 8-fold increase in the specific capacitance. As an electrode in the capacitive deionization unit, the modified graphene oxide shows high salt removal efficiency and satisfactory salt adsorption capacity, with high stability for both parameters. Overall, this study opens a new avenue for titanium oxide nanoparticles and nitrogen as effective dopants to improve the characteristics of carbonaceous materials to be exploited as efficient electrodes in capacitive deionization units.The Hummers method is a good chemical procedure for the preparation of graphene oxide nanosheets through a chemical route. Hydrothermal treatment of the prepared graphene oxide in the presence of titanium chloride and urea results in decoration of the carbonaceous sheets by TiO