The trend of the RSE values is in good agreement with the experimentally evaluated thermal tolerance of a series of mechanoresponsive RMs based on the bisarylcyanoacetate motif. Furthermore, the singly occupied molecular orbital (SOMO) levels clearly exhibit a negative correlation with σp within a series of RMs that are based on the same skeleton, paving the way toward the development of RMs that can be handled under ambient conditions without peroxidation.Radical-type mechanophores (RMs) are attractive molecules that undergo homolytic scission of their central C–C bond to afford radical species upon exposure to heat or mechanical stimuli. However, the lack of a rational design concept limits the development of RMs with pre-determined properties. Herein, we report a rational design strategy of RMs with high thermal tolerance while maintaining mechanoresponsiveness. A combined experimental and theoretical analysis revealed that the high thermal tolerance of these RMs is related to the radical-stabilization energy (RSE) as well as the Hammett and modified Swain–Lupton constants at the  Radical-type mechanophores (RMs) are attractive molecules that undergo homolytic scission of their central C–C bond to afford radical species upon exposure to heat or mechanical stimuli. Radical-type mechanophores (RMs), which undergo mechanical-stimuli-induced homolytic scission of their central C–C bonds to afford radical species, have been used to study micro-to-macroscale mechanical stimuli, strengthened materials based on sacrificial bonds, or subsequent polymer propagation.13–23The development of mechanoresponsive materials (MRMs) that exhibit a variety of macroscale functionalities, including coloring, fluorescence, self-healing, and self-strengthening, upon exposure to mechanical stimuli has been a research hotspot in materials science during the past few decades.18,24 In contrast, the central C–C bond of difluorenylsuccinonitrile (DFSN), which exhibits the highest thermal tolerance among hitherto reported RMs, does not dissociate up to 100 °C.22 The extraordinary thermal tolerance of DFSN inspired us to apply the radical-polymerization methodology to develop highly mechanoresponsive or shape-memory materials.15,25,26 However, despite the clear importance of decreasing the propensity of the central C–C bond to subdue to thermal scission while preserving its ability to scission upon exposure to mechanical impact, the relationship between both phenomena is still unclear, which limits the scope of RM-based materials.The nature of the central C–C bond of RMs exerts a great impact on the functionality of the resulting polymeric materials. For example, the central C–C bonds of diarylbibenzofuranone (DABBF) and tetraarylsuccinonitrile (TASN) readily dissociate even at ambient temperature, which makes it possible to endow the polymeric material with self-healing properties.i.e., the radical-stabilization energy (RSE) (27–31 as well as the Hammett and modified Swain–Lupton constants at the para-position (σp)  27–31 as ion (σp) .32 The Reactions . We simueactions . Meanwhitic part .σp in a series of representative radical molecules including derivatives of arylbenzofuranone (ABF), arylbenzothiophenone (ABT), arylindolinone (AI), diarylacetonitrile (DAAN), cyanofluorene (CF), aryldicyanomethane (ADCM), arylcyanoacetate (ACA), and arylmalonate (AM). Dimers of ABF, ABT, AI, DAAN, and CF radicals are radical-type mechanophores previously developed in our group.18,22,24,33,34 In order to elucidate the electron-donating or -withdrawing nature of these radicals, not only ADCMs, which are well-known stable radicals under ambient conditions that possess two electron-withdrawing cyano groups,35–45 but also ACAs and AMs were investigated as ADCM analogues.46,47 The relationship between RSE and σp is plotted in σp range. The RSE value clearly increases with increasing σp value in the region of σp < 0, where the electron-donating nature of the functional groups increases with decreasing σp value. In contrast, the RSE values did not show significant changes and almost saturated in the region of σp > 0, where the electron-withdrawing nature of the functional groups increases with increasing σp value. These trends were theoretically reproduced irrespective of the computational methodology used , with a series of functional groups at the para-position as novel RMs  levels52–54 and slow C–C bond cleavage under ambient conditions (vide infra). We conducted variable-temperature electron paramagnetic resonance (EPR) spectroscopic measurements to determine the bond dissociation ratio (BDR) of the central C–C bond of the RMs and their dissociation constant (Kd) at 100 °C (Table S1Kd (ln Kd) are plotted as a function of increasing RSE and σp values of the BiACAs. As expected, the BDR and ln Kd values decrease with increasing RSE and σp values. Specifically, BDR and ln Kd values of 158 × 10−3% and −16.7, respectively, were obtained for BiACA-1 , 12.1 × 10−3% and −21.6 for BiACA-2 , 1.37 × 10−3% and −26.3 for BiACA-3 , 0.41 × 10−3% and −27.3 for BiACA-4 , and 0.20 × 10−3% and −28.7 for BiACA-5 . Furthermore, BiACA-5 exhibited one of the highest levels of thermal tolerance among our previously reported RMs. These results indicate that the thermal tolerance of RMs can be correlated with the RSE values and, therefore, with the substituent effect associated with the σp value.To prove this hypothesis experimentally, we synthesized ACA dimers, ovel RMs . In cont Table S1. In Fig.55,56 According to this postulate, the energy barrier between reactants, i.e., RMs, and products, i.e., radicals generated from RMs, is amenable to the thermodynamic stability of the products. Thus, RMs that bear electron-withdrawing groups, which showed shallow RSE values and, in turn, low thermodynamic stability, exhibit a higher energy barrier for thermal dissociation into the corresponding radical products, indicating higher thermal tolerance. Although DFT is a powerful tool to evaluate the dissociation of the central C–C bond and the TS in RMs, the rotation of the central C–C bond, which leads to complicated rotamers, hinders the interpretability of the results. The present approach provides a simple method for the prediction of the thermal tolerance of RMs without complicated TS searches.The relationship between RSE/thermodynamic stability and the thermal tolerance of RMs can be explained by the Hammond–Leffler postulate , which dvia the atom transfer radical polymerization (ATRP) of methyl methacrylate using a bifunctional initiator consisting of the BiACA-4 skeleton   is 246 times higher than that obtained at 100 °C (0.41 × 10−3%), indicating that the mechanical stimulus (grinding) greatly enhances the C–C bond dissociation. These results indicate that the thermal tolerance of an RM can be increased without significantly deactivating its mechanochemical reactivity.To investigate the mechanochemical reactivity of BiACAs, a poly(methyl methacrylate) (PMMA) derivative, PMMA-BiACA-4-PMMA, was synthesized skeleton . The Br hy (GPC) . A compa58–68Fmax) of BiACAs vs. σp. The Fmax value increases in the region σp < 0 and was almost saturated in the region σp > 0, similar to the case of the plots of RSE vs. σp  calculations, which allow theoretically evaluating the mechanochemical reactivity .58–68FigE vs. σp  and 2. T54 In this study, the SOMO levels clearly show a negative correlation with the σp values within the series of radicals that share the same skeleton, reflecting the substituent effect at the para-position  skeletons with different electron density. Furthermore, not only the thermal but also the oxygen tolerance could be related to the SOMO level, and the oxygen tolerance can thus be tuned according to this parameter. Our results provide a systematic guideline for the design of RMs with various functionalities while maintaining good thermal tolerance.In conclusion, we have demonstrated a rational designing strategy of RMs with high thermal tolerance. According to the relationship between the RSE and 2207403). The NMR and EPR spectra and the results of DFT calculation and CoGEF studies are provided in the ESI.†The data generated in this study are available in the main text or the ESI.H. S., K. M. and H. O. conceptualized this work. H. S. designed the experiments and carried out DFT calculations. Y. L. performed experiments include ESR measurement. All authors wrote the manuscript. Y. L. and H. S. prepared the ESI.†There are no conflicts to declare.SC-014-D3SC02991C-s001SC-014-D3SC02991C-s002


Two-dimensional metals stabilized at the interface between graphene and SiC are attracting considerable interest thanks to their intriguing physical properties, providing promising material platforms for quantum technologies. However, the nanoscale picture of the ultrathin metals within the interface that represents their ultimate two-dimensional limit has not been well captured. In this work, we explore the atomic structures and electronic properties of atomically thin indium intercalated at the epitaxial graphene/SiC interface by means of cryogenic scanning tunneling microscopy. Two types of surfaces with distinctive crystalline characteristics are found: (i) a triangular indium arrangement epitaxially matching the (√3 × √3)R30° cell of the SiC substrate and (ii) a featureless surface of more complex atomic structures. Local tunneling spectroscopy reveals a varying n-type doping in the graphene capping layer induced by the intercalated indium and an occupied electronic state at ∼−1.1 eV that is attributed to the electronic structure of the newly formed interface. Tip-induced surface manipulation is used to alter the interfacial landscape; indium atoms are locally de-intercalated below graphene. This enables the quantitative measurement of the intercalation thickness revealing mono and bi-atomic layer indium within the interface and offers the capability to tune the number of metal layers such that a monolayer is converted irreversibly to a bilayer indium. Our findings demonstrate a scanning probe-based method for in-depth investigation of ultrathin metal at the atomic level, holding importance from both fundamental and technical viewpoints. The STM tip is used to visualize and to engineer the intercalated indium at the interface between graphene and SiC substrate with atomic precision. Together with 2D van der Waals (vdW) materials8,9 2D metals are considered as compelling material platforms for quantum and optoelectronic technologies10,11 as well as for the investigation of new physical effects.12Crystalline, atomically thin metals stabilized at the interface between epitaxial graphene (EG) and a silicon carbide (SiC) substrate exhibit remarkable physical properties owing to their distinct dimensionality and dominant quantum size effects.3 for metal atoms to spontaneously squeeze themselves under graphene and form large-scale ordered ultrathin crystals typically in a range between one and a few atomic layers.3,7 A graphene top layer stabilizes the 2D metals at the interface and protects them effectively against environmental degradation. Sandwiched between EG and the SiC substrate, ultrathin metal serves as part of the vdW heterostructure which is immediately available for advanced experimental investigations and future applications.3,13 Upon intercalation, metal at the EG/SiC interface tends to match epitaxially with the uppermost atomic layers of the SiC substrate, forming a highly ordered triangular lattice as demonstrated for Ga,3,7 Au,5,14 Ag,6 Sn,15,16 and Cu17 through a wide range of investigation techniques including low-energy electron diffraction (LEED), low-energy electron microscopy (LEEM), angle-resolved photoemission spectroscopy (ARPES), cross-sectional scanning transmission electron microscopy (STEM), first principles density functional theory (DFT) and scanning tunneling microscopy (STM). In order to understand and exploit the rich physics of these interfacial 2D metals, it is significantly important to understand their microscopic details as well as their interaction within the EG/SiC interface at the atomic level.The high energy of the EG/SiC interface provides a strong thermodynamic driving force18 (√7 × √3) In on Si (111) exhibits a nearly free electron 2D electron gas (2DEG) system19 or superconductivity behaviors;20 and ultrathin In confined at the EG/SiC interface shows an extraordinary nonlinear optical response.7 First, bias-dependent imaging is used to identify the atomic structure of In below graphene which is only sensitive to a specific bias voltage range. A differential tunneling spectrum (dI/dV) is used to probe the electronic structures of the EG/In/SiC interface revealing the electronic doping of graphene induced by the charge transfer from the intercalated metal and an occupied state arising from the newly formed interface. Second, tip-induced surface manipulation using a relatively large bias voltage pulse is applied between the sample and the tip to enable de-intercalation of the In atoms below graphene, thus facilitating an accurate measurement of the metal thicknesses inside the EG/SiC interface, and to further tune the atomic layer numbers of the In. Our results demonstrate microscopic evidence on the structural and electronic properties of the confined metal and feasibility to control the interfacial landscape with atomic precision using solely the STM tip.Here, we reveal the atomic and electronic structures, the intercalation thickness and the interfacial engineering of the 2D indium using a cryogenic STM system. Indium is a group 13 metal in which its 2D form has not been well studied, nevertheless exhibiting various intriguing physics. For example, triangular monolayer indium on SiC (0001) displays non-trivial topological band inversion induced by spin–orbit coupling;2.2.1via silicon sublimation at 1800 °C, following our previous works.3 Here, we utilize partial-buffer EG, where we grow sub-monolayer EG (∼90% coverage), yielding regions where the graphene buffer layer (GBL) is exposed, to enable intercalation of In without the need for the oxygen-plasma treatment. The GBL consists of a graphene-like honeycomb structure in which one third of the carbon atoms in this layer are covalently bound to the Si atoms of the SiC substrate.21 Thus, this layer does not display the typical Dirac cone in its band structure as for graphene. This sample preparation provides a route to reduce surface roughness following intercalation, thereby enabling STM measurements of the surface following In intercalation. Atomically thin In is achieved via the confinement heteroepitaxy (CHet) in a horizontal tube furnace fitted with a 1 inch outer diameter quartz tube. Indium pellets are placed in an alumina crucible ∼5 mm below an EG/SiC substrate, and CHet is performed at 800 °C in a 500 Torr Ar environment (Ar flow of 50 sccm) for 30 minutes.EG is first formed from SiC 2.2−10 mbar) at a sample temperature of ∼5 K. Differential conductance dI/dV  was measured by using a lock-in technique amplifier with a peak-to-peak bias modulation of 5–10 mV and a modulation frequency of 675 Hz. Prior to all measurements, the STM tip is carefully calibrated by performing conductance spectra in the field emission regime (FER) on graphene/SiC using a high positive bias voltage (ranging from 2 V to 10 V) applied between the tip and the sample  operating under UHV  and GBL in which the (6√3 × 6√3)R30° superstructure of the underlying SiC substrate is visible. The (6√3 × 6√3)R30° reconstructed structure in the GBL is formed by the 1/3 monolayer of Si adatoms on top of the SiC substrate and can easily be observed by STM images with high corrugation21,22,25 and in case of monolayer or bilayer EG/SiC without intercalation, this superstructure is also evidenced.26–29 Previous studies22,23 have reported the intercalation of In at the EG/SiC interface using the initially grown GBL in which the metal intercalation tends to start at the GBL areas, resulting in monoatomic and bilayer In domains. However, some parts of the initially grown sub-monolayer EG  and GBL areas are not intercalated, resulting a quite complex surface consisting of three different types of topography: (i) monolayer In, (ii) bilayer In and (iii) the un-intercalated GBL or monolayer EG. However, in our case, evidence of un-intercalated monolayer EG or GBL is not observed. This may be because of the fact that our STM is only able to probe nanosized areas, and thus misses the topographic information on the larger scale at the micron size or more, even though we have approached the STM tip on about twenty locations. Nevertheless, the dominant topographic characteristics shown in i.e. different bias voltages). Only a few step edges of larger apparent height (∼3 Å) are observed  and high (red curve) intercalated regions. Around the Fermi level, both spectra reveal the linear dispersion of the π and π* bands of the graphene top layer. This again confirms that the initially grown GBL and sub-monolayer EG surface have been transformed into mono and bilayer graphene, respectively due to the intercalation since the GBL does not exhibit a linear band structure as it lacks the π states in the vicinity of the Fermi level.25,34–36 This also indicates that the graphene structure is essentially unperturbed and stacks with the SiC substrate as a quasi-free-standing layer mediated by the In atoms to form an EG/In/SiC heterostructure. A closer inspection around the Fermi level can be viewed in the magnified dI/dV spectra shown in 17,37 Au,5,34 Ag,6 Li,38 and Pb39 which is attributed to the charge transfer from the intercalated metal to the graphene capping layer. However, the doping effect shown here indicates an interesting behavior which varies apparently due to different In thicknesses (that is ∼1.2 Å). As pointed out by Baringhaus et al.40 for Ge intercalation under EG/SiC, the graphene boundary edge between low and high intercalated regions having different carrier densities can be considered like a p–n junction. The origin of the doping difference observed in our system might be explained according to the DFT prediction41 which demonstrates that the n-doping of a graphene layer sitting directly above metals is increased when the metal–graphene separation reduces from 3.3 Å or less. In order to avoid any mistake in identifying the Dirac point, it is worth noting that the gap-like feature centered at ±67 meV around the Fermi level as highlighted by the shaded rectangle in 42 Apart from the linear band dispersion of graphene, a noticeable occupied state at ∼−1.1 eV (marked by the red triangle) in the red spectrum of I/dV spectra in the bias voltage range between −3 V and +3 V and did not identify any other state in both regions. Since the occupied state at −1.1 eV has not been observed for the GBL or pristine graphene on SiC without intercalation,35,39 this state should be attributed to the electronic structure emerging from the newly formed interface. It is unclear if this occupied state arises from the main electronic contribution of the In itself as it might develop its own band structure or associated by a complicated electronic coupling between In layers and the SiC substrate or graphene at the interface. This nevertheless exhibits a very interesting electronic structure for the ultrathin In. In order to precisely describe this newly emerging state, further theoretical calculation is needed.Two-dimensional indium forms its own electronic state at the interface and modifies the electronic structure of graphene capping layers 43 (7 × 7) reconstruction of Si (111) below monolayer graphene44 and SiC interface structure below monolayer graphene.45,46 Accordingly, when the two atomically indistinguishable surface regions shown in Height variation in the STM image is correlated with two distinct atomic phases of indium. A challenge for identification of the intercalated structures at the EG/SiC interface is that metal layers are covered by graphene preventing direct access to the confined atoms underneath. In the STM technique, the tip is sensitive to the density of states located several atomic layers below a surface that has been used to directly identify the buried atomic structures, for example, Si vacancies and domain boundaries in yttrium silicide surface,26 We image this area at different bias voltages and observe that the atomic structure of the 2D In is only visible in the bias range between 1.5 V and 2.2 V. At larger negative and positive voltages, the STM images appear very unstable without special features. Previous studies3,5,6,14–17 have demonstrated various metal elements intercalated at the EG/SiC interface in which the first intercalated metal layer tends to match (1 × 1) epitaxially to the SiC substrate such that each metal atom is bound directly to the topmost Si atom of the SiC substrate. The unit cell of 6H-SiC (0001) is known to be 3.08 Å46,47 which is considerably smaller than that of the intercalated In (5.4 Å) found here. Thus, such a (1 × 1) match cannot be considered for this In network in the low region. Indeed, we found that the In unit cell (5.4 Å) coincides with the (√3 × √3)R30° cell of the underlying SiC (that is √3 × 3.08 = 5.33 Å). Therefore, in order to match the two lattice planes, each In atom must bind to either Si atom, carbon atom (T4 site) or hollow (H3 site) of the SiC topmost layer. Among these sites, the T4 site is proved to be energetically more favorable than the H3 and the hollow sites in various epitaxial 2D systems formed on the SiC (0001), for example, in case of Si adatoms chemisorbed and formed the (√3 × √3)R30° reconstruction on the SiC surface48,49 and recent observations for In intercalated at the EG/SiC interface.22,23 Notably, the atomic characteristics of the intercalated In atoms in our case are very similar to that of Sn atoms adsorbed on the SiC (0001) substrate without the graphene capping layer.50,51 In this system, each Sn atom is found located at the T4 positions and energetically bound to its three adjacent Si atoms on the SiC substrate. Such observations lead us to conclude that the well-ordered In structure found in the low intercalated region is commensurate strictly with the (√3 × √3)R30° superlattice at T4 positions on the SiC substrate as illustrated in 18 it is still unclear under which circumstances the (√3 × √3)R30° triangular network of In is formed. Nevertheless, the ordered structure of ultrathin In provides strong evidence that the intercalated 2D In significantly interacts with the SiC substrate within the interface. In turn, the topmost atomic layers of the SiC substrate can be considered as a considerably important epitaxial template for 2D metal to be formed. It is also noted that the In lattice in this region has the same crystallographic orientations with the graphene top layer which implies that graphene coverlayer is only lifted up due to the intercalation and maintains its registry with the underlying SiC substrate. This behavior is consistent with the previous DFT calculation3 which revealed that adding the graphene to the intercalated In/SiC only affects the band filling without modifying the stability of the intercalated metal.Lower intercalated regions exhibit an ordered indium atomic array that epitaxially matches the (√3 × √3)R30° cell of the underlying SiC. The details of the low region are further shown in 21,27,29,52 However, since the graphene layer is lifted and becomes a quasi-free-standing layer due to the In intercalation, this supercell should have a different origin. In more detail, Forti et al.17 have demonstrated in case of Cu intercalated at the EG/SiC interface that the Cu atoms in a monolayer match epitaxially with the SiC substrate such that a (2 × 2) Cu cell (lattice constant of 2.66 Å) is commensurate with the (√3 × √3)R30° cell of the SiC substrate. In such an atomic arrangement, the (13 × 13) supercell emerges from the superposition induced by the two atomic planes between monolayer graphene and monolayer 2D metal. Within this cell, the underlying metal atoms register with the graphene network either directly below the hollow center of the graphene hexagon or below the A or B sublattice atom resulting in a superlattice varying potential. Since the In intercalated layer matches in a (1 × 1) ratio with the (√3 × √3)R30° superlattice of the SiC substrate , the potential modulation periodicity induced by the atomic registry between In and graphene is similar to that between Cu and graphene. In addition, this superstructure is also present in other intercalated metal systems at the EG/SiC interface such as Au5,14 and Cu37 showing consistency with our observation.Coexisting with the atomic structure of the In layer, a large superstructure arising as a result of the lattice mismatch between the monolayer graphene and the underlying In lattice is visible as faint corrugation lines indicated by a cyan rhombus in 50 Third, a bright atomic-like chain is also observed in some areas as indicated by the red square in A range of defect types exists in the low intercalated layer, enabling visualization of the ultrathin nature of 2D In. As can be seen in the STM images of 22 At this stage of analysis, we tentatively attribute these point defects to monovacancies of the intercalated In layers. However, in order to precisely conclude that these are vacancies in the In layer, further investigation should be considered. In addition, it is worth noting that the bright triangular defects coexisting with the atomic resolution of graphene are clearly displayed at 1 V or lower bias voltages as shown in 53,54 which results from local charge transfer between the defect and the three adjacent carbon atoms in the graphene lattice.55–57 These defects are attributed to be either monovacancies or foreign atoms substituted monovacancies in the graphene lattice.55Bias-dependent imaging is unable to verify the structure of “thick” indium intercalated regions which is hypothesized to be composed of more than one metallic layer preventing individual atoms to be resolved. In I/dV) spectroscopy is further used to elucidate the electronic signatures of the defects in 2D indium. I/dV spectra obtained with the tip placed above six defects marked from 1 to 6 in I/dV spectra. As clearly seen, the defects vary slightly in sizes and contrast. This implies that they do not sit on the equivalent lattice sites of the topmost In layer if one assumes that this layer is formed with a periodic structure. The dI/dV spectra taken on the defect 4 and 6 do not show any pronounced peak and have different contrasts in the STM images, notably those shown in Differential  and the applied bias voltages (4 V and 5 V). As a result, after the first pulse of 4 V, the first dark hole with a size of ∼5 nm and a depth of ∼1.4 Å is formed as can clearly be seen in the STM image in 25,35,45,58 This is an important indication that the area inside the hole is composed of graphene sitting directly above the 6√3 × 6√3 reconstructed SiC substrate without the intercalated metal (hereafter denoted as 6 × 6 graphene). We refer to this observation as the “de-intercalation” of the underlying In induced by the tip voltage pulse. This also reflects that following the de-intercalation, the topmost EG layer remains intact and stacks directly with the SiC substrate followed by the restoration of the underlying GBL. However, to understand the exact mechanism of how the GBL is recovered under the top EG layer, further investigation is required.Tip-induced surface manipulation enables direct visualization and engineering of In layers at the EG/SiC interface . This is because in our case the newly formed pristine graphene area is very small after the voltage pulse, the surrounding area is still intercalated by In atoms, and thus, we expect a different electronic coupling between graphene and the SiC substrate. We again exclude that this de-intercalated area is composed of the GBL because in this case, no linear band dispersion typical for graphene should be observed.45 Note that the Dirac point shift in this spectrum is irrelevant with the shift induced by In atoms in the low and high intercalated regions that has been discussed in I/dV spectra measured on such locations on both low and high intercalated regions .20,30 Thus, we now attribute the higher intercalated region to be composed of an In bi-atomic layer and infer the lower intercalated region as a monoatomic layer. Indeed, first-principles DFT calculations from Orimoto et al.32 pointed out that upon intercalation of a Cu monolayer at the GBL/SiC interface, the graphene capping layer is lifted by 2 Å which apparently supports our observation. The thickness of the In monolayer (2.13 ± 0.25 Å) in the low regions is higher than half that of the bilayer (high) region (that is 3.38 : 2 = 1.69 Å). This suggests interesting heterostructural strain-induced behaviors in both low and high intercalated regions. As demonstrated in previous STEM and DFT studies,7 the bonding characters in an EG/metal/SiC heterostructure for bilayer Ga and In evolves from covalent at the SiC/metal interface (with interspacing between SiC and the first Ga layer of 2.19 Å) to metallic between the first Ga layer and the second Ga layer (with a larger interspacing of 2.36 Å) and to nonbonded vdW-like at metal/graphene interface. Assuming that our first and second In layers stack with the SiC substrate and EG under similar conditions, one could expect that the apparent height of the bilayer region here must have a thickness at least larger than two times that of the low regions (that is 4.26 Å). Since the thickness of the In bilayer region is measured to be 3.38 Å, it suggests that the interaction of the interfacial In layers with graphene capping layers in the high region is stronger than that in the monolayer (low) region.Tip-induced interface manipulation is a powerful method to directly visualize and investigate the properties of 2D metals below the graphene capping layer. Upon de-intercalation, the metal thicknesses in the low and high intercalated regions can easily be explored by measuring the height of the atomic steps between the intercalated and de-intercalated (6 × 6 graphene) regions. As can be seen in 3 suggests that the strong metal–SiC covalent bonds play an essential role in providing a strong thermodynamic driving force for an In bilayer to be formed as the most stable atomic phase at the EG/SiC interface.Apart from the de-intercalation ability of metal layers below graphene, the tip-induced manipulation demonstrated here is a versatile tool to control the number of intercalated layers with atomic precision, through which the remarkable dynamic behavior of the interfacial In atoms is revealed. As already mentioned above, upon the tip voltage pulses, some areas around the tip location in the low intercalated region appear with a higher apparent height. In This finding is reinforced by the fact that when we carried out tip voltage pulse in the high intercalated region, the 2D In is only de-intercalated. No newly formed structures with a thickness other than a bilayer are found. In fact, we found that the monolayer In is very easily perturbed by changes in the tip conditions such as applied bias voltages, bias polarity or tunneling current while the bilayer region appears more stable. Therefore, the tip-induced manipulation not only de-intercalates the In material but can also be used as a versatile tool to engineer the intercalated layers at the EG/SiC interface.4.In conclusions, this work presents an experimental demonstration of the detailed structures of 2D indium stabilized at the EG/SiC interface using cryogenic STM. Two atomic phases corresponding to monoatomic and bilayer In structures stabilized under graphene are revealed. On the In monolayer, a triangular lattice matching epitaxially with the (√3 × √3)R30° periodicity of the underlying SiC substrate is identified. In contrast, no clear indication of the atomic arrangement is probed for the In bilayer. This observation clearly demonstrates the significant importance of the SiC substrate as an epitaxial template for 2D crystalline In to be formed. We have successfully used tip-induced manipulation as a versatile tool to either de-intercalate or reconstruct the intercalated materials while the graphene top layer remains unaltered. This allows microscopic insight into the ultrathin In at the EG/SiC interface with respect to intercalation thickness, good control over the number of metal layers and thermodynamic stability of In at the interface. Our results contribute to exploring the atomic and electronic structure of the confined metal, strongly emphasizing on tip-induced manipulation as a first demonstration for the intercalated metal at the EG/SiC interface.V. D. Pham designed, performed all the STM experiments, analysed the data and wrote the manuscript. J. A. Robinson and C. Dong designed and prepared the intercalation samples. All authors contributed critical revisions and gave final approval of the manuscript.There are no conflicts to declare.NA-005-D3NA00630A-s001


In recent years, the global population has increased significantly, resulting in elevated levels of pollution in waterways. Organic pollutants are a major source of water pollution in various parts of the world, with phenolic compounds being the most common hazardous pollutant. These compounds are released from industrial effluents, such as palm oil milling effluent (POME), and cause several environmental issues. Adsorption is known to be an efficient method for mitigating water contaminants, with the ability to eliminate phenolic contaminants even at low concentrations. Carbon-based materials have been reported to be effective composite adsorbents for phenol removal due to their excellent surface features and impressive sorption capability. However, the development of novel sorbents with higher specific sorption capabilities and faster contaminant removal rates is necessary. Graphene possesses exceptionally attractive chemical, thermal, mechanical, and optical properties, including higher chemical stability, thermal conductivity, current density, optical transmittance, and surface area. The unique features of graphene and its derivatives have gained significant attention in the application of sorbents for water decontamination. Recently, the emergence of graphene-based adsorbents with large surface areas and active surfaces has been proposed as a potential alternative to conventional sorbents. The aim of this article is to discuss novel synthesis approaches for producing graphene-based nanomaterials for the adsorptive uptake of organic pollutants from water, with a special focus on phenols associated with POME. Furthermore, this article explores adsorptive properties, experimental parameters for nanomaterial synthesis, isotherms and kinetic models, mechanisms of nanomaterial formation, and the ability of graphene-based materials as adsorbents of specific contaminants. The world’s main vegetable oils are derived from palm, soybean, coconut, olive, peanut, sunflower, cottonseed, and rapeseed . Palm oiSeveral techniques are available for POME treatment; however, not all are suitable as they have varying treatment efficiencies and costs . POME trIn recent times, nanotechnology has been widely employed in several applications, including wastewater treatment, as nanomaterials have been proven to be highly effective in eliminating inorganic and organic pollutants from wastewater effluents compared to conventional adsorbents due to their low toxicity and small size ,20,21. AThe utilization of nanosized GO as an adsorbent can affect the extent of oxidation via interactions and the presence of hydrogen bonds due to its complex structure . ResearcThe discharge of toxins from anthropogenic activities into the environment has resulted in one of the major challenges of this century, which is the scarcity of clean water. Phenol is frequently found in industrial wastewater generated by sectors such as oil, gas, medicine, and pesticide production. As it can be harmful, numerous environmental protection agencies have instituted stringent concentration limits for phenol in wastewater to safeguard human health and preserve ecological equilibrium . In ASEAPhenol, a white, crystalline, and volatile aromatic chemical compound, is widely found in wastewater from several industries, including oil, gas, medicines, and pesticides, and even at low concentrations, it can affect the taste and odor of water . Phenol Numerous studies have shown that phenol is becoming a concern due to its persistence in the environment . An uninAdsorption and advanced oxidative processes (AOPs) have been demonstrated as viable, practical, and environmentally friendly alternatives for removing phenol from water among the advanced remediation technologies. In recent years, tertiary treatments have been successfully used to increase phenol absorption and meet stringent water quality requirements. Advanced oxidation technologies and adsorption are reported as highly viable approaches among these tertiary methods. Adsorption is an advantageous technique for phenol removal due to its simplicity in construction and use, cost-effectiveness, and ability to handle low-concentration fluids . In addisp2 hybridized orbitals. As a result, graphene holds tremendous promise for various applications, such as field-effect transistors (FETs), gas and biomolecule sensors, transparent conductive films (TCFs), and graphene batteries, due to its outstanding properties. Furthermore, when carbon atoms are arranged in layers, graphene oxide (GO) is formed, which features oxygen-containing functional groups, such as =O, -OH, -O-, and -COOH, connected to the sides and edges of the plane [Graphene is a material that has been widely studied over the last few decades and has been dubbed the “material of the future” due to its exceptional properties. Graphene has a unique honeycomb lattice structure in a two-dimensional plane, where each carbon atom is bonded to three other carbon atoms through he plane . GO can 11H4O5 (corresponding to a C/O ratio of 2.2) [With the presence of multiple oxygen functional groups on its surface sites, GO can serve as a precursor for synthesizing various graphene derivative products, such as fluoro-graphene, bromo-graphene, and graphene. In addition, it is possible to synthesize graphene by thermally or chemically reducing GO. It can be noted that GO can also be applied to advanced applications, which include delivery of medicine, thermal conductivity, and materials for architecture. However, there are certain challenges with GO, such as toxicity and issues during mass production, that must be fixed and investigated in detail to utilize them for commercial applications. Thus, novel techniques for environment-friendly, inexpensive, and mass production of GOs are crucial. British chemist B. C. Brodie made the first effort to fabricate GO in 1859 while experimenting on the reactivity of flake graphite ,51. ThisFurther, Staudenmaier and co-workers altered Brodie’s procedure (ST-GO—graphene Staudenmaier’s oxide) by altering the introduction method of the chlorate and the addition of sulfuric acid to the mixture. In this method, potassium chlorate was gradually added to the mixture to reduce the risk of explosive byproducts and the evolution of heat. Furthermore, reaction time was reduced to increase the acidity of the environment, and the resultant GO closely resembles BR-GO in terms of its characteristics. Back in 1937, Hofmann synthesized graphene oxide (HO-GO) with a reduced oxygen content (C/O ratio of 2.5) by mixing non-fuming nitric acid and potassium chlorate. Jankovský et al. (2017) reported that nitric acid levels have a substantial impact on the degree of oxidation in the graphene-based product .2O2) to neutralize the excess potassium permanganate. The final product produced by the Hummers method has a C/O ratio comparable to BR-GO (2.25). However, the method is not environmentally friendly due to the presence of NOx during the reaction. Recently, Perumal et al. (2022) summarized the extracts of plants from different taxonomy groups for the novel synthesis of graphene via a green reduction approach. The authors mentioned that several plant species and major plant parts from each plant have exclusive potential to form GO and reduced GO [The Hummers method, also known as the permanganate procedure, is the most commonly used and efficient method for synthesizing GO to date, as developed by Hummers and Offeman in 1958. This method involves adding an excess of potassium permanganate, a low volume of sulfuric acid, and excess sodium nitrate to the reaction mixture, with a reaction time ranging from 8 to 12 h. This technique is safer than other traditional methods as it avoids the production of explosive chlorine oxide. The procedure is completed by adding a diluted solution of hydrogen peroxide (Hduced GO . However6Sn5) nanoparticles with reduced graphene oxide via a chemical reduction approach. The GO was synthesized via modified Hummers’ method using graphite, whereas bi-metal-GO nanocomposite was formed via chemical reduction method as shown in It is noteworthy that graphene oxides possess certain limitations, such as sturdiness and the inability to blend with other materials to form composites or alloys, especially for environmental applications . Thus, rRecently, Ullah et al. (2021) prepared size-controlled aluminum-doped graphene via a chemical vapor deposition (CVD) approach. In this study, the graphene was prepared with the help of low (23 Pa) pressure on the surface of a copper foil as a growth substrate in a horizontal tube furnace. The results showed that the CVD approach is beneficial for the preparation of high-quality aluminum-doped monolayer graphene with large areas of aluminum doped on the graphene lattice . In geneThe removal of phenol via adsorption has received attention in the past decade. In particular, the utilization of graphene-based adsorbents for the decontamination of these organic pollutants is gaining significant attention among researchers. The treatment of phenol using graphene-based composites has been reported in various studies, as summarized and presented in 2SO4 and HPO4 (named GO1) as well as H2SO4, H3PO4, and HNO3 (named GO2) for the treatment of phenol in water. The influence of various parameters, including initial phenol concentration, temperature, and pH, on the adsorption process was also studied. The authors reported the modeling of the equilibrium adsorption data and compared it with conventional models, such as Freundlich, Langmuir, Temkin, and Dubinin-Radushkevich adsorption isotherm models. It was noted that ultraviolet (UV) ray irradiation had a favorable influence on the adsorption process while increasing their adsorption capacity. It can be noted that the maximum adsorption capacities for GO1 and GO2 were 0.77 and 20.2 mg/g, respectively. It has been reported that the adsorption capacity reached its peak at pH 2 in the absence of UV radiation, whereas pH 6 was identified as optimal in the presence of UV irradiation. The adsorption rate increased with an increment in the initial phenol concentration, and an equilibrium point was reached where there was a drop in adsorption, after which it remained constant. The phenol concentration increment has resulted in increased pollutant uptake until equilibrium is attained, where the adsorption is reduced and has maintained a constant value. The maximum adsorptive capability of GO1 and GO2 has been elevated from 70.43 to 90.82% and 86.75 to 95.95% after UV exposure, respectively [3O4/PRd) for the adsorption of phenol in a batch process from a hydrous solution. The maximum adsorptive capability of GO composite was 191.0 mg/g at 15 min of equilibrium time. The adsorption behavior of phenol by the GO-Fe3O4/PRd adsorbent was described by Freundlich and Langmuir isotherm models with a pseudo-second-order kinetic model. The authors also revealed that the uptake of phenol by GO-Fe3O4/PRd was spontaneous, endothermic, and feasible [Recently, Al-Ghouti et al. (2022) reported the preparation of GO via two methods, such as the addition of Hfeasible .Bibi et al. (2022) developed GO modified with polyacrylic acid (GO-PAA) as a composite for the removal of phenol from real and synthesized effluents. The author utilized a variety of techniques, such as scanning electron microscope (SEM) and energy dispersive X-ray analysis (EDX), as well as Fourier transform-infrared (FTIR) and Brunauer-Emmet-Teller reaction (BET) techniques, to examine the GO and GO-PAA samples’ morphology, surface area, and surface chemistry, respectively. The adsorption parameters, such as temperature, pH, and initial phenol content, were also evaluated. The best adsorption was identified at a pH of 2 and a temperature of 25 °C. The results showed that the modification of the GO-PAA surface can lead to the aggregation of C=O groups, which eventually increases the adsorption ability of unmodified GO. The study also revealed that Langmuir’s isotherm model was best suited to describe the adsorption process of the GO sample, and the thermodynamics studies proved the exothermic and spontaneous nature of phenol adsorption processes. It was observed that the GO-PAA sample was able to remove 75% of the phenol from synthetic wastewater and 18% from real wastewater, respectively, at an optimal pH of 2 and a temperature of 25 °C . Further3O4 composite via a one-pot process for phenol uptake in batch adsorption. The composite exhibited a huge surface area and copious amino groups, and the maximum adsorptive capability, evaluated from the Langmuir model under optimum conditions, was 224.21 mg/g, with equilibrium reached in 60 min. The adsorption mechanism involved π-π interactions, hydrogen bonding, and electrostatic interactions between PVAm and phenols, and the composite was loaded with phenols that could be easily recycled using NaOH solution. Thus, the PVAm-GO-(o-MWCNTs)-Fe3O4 composite was an effective adsorbent for phenolic compound removal [3O4 hybrid material using sonication-assisted co-precipitation techniques to treat phenol. The adsorption kinetics showed that the phenol uptake efficacy was 68% in aqueous systems, with an outstanding adsorptive capability of ∼79 mg g−1 and an initial adsorption rate of 5.7 mg g−1 min−1. The process was initially governed by the boundary, then by surface diffusion, and finally via pore diffusion, as indicated by the kinetic models. According to the Langmuir isotherm, equilibrium isotherms showed that the adsorption was a spontaneous physisorption process with a maximum adsorption capacity of 658 mg/g [Zhou et al. (2020) synthesized a three-dimensional amino-poly(vinylamine) (PVAm)-functionalized GO-(o-MWCNTs)-Fe removal . In anot658 mg/g . Manna e658 mg/g . All theVerbena officinalis to reduce their toxicity towards the environment. The resultant GOs were encapsulated in sodium alginate via cross-linking to utilize the system for phenol treatment in olive mill wastewater. The study showed that these green synthesized GOs in sodium alginate were identified to possess 3.68 g/L of adsorbent dosage at a temperature of 25 °C and a pH of 4 with 135 min of adsorption time. In addition, the study also revealed that the GO system possesses 994 mg/g of phenol adsorption capacity for 4000 mg/L of initial concentration [The limitations of micro- and macro-sized graphene materials have led to the emergence of graphene nanomaterials and nanocomposites for the treatment of phenols via adsorption. Maio et al. (2020) prepared three-dimensional templates with graphene oxide-decorated polycaprolactone (PCL) via a one-pot process. In this study, the PCL solutions were coated on GO nanoparticles in ethanol through jet-wet electrospray or electrospinning on the surface of a stirred liquid collector. The phenol removal analysis showed that the GO-PCL nanoparticles possess a sorption equilibrium of 6 h via a pseudo-second-order model in fresh water with excellent robustness and recyclability . Even thntration . However2/h/bar, which is higher than individual graphene oxide membranes. The results revealed that the membrane can remove >75.8% of p-nitrophenol during a dead-end ultrafiltration process from wastewater [Othman et al. 2019) prepared novel copper-iron oxide nanoparticles that are supported on the surface of the reduced GO via a facile low-temperature approach. The synthesis has led to the decoration of copper-iron oxide nanoparticles on the surface of reduced GO sheets. The study showed that the composite possesses enhanced photocatalytic activity for phenol degradation. The results showed that the GO system can completely remove phenol from the contaminated water within 15 min at room temperature under photocatalytic conditions, as shown in stewater . All the019 prepaThe development of efficient, environmentally friendly, and cost-effective GO-based nanomaterials for micropollutant adsorption has gained considerable interest among researchers, industrial sectors, and policymakers . HoweverIn conclusion, the studies conducted on GO-based nanocomposites as adsorbents for phenol removal applications have demonstrated their high efficiency and effectiveness. These nanocomposites offer several advantages, including a high surface area, excellent mechanical strength, and unique chemical and physical properties, which contribute to their superior adsorption capacity for phenol and other organic contaminants. Furthermore, kinetic studies have shown that GO-based nanocomposites typically exhibit a pseudo-second-order reaction for phenol adsorption processes, indicating their ability to rapidly and efficiently remove phenol from wastewater. Moreover, the capacity of various GO-based composites with different modification types for adsorbing phenols has been extensively reported, showcasing their versatility and applicability in diverse wastewater treatment applications. It is noteworthy that the recovery of nanomaterials is the most significant aspect of their application in environmental applications. Recent research has highlighted the importance of developing efficient methods for the recovery and reusability of nanocomposites. Although specific percentages for nanomaterial recovery depend on the process, it is a critical area of focus for future investigations. Incorporating sustainable and environmentally friendly approaches to recover and recycle these materials will enhance the overall sustainability and cost-effectiveness of phenol removal processes. In summary, GO-based nanocomposites offer a sustainable and effective solution for the treatment of phenol-contaminated wastewater, providing a cost-effective and eco-friendly approach for pollutant removal. Further research efforts should aim to develop novel and efficient materials while also focusing on the recovery and reusability of nanomaterials to optimize their overall environmental impact and resource utilization.


Axon degeneration and Neuromuscular Junction (NMJ) disruption are key pathologies in the fatal neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). Despite accumulating evidence that axons and NMJs are impacted at a very early stage of the disease, current knowledge about the mechanisms leading to their degeneration remains elusive. Cytoplasmic mislocalization and accumulation of the protein TDP-43 are considered key pathological hallmarks of ALS, as they occur in ~ 97% of ALS patients, both sporadic and familial. Recent studies have identified pathological accumulation of TDP-43 in intramuscular nerves of muscle biopsies collected from pre-diagnosed, early symptomatic ALS patients. These findings suggest a gain of function for TDP-43 in axons, which might facilitate early NMJ disruption. In this review, we dissect the process leading to axonal TDP-43 accumulation and phosphorylation, discuss the known and hypothesized roles TDP-43 plays in healthy axons, and review possible mechanisms that connect TDP-43 pathology to the axon and NMJ degeneration in ALS. For many years, the molecular basis of neuromuscular junction (NMJ) disruption and motor neuron death in the fatal neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS) has remained elusive. Recent fundamental discoveries in axonal biology in health and disease have advanced our understanding of the local and independent subcellular mechanisms that sustain motor neuron axonal function and integrity. Several of those discoveries also highlighted the pivotal contribution of axonal degeneration to the development and progression of ALS pathology. These may mark a new era for the development of novel therapeutic strategies, as well as diagnostic tools for this lethal disease.TARDBP, were reported in about 0.5% of ALS patients  [TAR DNA Binding Protein TDP-43) is a DNA and RNA binding protein that belongs to a group of heterogenous nuclear ribonuclear proteins (hnRNPs). TDP-43 was first discovered in 1995 by Ou and colleagues as a protein that binds the pyrimidine-rich motifs within the TAR DNA of human immunodeficiency virus-1 (HIV-1) . Ever si is a DNAc forms) , 15.A primary interest in the non-nuclear roles of TDP-43 began shortly after discovering the presence of phosphorylated and ubiquitinated cytoplasmic inclusions of TDP-43 within motor neurons in the brains of patients with ALS and Frontotemporal Dementia (FTD) , 17. SomMotor neuron axons are the longest and most arborized ones in the nervous system. The volume of motor neuron axonal and synaptic cytoplasm  exceeds that of the cell body by several orders of magnitude, and thus also its protein content. It has now been established that local protein synthesis and axonal transport are two essential mechanisms by which motor neurons use to regulate their axonal and synaptic proteome . AlteratALS is a neurodegenerative disease that targets lower and upper motor neurons, with vast heterogeneity of proposed mechanisms that lead to toxicity in the disease. However, the existence of one common pathology has wide approval among experts, the presence of cytoplasmic inclusions of TDP-43 protein. Still, the notion that TDP-43 cytoplasmic inclusions can also accumulate in remote processes and facilitate local pathology in axons and synapses was until recently, a topic of controversy. This issue directly relates to the “dying back” theory, which depicts the NMJ and axons as the primary location for degeneration in the disease , 29. FolA key fact, which is often being neglected when discussing TDP-43 pathology in ALS, is that most of the existing data regarding human ALS patient tissues are of hyperphosphorylated TDP-43 cytoplasmic inclusions. Early reports for the presence of TDP-43 phosphorylation (pTDP-43) came together with the finding that TDP-43 is a common pathology of ALS . FurtherSeveral cellular mechanisms may explain the increase in local concentrations of proteins within axons: 1. Enhancement in cytoplasmic localization and axonal transport towards the nerve ending. 2. Decrease in axonal degradation and clearance. 3. Cell-to-cell protein transmission. 4. Local protein synthesis in axons. Although there is no concrete evidence as to which possibility is most likely in the case of TDP-43, several recent discoveries revealed few relevant pathways for this phenomenon in ALS  . TDP-43 Like other phosphorylated proteins, there is an enzymatic pathway coordinating TDP-43 phosphorylation, which is mediated by kinases and phosphatases. Five known kinases can directly phosphorylate TDP-43: Casein kinases 1 and 2 , Tau tubulin kinases 1 and 2  and Cell division cycle 7 (CDC7) kinase . Among tC.elegans models of TDP-43 proteinopathy, genetic deletion of calcineurin homolog gene tax-6 can lead to excess accumulation of pTDP-43 and motor dysfunction, while treatment with the calcineurin inhibitor tacrolimus rescued the motor phenotype [With regard to the opposite regulation of TDP-43 phosphorylation, the phosphatases Calcineurin, PP1 and PP2 have been shown to have a direct impact on TDP-43 phosphorylation . It was henotype . As for henotype .Still, regarding the activity of those kinases and phosphatases in the regulation of TDP-43 phosphorylation in axons and its functional outcomes, data is sparse at best. One possibility is that axonal kinase activity might be influenced indirectly from TDP-43 phosphorylation by itself. For example, CK2, a kinase known to phosphorylate TDP-43, was found to enhance axon growth by phosphorylating G3BP1 . G3BP1 pTwo pivotal questions which remain open are how TDP-43 is capable of mediating excessive damage to motor neuron axons and NMJs, and if there is higher vulnerability of those distal segments to TDP-43 condensation compared to cell bodies. To address those issues, it is first necessary to understand the unique properties of motor neurons and the roles TDP-43 has in supporting their growth and maintenance. Motor neuron axons are of the longest and most arborized axons in the human body. To coordinate complex spatiotemporal processes within their highly compartmentalized cytoplasm, neurons have adapted key mechanisms such as axonal transport of essential proteins , 82, orgOwing to the tremendous efforts invested in identifying TDP-43 interactors and TDP-43-bound mRNA, our knowledge of the cytoplasmic role of TDP-43 and more specifically in axons advanced in recent years. As TDP-43 is strongly associated with the formation of cytoplasmic and axonal inclusions in ALS, data from studies on these pathological forms assisted to reveal the normal context of TDP-43 at those subcellular environments. Various approaches were used to characterize the TDP-43-bound, and TDP-43-associated RNA populations in the nucleus, as well as in the cytoplasm , 97. SepNefl, Map1b) and mitostasis process  [Due to the spatial complexity of motor neurons, and considering the axonal pathology in ALS, a key unresolved question is identifying the RNAs associated with TDP-43 also in axons and NMJs. Alami and colleagues have identified that TDP-43 is a component of axonal mRNA granules where it binds and shuttles RNA . TDP-43  ATP5A1) . Moreove ATP5A1) . Recentl ATP5A1) , while o ATP5A1) , 100. Ci ATP5A1) . TDP-43  ATP5A1)  and was  ATP5A1) , 102. He ATP5A1) , 102–104Many publications in the recent decade have established that axons and synapses rely on local protein synthesis in order to develop, function, respond to changes, and maintain themselves , 106. YeA315T in motor neuron-like cortical neurons does not affect global protein synthesis, however, it does enhance the translation of specific transcripts, including Camta1 and Mig12, and that overexpression of mutant TDP-43A315T also enhances translation of Dennda4 mRNA [Drosophila Melanogaster models, Coyne et al., have shown that TDP-43 and drosophila FMRP colocalize together in PABP-positive stress granules in cultured neurons from TDP-43 overexpression model flies. The authors further show that overexpression of TDP-43 inhibits the translation of drosophila NMJ-associated futsch mRNA by its sequestration in these stress granules [Accumulating evidence indicates that these processes are controlled by the dynamic assembly and dissolution of RNP granules , 108. Thda4 mRNA . In Drosgranules . Hence, granules . Similargranules , hintingAxonal and synaptic mitochondria have been shown to depend on local protein synthesis of nuclear-encoded mitochondrial mRNAs in order to properly function , 117–119Following mitochondrial ones, transcripts encoding ribosomal subunits were also reported to occupy large proportions of axonal transcriptome , 122. ReAnother layer of regulation over translation is achieved by microRNAs (miRNA). miRNAs are short, 20–22 nucleotide non-coding RNA that mediate posttranscriptional silencing of their tens to hundreds of target mRNAs, some of which encode protein families that promote a specific function or pathway . As suchCollectively, this evidence marks cytoplasmic and axonal TDP-43 as a regulator of the axonal transcriptome and proteome. Yet, the mechanisms that regulate TDP-43 and control its ability to perform distinct properties over space and time in motor neuron axons and synapses remain to be determined. The post-transcriptional modifications which TDP-43 has been described to undergo, like phosphorylation, are one possible explanation for how TDP-43 switches between its different functions and localizations (nucleus/cytoplasm/axons), but may also cause neurodegeneration when are aberrantly made in space, time and intensity. It is highly important to understand how those switches occur, and even more so how we can interfere with them early enough to slow or prevent motor neuron degeneration in ALS.As years go by, scientific discoveries have promoted our understanding of ALS disease etiology, genetics, pathology and progression. Still, those achievements did not yet translate into the clinic. Therefore, it is necessary to ask, are we “looking under the lamp”?. Meaning, are current therapeutic interventions developed for ALS the most likely to succeed or the most available for testing. One key point regards the relative overlook of axonal projections and NMJs when trying to analyze ALS pathology. Until recently, the knowledge about ALS pathology was based mostly on histological sections of post-mortem CNS tissues, such as the brain and spinal cord , 30, 33.An important topic, which was discussed earlier, is the ability of TDP-43 protein to form cytoplasmic condensates. Still, little is known regarding the presence of those condensates in axons and their ability to form and dissolve in axons of ALS models. Over the last 10 years, a liquid-to-solid phase transition has become a central mechanism in ALS pathology –144. MulAdditionally, the function of RNP condensates in axons and synapses is also understudied. Gopal et al. revealed that axonal TDP-43 has influence on the solubility of RNP granules, and mutations in TDP-43 can influence its axonal transport . A recenTargeting motor neuron axons and NMJs with novel therapies early in the disease course might prove to be beneficial for some additional reasons. One possible advantage can be the increased pharmacological accessibility of peripheral nerves. Traditionally, it is difficult to develop and administer therapies for neurological diseases, due to the complex process of efficiently crossing the blood–brain barrier in order to reach adequate drug concentrations in the cerebrospinal fluid. If indeed the formation of pTDP-43 disease-causing condensates is initiated in peripheral axons and NMJs, it is possible that blocking this process can delay the disease or even prevent it from affecting the spinal cord where motor neuron cell bodies are located. There are already known compounds that were shown to inhibit TDP-43 phosphorylation and even to block condensate formation which might be used . HoweverThe novel finding regarding the presence of pTDP-43 pathology in peripheral motor neuron axons of ALS patients provides an exciting new perspective for understanding ALS pathology and etiology, as well as an avenue for developing new therapeutic approaches. Together with recent discoveries regarding the importance of TDP-43 in axonal health and the implications of its phosphorylation on RNA biology, local protein synthesis, and mitochondrial viability, an urgent need for timely research is at hand. One possible hypothesis which will need further proof is that the initial step in the disease is enhanced axonal localization of TDP-43 at distal motor nerves. This process can lead to a buildup of pTDP-43 condensates in the NMJ, which in turn facilitates repression in local protein synthesis, mainly of mitochondrial and ribosomal proteins. The following result is the failure of the NMJ to maintain enough functioning mitochondria and ATP, resulting in the accumulation of reactive oxygen species and NMJ degeneration, delivering a retrograde signal causing motor neuron cell death (Fig.


Uncontrolled human and industrial activities lead to the increase in demand for selective gas sensors for detection of poisonous gases in our environment. Conventional resistive gas sensors suffer from predetermined sensitivity and poor selectivity among gases. This paper demonstrates curcumin reduced graphene oxide-silk field effect transistor for selective and sensitive detection of ammonia in air. The sensing layer was characterized by X-ray diffraction, FESEM and HRTEM to confirm its structural and morphological features. Raman spectroscopy, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy was carried out to analyze the functional moieties present in the sensing layer. Curcumin reduced graphene oxide introduces sufficient hydroxyl groups in the sensing layer to provide high degree of selectivity towards ammonia vapors. The performance of the sensor device was evaluated at positive, negative and zero gate voltage. Carrier modulation in the channel through gate electrostatics revealed that the minority carriers (electrons) in p-type reduced graphene oxide plays a pivotal role in enhancement of sensitivity of the sensor device. The sensor response was enhanced to 634% for 50 ppm ammonia at 0.6 V gate voltage compared to 23.2% and 39.3% at 0 V and − 3 V respectively. The sensor exhibited faster response and recovery at 0.6 V owing to higher mobility of electrons and quick charge transfer mechanism. The sensor exhibited satisfactory humidity resistant characteristics and high stability. Hence, curcumin reduced graphene oxide-silk field effect transistor device with proper gate bias elucidates excellent ammonia detection and may be a potential candidate for future room temperature, low power, portable gas detection system. These demands call for intensive research towards fabrication of miniaturized, room temperature and low power gas sensors. In this context, sensing layers based on metal oxide semiconductors  are widely explored. These sensing layers are highly attractive due to their enormous sensitivity, but their functioning requires high operating temperatures . The high-power budget of metal oxides limits their usage in hand held, room temperature gas detection systems. For example, SnO2 based Taguchi sensors  that are available commercially, utilizes 200 mW power. Hence, efforts are required to develop room temperature, low power sensors. There are several attempts in this regard9, which aim to integrate the sensors with the matured CMOS platform. Certain gases like ammonia, hydrogen sulfide have low ignition temperature and are highly flammable. Therefore, researchers have focused towards synthesis of functionalized nanomaterials that operate at room temperatures. These materials include two-dimensional nanomaterials (graphene), transition metal di-chalcogenides , black phosphorus, metal organic frameworks, etc.Due to the rise of activities in chemical, food and automobile industries, there is a significant demand for development of hand held and battery operated10. Graphene is found to be highly sensitive towards different gas analytes. The two-dimensional honeycomb structure along with single layer of carbon atoms facilitate higher sensitivity towards different analytes. Pristine graphene which is free of defects, has poor adsorption energy towards different gases. Introducing defects and dopants in the graphene matrix shall enhance the adsorption energy level and facilitate better charge transfer between the target analytes and graphene matrix. Chemical exfoliation of graphene leads to graphene oxide, which on reduction produces reduced graphene oxide (RGO). Reduced graphene oxide consists of several defect sites along with various functional moieties, that introduces several active sites for adsorption of target gases. The biggest advantage of RGO is its ability to detect trace gases even at room temperature. This makes RGO an ideal candidate to be utilized in future generation room temperature, low power, portable gas sensors.Graphene, a two-dimensional nanomaterial has found significant attention due to its enormous surface area, thermal and mechanical stability, high mobility, and flexibility12. In case of prolonged exposure, severe health issues are observed in human beings, including death. According to OSHA , 15–28% ammonia concentration by volume in air is considered highly dangerous to health13. The flammable nature of ammonia demands fabrication of sensors that must operate at room temperature. Few chemo-resistive ammonia sensors based on graphene are developed21, however their performances are not satisfactory. RGO based sensors that are previously reported, suffer from large response and recovery, baseline drift, poor recoverability, and unsatisfactory selectivity. Earlier reports on graphene-based gas sensors lack analysis under humidity environments. In case of resistive sensors, once the sensing layer gets deposited, it cannot be further tuned or modulated. These factors boost the motivation towards development of Field effect transistor (FET) based gas sensors, where the channel can be modulated even after the sensor is fabricated. This strategy might facilitate “sensitivity enhancement” by controlling the number of effective carriers in the channel region22.In various industrial applications, ammonia is used as a common reagent. Ammonia is a highly pungent and toxic gas, which may cause severe effects on respiratory tracts, eyes, and skin, if exposed at higher concentrations (> 350 ppm)In the present work, a field effect transistor (FET) has been fabricated to detect ammonia vapors selectively. Curcumin reduced graphene oxide (Cur-rGO) is synthesized as the channel material and drop casted between the source and drain electrodes. Silk is used as the gate dielectric due to (i) high dielectric constant and (ii) ease of deposition. This reduces the fabrication cost and complexities. The current work demonstrates the play of gate voltage in tuning the type of charge carriers participating in the charge transfer/sensing mechanism. Even though RGO is known to be a p-type material, it is observed that application of proper gate voltage ensures electron participation for ammonia detection. During application of positive gate bias (0.6 V), the sensor exhibits enormous sensitivity of 634% at 50 ppm ammonia. When compared to zero gate bias (purely resistive-type) and negative gate bias, ~ 15 times enhancement in response is observed. The participation of electron enhances the rate of charge transfer, which is further reflected in response and recovery time analysis. The performance of these sensors under appropriate gate voltage reveals the significance of “optimization of gate electrostatics” in chemical sensors. The use of curcumin to reduce graphene oxide is also validated through Fourier transform infrared spectroscopy. The reduction through curcumin leads to complete removal of all other functional groups except hydroxyl (O–H bonds). These hydroxyl groups play a key role in determining the selectivity of the sensors towards ammonia. Cur-rGO was further characterized by FESEM, HRTEM, Raman spectroscopy and X-ray photoelectron spectroscopy to reveal its structural, morphological, and chemical properties. The Cur-rGO based silk FET exhibited good immunity against humidity and displayed good stability. Compared to the previous reports, its is evident that Cur-rGO silk FET with appropriate gate bias is highly efficient towards selective and sensitive detection of ammonia. These results prove that Cur-rGO silk FET based ammonia sensor can be used in future commercial low power, room temperature and portable gas detector systems.Morphological, structural, and compositional analysis of Cur-rGO was carried out through FESEM, HRTEM, XRD, FTIR, XPS and Raman spectroscopy. Figure 1 radiation (λ = 1.54 Å) equipped with a parallel beam diffractometer. Figure −1. The C=O bond at 1740 cm−1 relates to the carboxylic groups present at the edge of graphene sheet. The presence of C–O stretch at 1048 cm−1, C–O–C stretch at 1220 cm−1, C–OH stretch at 1373 cm−1 reflects the different functional moieties present in GO23. Thermal treatment of GO by curcumin leads to successful reduction of GO as confirmed by FTIR spectra. Apart from O–H stretching (centered at 3400 cm−1), all other functional groups are successfully removed from GO surface. The presence of this O–H group is highly influential towards selective detection of ammonia, compared to other VOCs.Examination of crystallographic orientation of the synthesized materials was carried out through XRD analysis having Cu Kα−1 and 1607 cm−1, however after reduction the D and G bands shifted to 1357 cm−1 and 1599 cm−1, which is in accordance with the previous reports25. The attachment of curcumin functionalities with the unsaturated carbon atoms leads to shifting of ‘D’ and ‘G’ bands to lower wave numbers. The electron donating curcumin leads to softening of phonons and shifts the fermi level27.Successful reduction through curcumin is further validated by Raman spectroscopy as shown in Fig. 3 molecules towards –OH groups.The curcumin reduced GO was further investigated by X-ray photoelectron spectroscopy (XPS) to further analyze the chemical states and functional groups. The deconvoluted XPS spectrum of C 1s region of Cur-rGO is shown in Fig. Reduction of GO in presence of curcumin leads to the formation of a highly sensitive ammonia sensing layer. The presence of field effect in the sensor devices allows modulation of charge carriers in the channel region. As reduced graphene oxide exhibits p-type characteristics, electrons play the role of minority carriers in all transport mechanisms. These electrons play a pivotal role towards highly sensitive detection of ammonia at low concentrations. It is predicted that charge transfer during sensing would be more efficient for electrons as compared to holes. Holes with a lesser mobility and higher effective mass would face larger hindrance towards fast charge transfer. Hence, it is focused to use electron as the main carrier for charge transfer by application of appropriate gate bias.28. The epoxy and hydroxyl groups in defect induced graphene sheets are found to have binding energies of be 0.219 eV and 0.840 eV respectively for ammonia28. This shows the importance of hydroxyl groups to be present over the sensor surface. When the applied gate voltage is negative or zero, holes are the carriers that take part in the sensing phenomenon. Ammonia molecules get bound to the hydroxyl groups and donates electrons to the Cur-rGO sensing layer as shown in Fig. Pristine graphene exhibits 0.114 eV adsorption energy towards ammoniad, gas and Id, air are the drain current upon exposure to ammonia and air respectively.Response is mathematically defined as:d-Vg characteristics (Vds = 1.5 V) in Fig. 5). With gate voltage less than 0.5 V, the device exhibits conductivity due to holes. However, after 0.5 V, the device exhibits electron conductivity. When the sensor is exposed to ammonia molecules, the hole current reduces (left of 0.5 V) but the electron current increases (right of 0.5 V). When the majority carriers are holes, then presence of ammonia molecules reduces the number of holes due to electron–hole recombination. Hence, a reduction in current is observed. With increased gate voltage above 0.5 V, the presence of ammonia donates electron in the channel. Now, electron being the majority carrier, its concentration increases and hence increase in current is observed.The I3 from 10 to 50 ppm. The concentration curve is shown at Vgs = 0.6 V, the best sensing condition. Figure gs and 39.3% with − 3 V Vgs. However, at Vgs = 0.6 V, there is tremendous increase in response to 634% for 50 ppm ammonia. This result signifies the essence of gate voltage optimization to increase the sensitivity of the sensing layer.Figure 3 molecules when compared to seven other VOCs . The current is observed to decrease with decrease in concentration, due to the n-type behavior of the channel at the desired gate voltage. Figure 3 at an optimized Vgs of 0.6 V. The sensor reaches the baseline current after the gas source is removed. The absence of baseline drift is a blessing in terms of calibration of the device.Figure gs = 0.6 V and Vgs = 0 V is illustrated in Fig. Response time is the time taken by the sensor response to reach 90% from 10% of the maximum value. Recovery time is the time taken by the sensor to recover from 90 to 10% of the maximum value. The response and recovery time of Cur-rGO at positive gate voltage (0.6 V) is lower compared to negative (− 3 V) and zero gate bias as shown in Fig. 3 is compared with previous works from literature as depicted in Table 3 at room temperature. The optimization of gate voltage to change the polarity of the channel and the presence of silk as dielectric ensures sensitive as well as low power operation. The proper reduction of graphene oxide by curcumin leaves behind hydroxyl groups, which facilitates selective ammonia sensing behaviour of the Cur-rGO Silk FET. Therefore, Cur-rGO Silk FET can be used in future commercial room temperature, low power ammonia gas detection systems.Hence, the effect of gate voltage towards enhancement of ammonia sensing performance for Cur-rGO introduces a new technique towards increasing of sensitivity in gas sensing domain. The performance of Cur-rGO towards NHGraphite powder, sodium nitrate, potassium permanganate, sulphuric acid, ammonia solution, ethanol, sodium carbonate, calcium chloride, formic acid and curcumin were obtained from Merck India pvt Ltd. and were used throughout the experiment without further purification. Commercially available Bombyx Mori silk was purchased from M/S Bombyx Mori Silks and Textiles, Srinagar, India. Millipore purification system was used for preparing de-ionized water.Widely known Hummer’s method was implemented towards synthesis of graphene oxide (GO) sheets by chemical exfoliation of graphite powder. For synthesis of curcumin reduced graphene oxide (Cur-rGO), solution of curcumin in ethanol was prepared by adding 5 mg curcumin to 10 ml ethanol. The solution was stirred for an hour to obtain a homogenous yellow solution. A beaker containing 100 ml GO dispersion (1 mg/ml) was constantly stirred and curcumin solution was added in dropwise manner to obtain a homogenous mixture. After stirring for 2 h, 20 μL of ammonia solution (27%) was added to attain a pH of 10. After further stirring for an hour, the solution was transferred to a blue capped reagent bottle. The solution was then heated at 85 °C in a hot air oven for 4 h. The resulting solution was cooled to room temperature and subjected to 2500 rpm centrifugation to eliminate unwanted impurities. The as prepared solution was termed Cur-rGO.29. Initially, a glue-like substance (Sericin), which binds the silk fibroins was removed. The fibroins were then dissolved to form a homogeneous solution. Sericin was removed from silk fibroins by treating it with 0.5% sodium carbonate-water solution at 110 °C for 1.5 h. In the next step, it was rinsed thoroughly with DI water and dried overnight. After the drying stage, the degummed silk was dissolved in a solution comprising of formic acid and calcium chloride (19:1), leading to regenerated silk fibroin solution. The presence of inorganic salts with formic acid facilitates dissolution of degummed silk. Calcium chloride penetrates the structure while formic acid enables swelling of silk. Penetration of these salts disrupts the hydrogen bonding between the fibroin protein chains and decomposes it to micro-sized fibrils and finally to nanofibrils. The solution was then casted onto a petri dish and dried at 60 °C. After drying, the film was immersed overnight in DI water to remove calcium chloride, as it causes detrimental effects on thin film properties. The film can be dissolved and reconstituted in formic acid directly, as, and when required.Dielectric silk solution was prepared by a method proposed by Sarkar et al.Fabrication of silk gate field effect transistor (Silk-FET) was carried out on p <100> type silicon wafer having resistivity 4–20 Ω-cm. A 200 nm thermal oxide (dry–wet-dry) was grown over silicon to achieve proper insulation. Deposition of 50 nm aluminum layer was carried out by thermal evaporation, which acted as the gate electrode. Dielectric layer of 200 nm silk solution was spin coated over the aluminum film. Source and drain electrodes were fabricated using DC sputtering, 20 nm titanium (Ti) adhesive layer was deposited followed by 100 nm gold (Au) through a shadow mask. Interdigit gap of 200 μm was achieved between the fingers. The width of the electrodes was 500 μm. The sensing material (Cur-rGO) was deposited onto the gaps by simple drop casting technique. The schematic of the fabricated Silk-FET device is depicted in Fig.