Ionic liquid (IL) combined with graphene additives have garnered extensive attention in the field of high-performance lubricating materials. However, the ambiguous mechanism of graphene influencing the load-carrying and anti-wear capacity of ILs needs further study. In this work, friction simulation shows that adding graphene causes friction coefficient to reduce by up to 88% compared with pure ILs, but lubrication performance is lost due to the destruction of graphene under high stress. Meanwhile, multilayer graphene has better friction-reducing performance and friction durability as compared to the monolayer structure, which is attributed to the easy-shear property and the reduction in the percentage of high tensile stress sites in multilayer graphene structure. In addition, it was found that excessively thick ILs film would form a three-body abrasive wear structure with graphene, which accelerated the structural destruction of graphene and caused a decline in its tribological properties. It is believed these findings can be valuable for designing of high-performance lubricating oil for practical engineering. Since the initial report published in 2001 ,5,6,7,8.Recently, extensive effort has been devoted to investigating the tribological properties of composite lubricants based on ILs and graphene. Functionalized multilayer graphene as additives can prominently enhance the anti-wear and friction-reducing ability of IL film under high vacuum, due to the lubrication synergy of ILs and multilayer graphene . Qi et aThe synergistic lubrication mechanism of graphene as additives for ILs has been studied from various aspects of tribochemical reactions and enhancing load-carrying capacity. However, since the scale of graphene as a low-dimensional material has entered the nanoscale, it is difficult to elucidate the microstructure and frictional properties of nano-additives only through experimental studies. Meanwhile, macroscopic experiments cannot observe the synergistic lubrication process as well as the structural changes of additives, and cannot obtain accurate lubrication mechanisms to efficiently guide the optimization of experimental processes. Therefore, the mechanism of graphene molecules as additives affecting tribology behavior of ILs is still unclear. The computer simulation method helps us to gain insight into the friction mechanism from the atomic scale. Hence, it has been widely used to observe the mechanism in tribology field.In this work, a molecular dynamics simulation (MD) approach is used to investigate the synergistic lubrication mechanism based on ILs and graphene. Diamond-like carbon (DLC) is employed as the friction substrate in this research due to its antifriction performance. It was found that their excellent lubrication performance, which has a significant dependence on the application environment, are dementedly influenced by the sp3-to-sp2 ratio . And the3. The IL is consisted of the cation  and the anion  (as shown in 2 carbon (C) atoms arranged in the hexagonal symmetry and H atoms passivating its edge . The open source software OVITO 3.8.0  was usedFn ; the corresponding normal forces, which are 44 nN, 220 nN, and 440 nN, respectively; IL amounts Ni , graphene amounts Ng ; surface roughness R ; and sliding velocity Vy , to investigate the tribology behaviors. The substrate rough surface was obtained by deleting the partial atoms adjacent to friction region according to the following sine function.z is the coordinate of the atoms in the direction of normal stress, R the amplitude of roughness, and y controls the distance between two rough micro-convex bodies. The Ff is calculated by summing the tangential force along the sliding direction of the rigid layer in upper DLC. The molecular average translational kinetic energy formula, Ek = 3kBT/2 [T) of the friction region. The coordination number of C atoms of DLC and graphene was calculated according to a cutoff length of about 1.9 Å, which corresponds to the value of the first minimum of the radial distribution function. The coordination number of C atoms with sp2 and sp3 hybrid states is three and four, respectively. The value of fsp3 was calculated by dividing the number of C atoms with the sp3 hybrid state by the total number of C atoms outside the rigid layer. The binding energy between multilayer graphene was calculated when two graphene sheets completely overlapped. The atomic stress tensor was calculated by multiplying the Voronoi stress tensor by an individual atom’s volume.In the simulation, considerations are given to different factors, such as normal stress = 3kBT/2 , was useFn) and amounts of graphene (Ng) on friction force (Ff). The short period (approximately 5 ps) after the beginning of sliding is defined as the running-in stage. In this stage, Ff is at a large value and then drops rapidly, which is due to the relative sliding of the friction pair required to overcome a large initial resistance. The running-in stage is followed by the stable friction stage. It can be seen from the illustration in Ff is kept at a small value in the whole sliding process in the condition of Fn ≤ 5 GPa. Such a phenomenon can be explained by the fact that the upper and lower layers of DLC are completely separated via composite lubricating film and there is no bonded interaction at the interface which resulted in extremely minute interfacial adhesion. When Fn continues to increase to 10 GPa, Ff increases more rapidly and the Ff at the final state is consistent with that of without graphene. It can be observed that Ng = 2, as shown in Ff increases rapidly at a large distance in the case of high Fn. For the multilayer graphene  between graphene layers. The Be between friction interfaces was calculated, as shown in Be with the upper and lower DLC substrates which makes the graphene move synchronously with the same-side substrate. The weak interlayer Be between graphene leads to the relative movement between the overlapped graphene layers, as shown in Be, which leads to the reduction in friction lubricated by multilayer graphene during the friction simulations. Meanwhile, the low Be between multilayer graphene ensures that the structure is more difficult to rupture [Fn [When the monolayer graphene is present  of C atoms in the graphene layers shown in Ff (Nb) between the graphene layer and the DLC film;, the simultaneously increase in Nb with Be is because of the dangling bonds produced by the bond breaking in the graphene layer and which form the C–C covalent bonds with the C atoms in DLC film across the contact interface. Meanwhile, the large amounts of Nb causes a sufficient adhesion force and thus lead to a remarkable increase in sliding resistant force. This result demonstrates that the good lubrication performance of graphene in low Fn disappeared in high Fn and this is ascribed to the rupture of graphene.In the simulation, it is observed that the layered structure of graphene is ruptured during the sliding process when wn in Ff c increasMultilayer graphene shows a persistent lubrication performance compared to monolayer graphene, which demonstrates that the multilayer graphene structure has better mechanical properties during sliding. In order to figure out the rupture process of the graphene layer, the normal stress state of C atoms in the graphene layer on the sliding plane was calculated during sliding. The local structural evolution of the graphene layer is shown in x–y plane normal stress distribution of C atoms when Fn = 10 GPa. For Ng = 2, we snapshot a piece of graphene with a monolayer structure as a reference, and snapshot a part of the graphene overlapped with multilayer structure in the case of Ng ≥ 4. It can be observed that there are more C atoms subjected to high tensile stress in the monolayer graphene. The distribution of normal stress of C atoms is almost identical for the multilayer structure between Ng = 4 and Ng = 6; most of the C atoms in the graphene layer are subjected to compressive stress and only few atoms are under tensile stress. To quantify this, we plotted the histogram of normal stress of C atoms with different amounts of graphene. The histogram has larger quantity at low-magnitude normal stress and lower quantity at high-magnitude normal stress. It should be noted that the highest frequency of the high-magnitude tensile stress range is in the case of Ng = 2, while the lowest frequency is in the case of Ng = 6. In both cases of Ng = 4 and Ng = 6, the distribution frequency of atomic normal stress is almost identical. This is because all graphene exist in lubricant as overlapping-layer structure in the two cases. The above phenomenon is consistent with the normal stress distribution of C atoms with different layered structure of the graphene, as shown in To further quantitatively investigate the effect of layer structure on rupture behavior of graphene, we performed normal stress sites analysis for different layered graphene structures. Fn and ionic liquids amounts (Ni) on the performance of IL-based lubricants by employing constant graphene amounts (Ng = 4). In the case of small amounts of IL (Ni ≤ 20), the Ff remains at a small value after sliding which is attributed to the separation effect of IL-based lubricants on the DLC friction pairs and low interfacial adhesion between the friction interface. Ni = 10, 20 and 40, we define them as “insufficient”, “appropriate” and “excessive” amounts, respectively. Different Ni represent different thicknesses of the lubricating film. When the Ni ≤ 20 and Fn = 10 GPa, the IL-based lubricants show more persistent lubrication performance with the amounts of IL increasing from insufficient to appropriate; this is because the increase in Ni enhance the dispersion of graphene. On the other hand, in the case of excessive amounts of ILs (Ni = 40), the Ff increases rapidly after sliding a short distance and the increasing rate of Ff is more obvious under higher Fn. The high Ff demonstrates that excessive amounts of ILs may result in the immediate invalidation of the performance of IL-based lubricants.For the three lubrication conditions of Ff at high graphene concentration in our research.In previous studies, the decrease in fluidity of graphene caused by sheets aggregation was used to interpret the poor lubrication at high graphene concentration . In ordeFf, as shown in Ni = 10. This loose configuration of lubrication film comprises most of the load acting on the lubricating film loaded by the graphene layer which make the stress on the graphene greater than the theoretical value (Fn = 10 GPa). However, for Ni = 20, this IL-based lubricating film is dense during sliding simulation which makes the stress on graphene closer to the theoretical value. The average normal stress (Sn) value of the C atom in the graphene layer on the z-direction was shown in Fn, the Sn of graphene decreases with the increase in Ni in the sliding process. The increased amounts of IL is responsible for the normal stress on the graphene, which guarantees the stability of the graphene structure.In this work, the normal stress state of C atoms in the graphene was calculated to explain this high Ni = 40), the Ff increases sharply in the initial stage of sliding. Pe) of graphene starts to increase remarkably in the initial stage of sliding. The increase in the Pe of graphene is due to the transition of C atoms from the sp2 hybridized state to sp3 hybridized state which indicates that the regular hexagonal structure of graphene has been ruptured during sliding. And the process of graphene structural rupture is shortened with the increase in stress. Ff. To investigate the mechanism of the invalidation of lubricating performance caused by excess ILs, the stress state of graphene during sliding was calculated, as shown in In the case of excess amounts of IL , the upper and lower surfaces of the DLC can contact directly and form a large number of covalent bonds, as shown in f. Hence, in the case of different roughness, the number of C–C covalent bonds between lubrication interfaces is the leading factor affecting the different Ff.The effect of substrate surface roughness on the lubrication performance of IL-based lubricants were studied under the condition of constant amounts of ILs and graphene layers, as shown in Fn and forms a covalent bond with the substrate which makes graphene have a great adhesion to the substrate, while there is almost no covalent bonds between graphene layers due to the strong van der Waals force, as shown in Ff at a relatively small value during sliding. Ff is still higher in the case of surface roughness than in the case of smooth plane; it indicates that the separation of the graphene layers to DLC pairs and the enhancement of the diffusion performance of compound lubricant weaken the Ff to some extent, but the bonding between DLC substrates still plays a dominant role.The surface roughness will also affect the fluidity of lubricants. The MSD of graphene increases obviously when the surface changes from smooth to roughness, as shown in Vy) on the lubrication performance of IL-based lubricants was studied under the condition of constant Ni and graphene layers, as shown in Ff has a short process of rising and falling at the beginning of sliding which corresponds to the activation of lubricants flow under shear stress, and the amplitude increases with the Vy. At a low sliding speed (Vy = 1 Å/ps), IL-based lubricants can maintain more persistent lubrication performance. The analysis of the bonding between graphene and DLC shows that the amounts of covalent bonds between the interfaces increases significantly with the sliding velocity. This is because high sliding velocity induces the formation of wrinkles in the graphene, which can significantly drop the load-carrying capacity  by almost an order of magnitude [Vy.The effect of sliding velocity (agnitude . TherefoFf in high sliding velocity (Vy ≥ 1.5 Å/ps), the friction results show that the increase in velocity reduces the Ff in the stable stage, which can be explained by the high friction temperature caused by high speed. The failure of lubricant leads to the direct formation of C–C covalent bonds between the upper and lower DLC films and results in mechanical mixing at the contact interface. In this condition, the Ff depends on the composition and structure of the mixing interface. 3C bonding atoms. In the early stage of friction, the decrease in fsp3 corresponds to the sp3–sp2 transitions (graphitization) in the DLC films which is induced by the high temperature in the friction region, as shown in fsp3 is because the sp2-bonded C atoms in graphene bonded with C atoms in DLC films to form the sp3-bonded C atoms during the process of structural damage of the graphene. After the stabilization of fsp3, the fps3 in the case of Vy = 2 Å/ps is lower than that of Vy = 1.5 Å/ps. This is because the high temperature in the friction region caused by high Vy promotes the graphitization of C atoms at the friction interface. It has been demonstrated by many previous studies that high sp2C content can lead to smaller Ff. The graphitization caused by high temperature results in the easy-shear properties and loss of the structural mechanical strength of DLC friction interfaces [Ff in the stable stage.After the stabilization of terfaces , and thuVy, which makes it easier for the graphene to promote the common sliding of IL molecules along the sliding direction, thus enhancing the fluidity of the lubricants. In the initial stage of friction (Time < 1 ps), the flow of lubricant is activated due to the shear stress of the upper DLC film, as shown in Ff to activate the lubricant films at the initial stage of friction. As the velocity of the lubricant molecules reaches a stable level, the fF decreases to a stable value.The increase in speed enhances the fluidity of the IL-based lubricants, as shown in Fn, the lubricating performance of the graphene-added lubricants is superior to that of pure IL lubricants, which is due to the fact that intact graphene prevents direct contact of the friction pairs interface. As the Fn increases, graphene is subjected to high stress to make its structure unstable and C atoms with high chemical activity appear in the graphene. These atoms form covalent bonds with the C atoms in the DLC to rupture the 2D structure of graphene and cause an increase in Ff. The simulation experiments show that the lubricant is ineffective due to the structural destruction of the graphene when Ff is large enough, as evidenced by the comparison of tribological properties with pure ILs at high Fn.Graphene is an excellent IL lubricant additive owing to its high load-carrying capacity and its ability to separate the surface of the friction pairs. At low Fn [Previous experiments have shown that the worse tribological performance of pure ILs lubricants is due to the poor load-carrying capacity in high Fn ,45. For Previous studies have shown that the performance of graphene as additives for IL lubricates is connected with the concentration and the presence of the layered structure of graphene. He et al. found that low concentrations of graphene oxide sheets as additives for lubricants has poor lubricating effects . Saurin Ff, which was ensured by the synergistic effect of graphene and ionic liquids. On one hand, previous research have shown that graphene is easily torn by the frictional substrate [Under all the conditions given in this study, the addition of graphene resulted in a significant reduction in ubstrate , and theubstrate . Graphen(1)Graphene as lubricant additives can improve the lubrication performance of ILs at all given conditions. The reduction reach up to 88%. The improved tribological properties of composite lubricants are attributed to the fact that intact graphene prevents direct contact of the friction pairs interface, but fail under high normal stress due to structure rupture.(2)Multilayer graphene has better friction-reducing performance and better friction durability compared to monolayer structure. The antifriction performance is ascribed to the easy-shear property between the multilayer graphene layers with super-low binding energy, which is 5% of the binding energy between the DLC and the graphene. The friction durability is due to the decrease in the percentage of high tensile stress sites which ensures that the multilayer graphene has a more stable structure in lubrication.(3)Ni. Void space occurring under insufficient Ni in lubrication film makes the Fn acting on graphene higher than the theoretical value, which then makes graphene prone to rupturing under the same theoretical pressure. Over-thick IL film and graphene form a three-body abrasive wear structure with stress concentration in the graphene, and eventually lead to degradation of tribological performance of the compound lubricant.The lubricating performance of the compound lubricating oil is sensitive to (4)Both the rough surface of the substrate and the high sliding speed affects the lubricating properties of the lubricant, which is caused by the excessively concentrated stress in the graphene plane, promoting the bond formations between graphene and DLC substrates. In addition, it is found that graphitization caused by high temperature effectively reduces friction in the stable stage in high speed.(5)The results of this study explain the basic friction mechanism of graphene additives in ionic liquid lubricants, and provide a reference for the development of new fluid lubricants by adjusting the content of graphene, normal stress and the thickness of ionic liquid lubrication film to meet potential applications.In this paper, an atomic configuration comprising two DLC substrates and IL lubricants with graphene additives was constructed to evaluate the tribological behavior of the lubricants using MD simulation. Through the systematical analysis of the evolution of friction interface, dynamic trajectory of the lubricant, and stress state of the additives and friction property, we arrived at the the following conclusions:


A reconfigurable passive device that can manipulate its resonant frequency by controlling its quantum capacitance value without requiring complicated equipment has been experimentally investigated by modifying the Fermi level of large-area graphene using an external electric field. When the total capacitance change, caused by the gate bias in the passive graphene device, was increased to 60% compared to the initial state, a 6% shift in the resonant frequency could be achieved. While the signal characteristics of the graphene antenna are somewhat inferior compared to the conventional metal antenna, simplifying the device structure allowed reconfigurable characteristics to be implemented by using only the gate bias change. Graphene is a monolayer material but acts as an interface for bonding with other materials. Consequently, the primary area of study regarding the constant voltage condition has been the analysis of the resistance change of a device due to the interface charging phenomenon. For radio frequency (RF) applications, the inductance and capacitance of the device greatly affect the operating characteristics. Moreover, unlike metal, most signals applied from the outside would be transmitted, rather than absorbed or reflected, owing to graphene’s thinness  and possibly being due to the frequency dependence of skin depth. This suggests that the material with a thickness in nm is more reactive in the THz region ,2,3.Early studies regarding the RF characteristics of graphene devices primarily focused on applied research, using a high cutoff frequency and the frequency-doubling effect in field-effect transistor (FET)-type active devices ,5,6,7,8.In this work, two dipole antennas integrated on the same plane were fabricated using a four-inch wafer-scale graphene sheet. When graphene was used as an antenna, a 2% level of resonance-frequency modulation was observed, according to the 10% change in capacitance. In addition, when the graphene was used as a ground plane, a 6% level of resonance-frequency modulation was successfully achieved due to the 60% capacitance change achieved via the quantum capacitance of the graphene sheet. For reconfigurable passive devices requiring resonant frequency control ,25, a re2 (300 nm)/Si substrate using a reactive ion-etching process. Subsequently, a 50 nm Pt electrode was deposited inside the trench by using an E-beam evaporation system to form a buried-gate electrode with significant advantages over the other structures in terms of the uniformity of the electric field applied to the channel region. To achieve planarization of the gate electrode, using chemical and mechanical polishing processes, 20 nm of Al2O3 was deposited using an atomic layer deposition (ALD) process and a 300 °C vacuum-annealing process for 1 h. Subsequently, the 4-inch-scale CVD graphene sheets  were transferred using a vacuum-transfer method to ensure a stable and consistent device operation, considering that this method can reduce impurities, such as oxygen and moisture, between the graphene and the substrate [2 plasma, the graphene area was patterned via a photolithography process using a metal hard mask (Au). Finally, the top electrode (Au) was formed using an E-beam evaporation process and wet etching.To create the graphene-antenna device, a 50 nm oxide trench pattern was formed on a SiOubstrate . To prev2O3 gate dielectric was 20 nm (dielectric constant = 6). The antenna itself was simply designed to operate at approximately 660 GHz, with resonance at 10–20 GHz. The ground plane was made of metal and platinum (Pt) to control the Fermi level of the graphene. The spacing between the transmitter and the receiver was alternated between 10 and 150 μm to observe the changes in signal-transmission characteristics. The Fermi level of the graphene could be controlled using the bias applied to the Vg contact shown in A passive device with a transmitter and receiver of two basic dipole antennas integrated on the same plane was fabricated to study the reconfigurable passive device using graphene. Device 1 consisted of 100 μm pitch ground–signal–ground (GSG) pads, as shown in d–Vg (drain current–gate voltage). In a passive device, such as Device 1, a DC current does not flow. Hence, the gate voltage value for accurate Dirac point setting was unknown during the RF measurements. Moreover, Dirac point analysis in graphene devices is possible using C–V measurements instead of Id–Vg measurements. However, reconfiguring the four electrodes into one electrode for correct C–V measurements would be necessary, as two dipole antennas were patterned with graphene during the formation of Device 1. A capacitance measurement was carried out using the top electrode and buried electrode on the same substrate of the antenna device. Consequently, the Fermi level change in the graphene at the S-parameter measurement was set to a point where the Dirac point appeared at a gate voltage of 0 V. In this case, the estimated carrier density existing at the charge neutrality point was 3.1 × 1011cm−2. This value is very important because the quantum capacitance of graphene is generally more clearly observed when it has a lower carrier density than the classical capacitance of the device, and this is the ultimate reason for the tunability of the resonance frequency in these devices [Its characteristics in the RF domain were analyzed using a network analyzer (Agilent 8510C) and a two-port probe system in the 1–40 GHz range. The S-parameters, reflections , and transmissions  were measured using a network analyzer. A constant voltage applied to the ground plane modulated the Fermi level of the graphene. Then, the S-parameters were measured as a function of the Fermi level of the graphene. First, we observed the appearance of signal reflections and transmissions when the spacing between the three antennas physically changed. The Fermi level of the graphene for the graphene antenna was assumed to be at the Dirac point. The observation of the Dirac point, based on the doping condition of graphene devices, can generally be obtained by measuring the I devices .In the case of S12, as shown in The S-parameter measurements of the RF operating characteristics of Device 2 did not exhibit resonance at a specific frequency as they did in Device 1 or in the reference device. For Device 2, analysis was conducted based on the characteristic changes at 4.5 GHz. When the distance between the metal antennas was increased in Device 2, a change in the S-parameter value over the entire frequency range, rather than a resonant frequency shift, was observed. In particular, S12 or S21 showed signal transmission attenuations of more than 15 dB when the distance was increased. Simultaneously, in S11 or S22, the reflection amount increased as the distance between the antennas increased.Next, the RF characteristics were investigated while applying the external bias to the graphene to modulate the Fermi level of the graphene. Using a gate electrode as a ground plane, measurements were made at −5, −3, −1, 0, 1, 3, and 5 V; different polarities were applied to observe the changes in the graphene carrier types in the graphene layer.g increased, the resonant frequency shifted towards the lower frequencies. The frequency shift range was approximately 150 MHz. The change in characteristics due to the Vg was shown in that the degree of the shifting of the resonant frequency was proportional to the absolute value, regardless of the polarity of the Vg, i.e., when the Fermi level of graphene changed via the gate bias, the carrier type and carrier concentration changed based on this value and the polarity of the gate bias. However, as seen from the Id–Vg measurements of the graphene device, the same conductance could be seen even if the absolute value of the gate bias was the same, regardless of the carrier type. Therefore, as shown in As resonance was most apparent when the antenna spacing was at 100 μm, the characteristics based on the gate bias change were observed in the device at that spacing. The results, based on changing the gate bias, did not differ much compared to those obtained when the antenna spacing was changed, as shown in g increased, the signal transmission improved by 3 dB and the reflectance reduced by 1 dB, as shown in Although Device 1 and the graphene RF device exhibited similar resonance characteristics to those of the reference passive device under its operating conditions, their transmittance values were low and their reflectance values were very high, possibly due to the significantly high contact resistance between the graphene and the graphene–metal contact. Therefore, improving the structure of the passive device would be necessary to secure its basic performance while facilitating the movement of resonant frequency by controlling the quantum capacitance of the graphene. The changes in the resonant frequency and measured S-parameter values due to changes in the Fermi levels of graphene, used as a ground plane by the gate bias, were more apparent than those in Device 1. As the VUnder the same conditions, the measured values of Device 1, Device 2, and the reference device were compared to analyze the cause of different signal transmissions and reflection characteristics, as shown in Conversely, in the case of signal transmission , the impedance value of the passive device itself and the space for transmitting the RF signal were important . The RF g was compared, as shown in g could be observed. However, a change in resonant frequency could be seen when the graphene was used. For S11, when Device 1 was exposed to a larger Vg value, an approximately 100 MHz shift towards the lower frequency was observed. However, for Device 2, the resonant frequency increased by approximately 200 MHz. The analysis results of g occurring approximately 1.5 times more, i.e., Device 1 and Device 2 exhibited a resonant frequency change of approximately 200 MHz and 300 MHz, respectively.Consequently, the degree of resonant frequency variation regarding the reflectance and transmittance due to the VIncreasing the carrier concentration of graphene in passive devices improved the RF signal transmission, and the quantum capacitance changes induced a change in the resonant frequency. The capacitance value of each passive device was measured to examine the correlation between the S-parameter characteristics and the capacitance changes in the passive device, as shown in g = 0 V. As shown in In this experiment, we changed the resonant frequency in a passive device using graphene. The relationship between the rate of resonant frequency shift and the rate of capacitance change was investigated based on Vg. When graphene was used as an antenna, characteristics similar to those of a passive device using a conventional metal film were observed, suggesting that graphene could be applied as a passive device based on the patterning. However, when a graphene ground plane was used, small-signal RF transmission was compromised and an accurate passive device operation could not be performed. Nonetheless, changes in quantum capacitance and carrier concentration due to graphene Fermi level changes affected the resonant frequency control and signal propagation. For reconfigurable passive devices requiring resonant frequency control, a resonant frequency change rate corresponding to 2.5~9.6% of the capacitance change rate in the passive graphene device was possible. Although these changes are insufficient for the practical application of graphene in reconfigurable passive devices, they indicate the potential to explore and positively contribute to their development.To design a reconfigurable passive device using graphene, we fabricated integrated passive devices with two dipole antennas and analyzed their RF characteristics, aiming to analyze the use of graphene in passive devices and the amount of resonant frequency change possible based on the changes in V


TARDBP, the gene encoding TDP-43, can cause ALS and cluster in the C-terminal prion-like domain (PrLD), where they modulate the liquid condensation and aggregation properties of the protein. TDP-43-positive inclusions are also found in rimmed vacuole myopathies, including sporadic inclusion body myositis, but myopathy-causing TDP-43 variants have not been reported. Using genome-wide linkage analysis and whole exome sequencing in an extended five-generation family with an autosomal dominant rimmed vacuole myopathy, we identified a conclusively linked frameshift mutation in TDP-43 producing a C-terminally altered PrLD (TDP-43p.Trp385IlefsTer10) (maximum multipoint LOD-score 3.61). Patient-derived muscle biopsies showed TDP-43-positive sarcoplasmic inclusions, accumulation of autophagosomes and transcriptomes with abnormally spliced sarcomeric genes (including TTN and NEB) and increased expression of muscle regeneration genes. In vitro phase separation assays demonstrated that TDP-43Trp385IlefsTer10 does not form liquid-like condensates and readily forms solid-like fibrils indicating increased aggregation propensity compared to wild-type TDP-43. In Drosophila TDP-43p.Trp385IlefsTer10 behaved as a partial loss-of-function allele as it was able to rescue the TBPH (fly ortholog of TARDBP) neurodevelopmental lethal null phenotype while showing strongly reduced toxic gain-of-function properties upon overexpression. Accordingly, TDP-43p.Trp385IlefsTer10 showed reduced toxicity in a primary rat neuron disease model. Together, these genetic, pathological, in vitro and in vivo results demonstrate that TDP-43p.Trp385IlefsTer10 is an aggregation-prone partial loss-of-function variant that causes autosomal dominant vacuolar myopathy but not ALS/FTD. Our study genetically links TDP-43 proteinopathy to myodegeneration, and reveals a tissue-specific role of the PrLD in directing pathology.Neuronal TDP-43-positive inclusions are neuropathological hallmark lesions in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Pathogenic missense variants in The online version contains supplementary material available at 10.1007/s00401-023-02565-1. TARDBP, is an evolutionary highly conserved and ubiquitously expressed RNA- and DNA-binding protein . It is iewed in , 44. Theiewed in .TARDBP , even being at equal levels compared to control muscle tissue. These data suggest that accumulations in TDP-43p.Trp385IlefsTer10 muscle tissue are primarily composed of mutant protein, while pTDP-43[Ser409/410]—originating from the wild-type allele—remains at baseline levels in a soluble state.While neuropathological and genetic studies have established the crucial involvement of TDP-43 proteinopathy in the pathogenesis of ALS/FTD and related neurodegenerative disorders , increasopathies . Of intep.Trp385IlefsTer10 clearly differs from the ALS-causing missense variants which are present in an otherwise normal PrLD. Indeed, a major and unexpected finding in this study was the mainly chronic and slowly progressive myopathic clinical manifestation associated to TDP-43p.Trp385IlefsTer10. Although milder neurogenic signs were also present in some patients, ALS and/or FTD phenotypes were never observed. The specific nature of our truncating variant therefore likely holds the key as to how TDP-43p.Trp385IlefsTer10 leads to a muscular rather than a neurodegenerative condition. Interestingly, recent reports have linked a similar truncating TDP-43 variant, TDP-43p.Y374Ter, to a typical ALS phenotype  phosphorylated TDP-43 are maintained in muscle tissue, apparently at the expense of unmodified full-length WT TDP-43 from the normal allele, which could further contribute to a depletion and loss-of-normal function of WT TDP-43. Remarkably, this phenomenon was not observed in fibroblasts, reinforcing the idea that TDP-43 undergoes muscle-specific processing and modification including phosphorylation, thereby providing a possible explanation of the myopathy phenotype associated with TDP-43p.Trp385IlefsTer10. Further evidence for a tissue-specific function of TDP-43 was recently obtained in mouse muscle and neuronal cells showing cell-type-specific differences in TDP-43-mediated pre-mRNA processing [Despite our functional studies suggesting that TDP-43ocessing .p.Trp385IlefsTer10, our transcriptomic analysis of TDP-43p.Trp385IlefsTer10 muscle tissue provided evidence for a nuclear loss-of-function. Through analysis of alternative pre-mRNA processing patterns, a nuclear process TDP-43 is tightly involved in, we identified a number of transcripts affected by AS. Interestingly, the great majority of these AS targets corresponded to muscle-specific structural/contraction-associated proteins. Indeed, 5 out of the top 10 transcripts with the most AS events corresponded to sarcomeric genes, including TTN and NEB, indicating that abnormal nuclear pre-mRNA splicing of functionally important sarcomeric genes could contribute to the pathogenic mechanism of the myopathy in our family.In line with the proposed partial loss-of-function nature of TDP-43TTN and NEB, within sarcoplasmic myo-granules during muscle development/regeneration [p.Trp385IlefsTer10, the attempted and failed regeneration by the muscle fibres would result in an upregulation of myogenic pathways and certain sarcomeric mRNAs, which is supported by the observed upregulation of developmental myosin isoforms [p.Trp385IlefsTer10 resulting in autophagic myodegeneration over time [In mice, TDP-43 has been shown to associate with sarcomeric mRNAs, such as neration , and is neration  showed tisoforms . Furtherver time .Finally, this finding adds TDP-43 to the growing list of RNA-binding proteins as genetic causes of myopathies. Interestingly, similar to TDP-43, frameshift mutations in the PrLDs of HNRNPA2B1 and HNRNPA1 that change the prion-like behaviour of the mutant proteins have been described recently as the genetic cause of myopathies , 29, whep.Trp385IlefsTer10 is in early stages, the findings we report here will help to understand a muscle-specific function for TDP-43 and how it can be related with the development of myopathies. Furthermore, our work points towards a broadening of the disease spectrum of TDP-43 proteinopathies, expanding from pure neurodegenerative to muscular disorders.Although work on TDP-43p.Trp385ilefsTer10 variant was identified. Individuals identified with “m/-“ are affected individuals that have either been genotyped or identified through reconstruction of the pedigree. Individuals that were genotyped are identified with an asterisk(*). Individuals in white are untested but assumed to be unaffected.Figure S2. TEM images and haplotype analysis. a) TEM images of different features of the patient skeletal muscle: i. Intact motoric end-plate without any signs of degeneration; ii. Necrotic muscle fibre with apoptotic bodies (arrows) and abnormal mitochondria (arrowheads); iii. Filamentous material found within the nuclei of the muscular sarcolemma. b) TEM of patient-derived cultured myocytes. i. Presence of multiple vacuolar bodies (arrowheads) and enlarged autophagic compartments (arrow); ii. Close-up of a “zebra” pattern autophagosome containing electron-dense material and “fibre-like” material. c) Haplotype analysis of the members of the family in which the TDP-43p.Trp385IlefsTer10 variant was identified . The 4.7 Mb genomic region shared between all affected individuals (rs10864439-rs10927466) is shown in the red box. The disease haplotype is marked as number 1 (highlighted red). Figure S3. Genome-wide multipoint LOD-scores.Figure S4. IGV view of RNA-seq data in patient muscle tissue. RNA-seq-derived bridged reads spanning the 11 bp deletion in the TARDBP gene on patient muscle samples (red box).Figure S5. Automated microscopy and survival analysis. Rodent primary neurons were dissected, cultured, and transfected with plasmids encoding fluorescent proteins. The cells were then imaged using an automated epifluorescence microscopy platform allowing repeated and chronological visualization of individuals neurons. After 10d of imaging, neurons were identified by customized scripts in python and Fiji (green outline), assigned a unique number (yellow), and their time of death determined based upon the loss of fluorescence, soma rounding, neurite retraction, and/or cellular blebbing (red outline and number). (PDF 28047 kb)Supplementary file1 Figure S1. Expanded pedigree of the TDP-43p.Trp385IlefsTer10 family. Expanded pedigree of the family in which the TDP-43Below is the link to the electronic supplementary material.


To address this limitation, this study introduces a modified transfer process that utilizes a temporary UV-treated SiO2 substrate to adsorb impurities from graphene before transferring it onto the final substrate. Optical microscopy and Raman mapping confirmed the adhesion of impurities to the temporary substrate, leading to a clean graphene/substrate interface. The retransferred graphene shows a reduction in electron–hole asymmetry and sheet resistance compared to conventionally transferred graphene, as confirmed by the transmission line model (TLM) and Hall effect measurements (HEMs). These results indicate that only the substrate effects remain in action in the retransferred graphene, and most of the effects of the impurities are eliminated. Overall, the modified transfer process is a promising method for obtaining high-quality graphene suitable for industrial-scale utilization in electronic devices.Graphene has immense potential as a material for electronic devices owing to its unique electrical properties. However, large-area graphene produced by chemical vapor deposition (CVD) must be transferred from the as-grown copper substrate to an arbitrary substrate for device fabrication. The conventional wet transfer technique, which uses FeCl However, these processes introduce two types of impurities into the graphene layer: polymer residues on the graphene surface and interfacial impurities [The integration of graphene into optoelectronic applications requires a large, homogeneous graphene sheet that is typically produced by chemical vapor deposition (CVD) on metal catalysts such as Cu or Ni, producing diverse outcomes from monolayer to multilayer graphene ,2,3. Forpurities ,7,8,9,10purities .3 as a metal etchant can lead to the formation of complex nanoparticles at the interface between graphene and the substrate. Kraus et al. reported that FeCl3 etching of copper resulted in cubic Fe0.942O nanoparticles under graphene, indicating the presence of uncontrolled phases of iron oxides such as FeO, Fe2O3, Fe3O4, etc. These iron oxides can exhibit n- or p-type behaviors depending on the phase species [The use of FeCl species ,13,14. S species , mechani species , and RCA species . However species .2 substrate to achieve high efficiency in eliminating impurities from the graphene/substrate interlayer. In this technique, the graphene layer is first transferred onto a UV-irradiated hydrophilic substrate, allowing water to intercalate at the interface between the graphene and the substrate. In this process, interfacial impurities are retained on the temporary substrate, and the cleaned graphene can be retransferred to a target substrate without physical damage. Using optical microscopy and Raman spectroscopy, we confirmed that all interfacial impurities in the retransferred graphene were eliminated. Furthermore, we demonstrated significant improvements in electrical measurements using the transmission line model (TLM) and Hall effect measurements (HEMs), such as a reduction in sheet resistance and electron–hole asymmetry in the retransferred graphene. We suggest that this modified wet transfer method can be implemented as an efficient method for obtaining a clean interface during the transfer of all thin films and 2D materials, as well as for devices that are sensitive to interfacial properties [In this study, we introduce a modified wet transfer method that uses a temporary SiOoperties ,20,21,22TM) in a tube-type CVD. The Cu foil was placed inside the CVD chamber and annealed for 1 h with 200 sccm of flowing argon gas, raising the chamber temperature to 980 °C. Subsequently, 80 sccm of hydrogen gas was introduced, and the sample was annealed at the remaining temperature for 1 h. Following annealing, 1.5 sccm of methane gas was introduced to grow graphene for 2 h, resulting in continuous single-layer graphene. Following the growth process, the graphene was rapidly cooled by moving the furnace to the cooling zone.Graphene was grown on commercial copper foils /Si substrates were prepared using various surface treatments, including blowing N2 gas, sonication in deionized (DI) water (40 min), sonication in acetone (20 min) and isopropyl alcohol (20 min), UV treatment (60 s), and annealing at 120 °C for 30 min in high vacuum, to optimize the substrate cleaning conditions for electrical property measurements [Thermally oxidized SiOurements .Raman spectroscopy was performed using a confocal spectrometer  at the CORE Facility Center for Quantum Characterization/Analysis of Two-Dimensional Materials and Heterostructures. The measurements were recorded with a 532 nm laser with 5 mW power and a 100× objective, resulting in a laser spot size of approximately 2 µm.2/Si substrates with pre-deposited Cr/Au (5 nm/30 nm) electrodes. The electrode and graphene channels were defined using laser lithography  with KL5305  as the photoresist and PMMA 950 A6  as the buffer layer.Using the conventional wet transfer method and our modified retransfer method, graphene was transferred onto SiO2 plasma was performed. The Cu foil was etched by floating it on an iron chloride (FeCl3) solution followed by rinsing it with DI water. The general wet transfer process typically involves transferring the PMMA/graphene structure to the final target substrate and removing the PMMA after drying. However, we modified the process by transferring the PMMA/graphene structure onto a temporary SiO2 substrate that had been treated with UV light. Several treatments were tested to determine the highest hydrophilicity for the temporary substrate, among which UV treatment was the most efficient  to the 2D and G peaks (I2D/IG). Compared with the optical microscopy results, the local impurity points and line-shaped graphene folds have high intensities in the ID/IG mapping but low intensities in the I2D/IG mapping. Generally, measuring Raman spectra of graphene on a metal substrate is the most challenging task because the scattering of the metal surface hinders the identification of the distinct peak information of graphene. If graphene is transferred onto a partially metal-containing substrate, that is, onto metal nanoparticles, the background signal increases and shades the graphene spectrum [D/IG and I2D/IG, instead of ID and I2D alone. In general, ID (~1350 cm−1) indicates a defective non-sp2 domain, and some iron oxide phases exhibit a distinct peak near ~1320 cm−1 [D/IG can be used as an index that shows the position of the residual impurities and their structural effects on graphene. I2D/IG is used as an index not only for confirming that the graphene is single-layer, but also for determining the amount of p-doping owing to the increase in the G-peak and decrease in the 2D-peak on p-doped graphene [To check for the invisible quality, we performed Raman analysis of the transferred and retransferred graphene. Raman analysis was conducted by mapping an area of 50 × 50 μmspectrum ,29. To c320 cm−1 . Therefographene .Si) to 1, we could observe changes in both the Raman intensity and intensity ratios. Interestingly, in contrast to the D-peak intensity, which increased due to local defects, the G- and 2D-peak intensities also increased as a result of the impurities. This is explained in We compared the positions, distributions, and electrical effects of the impurities between the two methods. Three points were selected for each Raman mapping result. In the transfer result a, the im−1, 2676.7 cm−1) [To quantitatively analyze the improvement in strain and doping, we used a vector decomposition method based on the quasi-linear change in phonon energy with strain and doping . In this.7 cm−1) . The OX→To determine the strain and doping, we used the Jacobian transformation (Equation (1)) .(1)OX→=D/IG analysis, and are visible in the tensile strain analysis, exhibit n-type doping characteristics, indicating electron donation. This may be attributed to the n-type behavior of certain iron oxide phases [Using Equation (1), we reconstructed Raman mapping images of the disentangled strain and doping of the transferred and retransferred graphene b. Interee phases . Overall2 substrate. Laser lithography was used with a photoresist (PR) and PMMA heterostructure to pattern the electrodes and etch graphene to define the channel dimensions. In general, e-beam lithography is preferred for graphene device fabrication because of its many advantages in fabricating submicropatterns and effective elimination of the PMMA layer. Although laser lithography is faster for fabricating large-scale patterns, an effective cleaning method for PR residues on graphene has not yet been developed. Therefore, we spin-coated PMMA onto the graphene surface and sequentially deposited PR. After developing the desired pattern on the PR, the PMMA and underlying graphene layers were etched using O2 plasma. Subsequently, the remaining PR/PMMA layers were removed using acetone/IPA solvent.The electrical properties of graphene were investigated to confirm the effect of the retransfer process using transmission line model (TLM) devices with a width of 50 μm and a total length of 800 μm. Larger channel dimensions were chosen to avoid sample-to-sample variations due to the impurity size, and the distances between the impurities were at the micro and sub-micro scales. As shown in D-VG curves for the transferred and retransferred graphene devices and their respective channel lengths are presented in e/μh) of transferred graphene FET, which averages 0.76 from 10 devices. In D-VG curve for the retransferred graphene, which showed relatively stable Dirac voltages and drain current levels. This suggests that the interfacial doping effects were significantly reduced, and only the SiO2 substrate effects remained active [2 tends to undergo p-doping. For the retransferred graphene, the ratio between electron and hole mobilities (μe/μh) averaged 0.85.The Id active . GeneralD-VD measurements were performed with VD = 10 mV. The resulting sheet resistance (RS) is shown as a function of channel length in Total = 2Rc + Rs L/W was used to fit the data, where RC is the contact resistance, L is the length, and W is the channel width. We also examined these effects using Hall effect measurements with the van der Pauw method to investigate the electrical properties of the graphene sheet, such as the RS, carrier concentration, and Hall mobility [D/dVG) (Lch/WchVDCox) [2 capacitance is Cox = 11.51 × 10−9 F/cm2. The black and red circles indicate the spread areas of the hole mobilities versus the Dirac voltages for the transferred and retransferred devices, respectively. Comparing the circles, the retransferred sample exhibits a larger improvement than the transferred graphene. We found that the impurities in the graphene–substrate interface consist of p- and n-type dopants with random distributions, resulting in wide spreads in the Dirac voltage and carrier mobilities of the transferred graphene.To examine the changes in the conductance and scattering effect of the TLM devices, Imobility . (See SuchVDCox) , where S2 substrate and UV treatment, our retransfer process enabled the removal of metallic impurities and reduced the interfacial doping effects in graphene. As a result, the retransferred graphene showed significantly improved electrical properties, including reduced sheet resistance and stable electron–hole asymmetry distributions. These findings provide valuable insights for the development of high-performance graphene-based electronic devices, highlighting the importance of interfacial purity in achieving optimal device performance.Our study demonstrated the effectiveness of a modified wet transfer technique for eliminating interfacial impurities in CVD-grown graphene. Using a temporary SiO


Amyotrophic lateral sclerosis (ALS) associated with TAR DNA-binding protein 43 (TDP-43) aggregation has been considered as a lethal and progressive motor neuron disease. Recent studies have shown that both C-terminal TDP-43 (C-TDP-43) aggregates and oligomers were neurotoxic and pathologic agents in ALS and frontotemporal lobar degeneration (FTLD). However, misfolding protein has long been considered as an undruggable target by applying conventional inhibitors, agonists, or antagonists. To provide this unmet medical need, we aim to degrade these misfolding proteins by designing a series of proteolysis targeting chimeras (PROTACs) against C-TDP-43.C. elegans by motility assay and confocal microscopy. The impact of TDP-43 PROTAC on C-TDP-43 oligomeric intermediates was monitored by fluorescence lifetime imaging microscopy and size exclusion chromatography in the Neuro-2a cells co-expressing eGFP-C-TDP-43 and mCherry-C-TDP-43.By applying filter trap assay, western blotting, and microscopy imaging, the degradation efficiency of C-TDP-43 aggregates was studied in Neuro-2a cells overexpressing eGFP-C-TDP-43 or mCherry-C-TDP-43. The cell viability was characterized by alarmarBlue assay. The beneficial and disaggregating effects of TDP-43 PROTAC were examined with the YFP-C-TDP-43 transgenic 2 decreased C-TDP-43 aggregates and relieved C-TDP-43-induced cytotoxicity in Neuro-2a cells without affecting endogenous TDP-43. We showed that PROTAC 2 bound to C-TDP-43 aggregates and E3 ligase to initiate ubiquitination and proteolytic degradation. By applying advanced microscopy, it was further shown that PROTAC 2 decreased the compactness and population of C-TDP-43 oligomers. In addition to cellular model, PROTAC 2 also improved the motility of transgenic C. elegans by reducing the C-TDP-43 aggregates in the nervous system.Four PROTACs with different linker lengths were synthesized and characterized. Among these chimeras, PROTAC 2 against both C-TDP-43 aggregates and oligomers to reduce their neurotoxicity, which shed light on the potential drug development for ALS as well as other neurodegenerative diseases.Our study demonstrated the dual-targeting capacity of the newly-designed PROTAC The online version contains supplementary material available at 10.1186/s12929-023-00921-7. With the increase in the aging population, the misfolded proteinaceous agents associated with neurodegenerative diseases have drawn increasing attention , 2. The Recently, proteolysis targeting chimeras (PROTACs) have received increasing attention due to their potential abilities to induce targeted protein degradation including the neurodegenerative disease associated proteins –17. StruNowadays, PROTACs have been developed to degrade a wide range of proteins, such as androgen receptor , bromodo1–4  also rescued the C-TDP-43-mediated motility defects in nematode C. elegans.To provide a proof-of-concept examination, we developed “TDP-43 PROTAC” as a novel therapeutic strategy for reducing the C-TDP-43 cytotoxicity in ALS and other TDP-43 proteinopathy Fig. A. A TDP-2Cl2) was distilled from CaH2, and tetrahydrofuran (THF) was distilled from sodium. All nonaqueous reactions were performed in oven-dried glassware under a slightly positive pressure of argon unless otherwise noted. Reactions were magnetically stirred and monitored by thin-layer chromatography on silica gel using phosphomolybdic acid (PMA), p-anisaldehyde, KMnO4, ninhydrin or iodine vapor as visualizing agent. Flash chromatography was performed on silica gel of 60–200 μm particle size. Yields are reported for spectroscopically pure compounds. Melting points were recorded on a Yanaco micro apparatus or Electrothermal MEL-TEMP 1101D apparatus in open capillaries and are not corrected. Infrared (IR) spectra were recorded on Thermo Scientific Nicolet is-5 FT-IR spectrometer. Nuclear magnetic resonance (NMR) spectra were obtained on Bruker AVIII 500 (500 MHz) and Bruker AVIII 400 (400 MHz) spectrometers. Chemical shifts are given in δ values relative to tetramethylsilane . Internal standards were CHCl3 (δH = 7.24), CDCl3 , CD2HOD (δH = 3.31), CD3OD (δC = 49.15), (H3C)2SO , or (CHD2)2SO . The splitting patterns are reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (double of doublets), td (triple of doublets) and br (broad). Coupling constants (J) are given in Hz. Electrospray ionization high-resolution mass spectra (ESI-HRMS) were recorded on a Bruker Daltonics BioTOF III high-resolution mass spectrometer. UV–Vis absorption spectra were recorded on a PerkinElmer Lambda 35 spectrometer. Fluorescence spectra were recorded on an AMINCO-Bowman Series 2 luminescence spectrometer.All the reagents and solvents were reagent grade and used as purchased without further purification unless indicated otherwise. All solvents were anhydrous grade unless indicated otherwise. Dichloromethane . All compounds are > 95% pure by HPLC analysis.New compounds were characterized by their physical and spectroscopic properties  in anhydrous acetonitrile (75 mL) at 65 °C for 1.5 h to give 2-bromo-6-methoxybenzo[d]thiazole (5) thiazol-2-yl)-N,N-dimethylaniline (7) thiazol-2-yl)-N,N-dimethylaniline compound (S2a) . C19H21N5O2S; yellow solid; mp 96.0–97.0 °C.To a solution of compound ulfonate   in THF (3 mL) was stirred with PPh3  and H2O  at room temperature for 24 h. The mixture was concentrated under reduced pressure, and purified by flash chromatography on a silica gel column with elution of CH2Cl2/MeOH (10:1) to give the amino compound 9a . C19H23N3O2S; yellow solid; mp 66.5–67.5 °C; TLC (CH2Cl2/MeOH = 7:1) Rf = 0.13; IR νmax (neat) 3358, 3190, 2919, 2849, 1659, 1653, 1634, 1470, 1423, 1264, 828 cm–1; 1H NMR  δ 7.83 , 7.80 , 7.26 , 7.00 , 6.65 , 4.10 , 3.77 , 3.52 , 2.95 , 2.83 , 1.91 ; 13C NMR  δ 166.4, 156.0, 151.7, 148.9, 135.6, 128.3 (2 ×), 122.5, 121.3, 115.2, 111.5 (2 ×), 105.2, 73.3, 69.3, 67.9, 41.5, 39.9 (2 ×). ESI-HRMS calcd for C19H24N3O2S: 358.1584, found: m/z 358.1570 [M + H]+.A solution of azido compound 2O  in AcOH (3 mL) was heated under reflux for 12 h. The mixture was concentrated under reduced pressure, and purified by flash chromatography on a silica gel column with elution of CH2Cl2/MeOH (100:1) to give 2--4-fluoroisoindoline-1,3-dione (10) +.A mixture of compound 1, the substitution reaction of 10  with 9b  gave a crude product, which was purified by flash chromatography on a silica gel column with elution of EtOAc/CH2Cl2 (1:1) to give the desired compound 2 . The purity of compound 2 was 97.0% as shown by HPLC on a silica column , elution: EtOAc/hexane = 3:1 at a flow rate of 3.0 mL/min, tR = 17.9 min. C34H35N5O7S; yellow solid; mp 128.0–129.0 °C; TLC (EtOAc/ CH2Cl2 = 1:1) Rf = 0.5; IR νmax (neat) 3357, 3197, 2920, 2849, 1653, 1632, 1471 cm–1; 1H NMR  δ 8.35 , 7.88 , 7.82 , 7.41 , 7.29 , 7.01–6.99 , 6.84 , 6.71 , 6.44 , 4.85 , 4.15 , 3.86 , 3.78–3.64 , 3.40 , 3.01 , 2.87–2.59 , 2.09–2.00 ; 13C NMR  δ 171.1, 169.2, 168.4, 167.6, 166.6, 156.2, 151.9, 149.0, 146.7, 136.0, 135.6, 132.4, 128.5 (2 ×), 122.6, 121.5, 116.7, 115.4, 111.7 (2 ×), 111.6, 110.2, 105.4, 70.9, 70.7, 69.8, 69.5, 68.1, 48.8, 42.3, 40.14 (2 ×), 31.3, 22.7. ESI-HRMS calcd for C34H36N5O7S: 658.2330, found: m/z 658.2307 [M + H]+.By a procedure similar to that for compound 1, the substitution reaction of 10  with 9c  gave a crude product, which was purified by flash chromatography on a silica gel column with elution of EtOAc/CH2Cl2 (1:1) to give the desired compound 3 . The purity of compound 3 was 95.1% as shown by HPLC on a silica column , elution: EtOAc/hexane = 9:1 at a flow rate of 3.0 mL/min, tR = 15.9 min. C36H39N5O8S; yellow solid; mp 87.5–89.0 °C; TLC (EtOAc/CH2Cl2 = 1:1) Rf = 0.38; IR νmax (neat) 3358, 3197, 2919, 2849, 1661, 1645, 1622, 1471, 1407 cm–1; 1H NMR  δ 8.55 , 7.86 , 7.81 , 7.39 , 7.27 , 7.05–6.96 , 6.82 , 6.68 , 6.42 , 4.86 , 4.13 , 3.84 , 3.72–3.67 , 3.67–3.59 , 3.38 , 2.99 , 2.80–2.60 , 2.04 ; 13C NMR  δ 171.2, 169.2, 168.5, 167.5, 166.5, 156.2, 151.9, 148.6, 146.7, 135.9, 135.4, 132.4, 128.5 (2 ×), 122.5, 121.2, 116.7, 115.4, 111.7 (2 ×), 111.5, 110.1, 105.4, 70.7, 70.6 (3 ×), 69.6, 69.4, 68.1, 48.8, 42.3, 40.1 (2 ×), 31.3, 22.6. ESI-HRMS calcd for C36H40N5O8S: 702.2592, found: m/z 702.2589 [M + H]+.By a procedure similar to that for compound 1, the substitution reaction of 10  with 9d  gave a crude product, which was purified by flash chromatography on a silica gel column with elution of EtOAc/CH2Cl2(2:1) to give the desired compound 4 . The purity of compound 4 was 99.3% as shown by HPLC on a silica column , elution: EtOAc/MeOH = 99:1 at a flow rate of 3.0 mL/min, tR = 11.3 min. C38H43N5O9S; yellow solid; mp 77.5–79.0 °C; TLC (EtOAc/ CH2Cl2 = 2:1) Rf = 0.38; IR νmax (neat) 3356, 3197, 2921, 2851, 1653, 1634, 1470, 1456, 1368, 742, 701 cm–1; 1H NMR  δ 8.66 , 7.85 , 7.80 , 7.40 , 7.27 , 7.05–6.96 , 6.83 , 6.68 , 6.42 , 4.85 , 4.14 , 3.84 , 3.73–3.67 , 3.67–3.57 , 3.38 , 2.99 , 2.85–2.60 , 2.07–1.97 ; 13C NMR  δ 171.3, 169.1, 168.5, 167.5, 166.5, 156.1, 151.8, 148.7, 146.7, 135.9, 135.5, 132.4, 128.4 (2 ×), 122.5, 121.3, 116.7, 115.4, 111.7 (2 ×), 111.5, 110.1, 105.3, 70.8, 70.6, 70.5 (2 ×), 70.4 (2 ×), 69.6, 69.3, 68.0, 48.7, 42.2, 40.1 (2 ×), 31.3, 22.6. ESI-HRMS calcd for C38H44N5O9S: 746.2854, found: m/z 746.2888 [M + H]+.By a procedure similar to that for compound S-transferase (GST) antibody labelled with europium cryptate which binds GST-tagged human cereblon/DDB1 protein. This assay can be used to detect competitive ligand that replaces the binding of thalidomide to human cereblon/DDB1 protein. GST-tagged human cereblon and DDB1 protein was co-expressed by using baculovirus expression system. The GST-tagged protein complex was purified by using Glutathione Sepharose (Cytiva LifeSciences) and the purity of the recombinant protein was confirmed by SDS-PAGE. The competitive binding of a given compound was measured by incubating various concentrations of compounds with 100 nM cereblon/DDB1 in a buffer containing 50 mM Tris at pH 7.5 and 200 mM NaCl. Final concentration of DMSO was kept at 2.5%. Subsequently, the reaction was added 20 μL of 10 nM XL-665-labelled-thalidomide and 100 nM europium cryptate-labelled GST antibody. All assays were performed in 384-well plates (Geiner Bio-One) and the signals were measured using a Pherastar (BMG) plate reader with excitation at 337 nm and emission at 665 nm/620 nm for detection. The ratio of the acceptor  and the donor  emission signals were used for calculation of EC50 values by using a nonlinear fit model (GraphPad Prism Software). Data was presented as means ± standard deviation (n = 3).A cereblon TR-FRET binding assay was developed by using XL-665-labelled thalidomide (Perkin Elmer) and a specific glutathione 208–414, C-TDP-43) were constructed into pEGFP-C3 vector , Academia Sinica). To get the mCherry-C-TDP-43 and FLAG-C-TDP-43 plasmid, C-TDP-43 was subcloned from pEGFP-TDP-43208–414 construct into pcDNA3.1-mCherry and pCMV-tag2B vector, respectively. Either eGFP, mCherry, and FLAG was fused N-terminally of TDP-43208–414. The pEGFP-C3 and pcDNA3.1-mCherry plasmid were used as control plasmids for FLIM-FRET experiments.The cDNA encoding N-terminal truncated TDP-43  at 37 °C in a humidified incubator containing 5% CO2.Neuro-2a cell is the mouse neuroblastoma cell line from Dr. Yijuang Chern . Neuro-2a cells were cultured in Dulbecco’s modified Eagle’s medium  containing 2 × 10g at 4 °C for 40 min . The supernatant (RIPA-soluble fraction) was carefully collected and the pellet of C-TDP-43 aggregates was further washed with RIPA buffer. During the washing step, the pellet was resuspended by 200 μL RIPA buffer and centrifuged at 70,000 g at 4 °C for 10 min. After centrifugation, the supernatant was carefully removed. Then, the washing step was repeated to remove most of the remaining RIPA-soluble protein. Lastly, 50 μL 1% sarkosyl buffer (1 g sarkosyl in 50 mL PBS buffer) was added into the pellet tube, followed by intense pipetting to resuspend the pellet (sarkosyl-soluble fraction). Both the RIPA-soluble and insoluble samples were further processed for detection by using western blot.This method demonstrated how to collect the RIPA-insoluble C-TDP-43 aggregates for SDS-PAGE analysis. RIPA buffer-lysed C-TDP-43 expressing Neuro-2a cells were centrifuged at 70,0004 cells/well and incubated overnight. The attached Neuro-2a cells were then transfected with the eGFP-C-TDP-43 plasmid (1.1 μg) using TurboFect™ transfection reagent (Invitrogen) according to manufacturer’s recommendations. After 2 h transfection, Neuro-2a cells were further treated with or without 5 μM PROTAC (compounds 1–4). The cell viability indicator, AlamarBlue (Invitrogen), was added after 42 h of incubation, and the mixture was incubated for another 6 h . Two-hundred μL conditioned medium was transferred to a 96-well plate, and cell viability was determined by the increased fluorescence intensity .Neuro-2a cells were seeded in a 24-well plate at a concentration of 8 × 105 cells/well and incubated overnight. Then, Neuro-2a cells were transfected with the eGFP-C-TDP-43 plasmid (2.2 μg) using Turbofect transfection reagent (Invitrogen) and treated with or without 5 μM PROTAC molecules after 2 h transfection. After incubation for another 22 h, the transfected cells were harvested by RIPA buffer containing protease inhibitor (Roche) and sonicated on ice for 10 s. Extracts were centrifuged at 14,000 rpm for 10 min at 4 °C, followed by measurement of protein concentration using a bicinchoninic acid (BCA) assay. For filter trap assay, 300 μL sample  were passed through 0.2 μm cellulose acetate (CA) membranes  membranes using a 48-well slot-blot apparatus. Aggregated eGFP-C-TDP-43 protein retained on CA membranes was determined by immunoblotting with TDP-43  antibody . Amersham™ protein® Nitrocellulose (NC) membranes  were applied to slot blot assay for soluble protein lysate analysis (endogenous TDP-43 and loading control).Neuro-2a cells were seeded in a 6-well plate at a concentration of 2 × 106 (blank control) or 2 × 106 (mCherry-C-TDP-43 overexpression) cells/mL. After overnight incubation, Neuro-2a cells were transfected with the mCherry-C-TDP-43 plasmid (10 μg) using Lipofectamine® 3000 (Invitrogen) and incubated for another 24 h. The transfected cells were then harvested with RIPA buffer containing complete protease inhibitor (Roche) and sonicated on ice . Next, PROTAC 2 at various concentrations  was added to each extract . The mixtures (PROTAC 2 + cell lysate) were gently shaken at 4 °C for 2 h. After that, the mixtures were fractionated and the RIPA-insoluble fraction were resuspended with RIPA buffer and loaded on CA membrane by applying filter trap assay. The fluorescent PROTAC 2 retained on C-TDP-43 aggregates was detected by Typhoon9410 Variable Mode Imager  .Neuro-2a cells were seeded in 10 cm dishes at a concentration of 1 × 106 cells in sterile 35 mm µ-Dish ] were transfected with mCherry-C-TDP-43 (1.1 µg) by TurboFect transfection reagent (Invitrogen). For quantifying the C-TDP-43 aggregates in the presence or absence of PROTAC 2 or MG132, mCherry-C-TDP-43 transfected cells were treated with PROTAC 2 (5 µM) after 2 h of transfection. For the group intended to block proteasome activity, MG132 (2 μM) was pre-treated 1 h before adding PROTAC 2. After total 24 h incubation, the Neuro-2a cells were fixed  and subsequently imaged with NIKON TiE microscope. Epifluorescence images were illuminated with an ultrahigh pressure mercury lamp (130 W) for UV excitation or using a 488 nm laser light source. Filters were used to collect fluorescence emission including excited eGFP  and mCherry  cubes. Cellular images were captured with an Andor iXon3 888 back-illuminated high-sensitivity EMCCD camera. Images were edited and cropped using Nikon NIS element software.Neuro-2a cells . The eGFP-C-TDP-43 excitation sources came from 488 nm (5 mW) sub-nanosecond modulated pulsed laser at the fundamental frequency of 20 MHz was controlled by ISS VistaVision software. The photon counts of eGFP were collected by GaAs photomultiplier tube (PMT) detector with EM1 filter (530/43 nm bandpass filter). To precisely obtain the lifetime value, the calibration of the system was operated by measuring fluorescein, a fluorophore with a single exponential lifetime around 4 ns in ddH2O, every time before the measurement.To study the EFRET maps and per-pixel distribution histogram (the population of each pixel corresponding to the EFRET maps)  Fig. B.2 would not form crosstalk with the eGFP donor .To fairly judge the E6 cells/dish) and treated after 2 h with PROTAC 2 (5 µM) or both PROTAC 2 (5 µM) and MG132 (2 µM). After 48 h incubation, the transfected cells were harvested in 1000 μL of ice-cold RIPA buffer containing protease inhibitor cocktail (Roche) and sonicated on ice for 1 min. Extracts were centrifuged at 14,000 rpm for 10 min at 4 °C and the protein concentrations were determined using BCA assay. Samples containing 300 μg of total proteins in a volume of 500 μL were filtered with a 0.22 μm filter (Millipore) and fractionated on a Superdex 200 10/300 column  at a flow rate of 0.3 mL/min. Each fraction (1 mL volume/fraction) was collected and subjected to western blot and slot blot analysis.Neuro-2a cells harboring eGFP-C-TDP-43 (10 µg) were seeded in 10 cm dishes ] and IW62 [Psnb-1::YFP(iwIs25)], respectively . The strains of nematodes were maintained with standard procedure and grown at 20 °C [2HPO4, 871 mL 0.05 M KH2PO4, 5.85 g NaCl) for overnight. For drug treatments, PROTAC 2 or/and DMSO were solely or along with MG132 applied to fresh NGM plates prior to installing the synchronized C. elegans. The body bends of the corresponding treatment in a duration of 30 s of the various strains were documented through SMZ800N stereomicroscope equipped with a CCD camera (Nikon). A body bend was counted as the head of one-day adult C. elegans travels across the mid-body in 1 × PBS buffer. Then, the bending videos of C. elegans were analyzed by ImageJ with the wrMTrck plugin [2 on C-TDP-43 accumulation in L4 worms, the confocal images were captured with LSM 780 (Zeiss).The YFP-C-TDP-43 and YFP transgenic strains of at 20 °C . For lark plugin . For monStatistical comparison of multiple independent groups was conducted by one-way ANOVA with Tukey or Dunnett post-hoc test. Two-way ANOVA with False Discovery Rate post-hoc test was used to determine the effect of two nominal predictor variables on a continuous outcome variable. Statistical comparison of two independent groups was done by two-tailed unpaired t-tests. Significance was accepted at p < 0.05. ns is not significant. All the statistical figures and analysis were done by GraphPad Prism9 software.7 phenylboronic acid (6) underwent Suzuki coupling reaction by the catalysis of Pd(dppf)Cl2 to form anisole 7 as the core structure of TDP-43 binder [7 was treated with BBr3 at 0 °C to obtain the 6-hydroxy substituted BTA (8). As an example, BTA 8 was subjected to alkylation with a PEG linker N3CH2CH2(OCH2CH2)OMs in the presence of K2CO3, followed by reduction of the azido group to amino group, to afford compound 9a. The conjugation of 9a with a pomalidomide analog 10, which bears a fluorine atom at the 4-position of the isoindoline ring, was conducted in 1-methyl-2-pyrrolidone (NMP) [1 (JMF4605). Other PROTAC molecules 2–4  having different PEG units were similarly synthesized (Supplementary Scheme S1 and S2). The successful conjugation reaction should not be performed under alkaline conditions in order to retain the imide groups in the pomalidomide structure.Bearing the linker optimization issue in mind, we synthesized PROTACs 3 binder . Anisolene (NMP) , an apro1–4 can induce C-TDP-43 aggregates degradation by expressing eGFP-TDP-43208–414 (hereafter referred as eGFP-C-TDP-43) in Neuro-2a cells  membrane  markedly reduced the eGFP-C-TDP-43 aggregates  and other PROTAC-treated groups did not significantly affect endogenous TDP-43 protein level compared to mock . PROTACs with shorter linker (PROTAC 1 and 2) showed neglectable cytotoxicity compared to mock group of Neuro-2a cells   [2 treatment. According to the western blot results, GSPT1 level remains stable irrespective of PROTAC 2 treatment at various concentrations  , we also , we als1–4 to CRBN with an in vitro time-resolved fluorescence resonance energy transfer (TR-FRET) assay  with the EC50 values in the range of 0.6–2.6 μM . We also examined whether PROTAC 2 can bind to C-TDP-43 aggregates by in vitro protein binding assay  colocalized with mCherry-C-TDP-43 aggregates . Both RIPA-soluble and insoluble fractions were further analyzed by SDS-PAGE with immunoblotting.Both ubiquitin–proteasome system (UPS) and autophagy pathway may account for the clearance of misfolded TDP-43 . General2 reduced the amount of insoluble C-TDP-43  (72%) compared to PROTAC 2 (33%) and the mock (59%)  was applied to monitor the population of C-TDP-43 oligomer. Since FLIM-FRET reflects the energy transfer through dipole–dipole coupling of a fluorescent donor and acceptor [In addition to TDP-43 aggregates, cumulative studies have also disclosed the pathological role of oligomeric TDP-43 in neurodegenerative diseases . As PROTacceptor , we furt2  lifetime fitting analysis (frame model) to obtain the efficiency of FRET (EFRET) maps and per-pixel distribution histograms from the FLIM image  in Neuro-2a cells  value in the cytosol   had similar expression yield . According to the FD lifetime fitting data of Additional file 2 significantly decreased the EFRET of the C-TDP-43 oligomeric intermediates from 48.92% to 40.11%  to the soluble ones (oligomers + monomers). By setting the threshold of the photon counts, we defined the 2FP-C-TDP-43 species which emitted high photon counts (red pixels) as “aggregated C-TDP-43”  transgenic C. elegans strains, which ectopically express either YFP alone or the YFP fused with a 25 kDa C-terminal fragment of human TDP-43 in the pan-neuron system [2, we recorded the cytosolic aggregates and kept tracing the thrashing frequency of YFP-C-TDP-43 C. elegans in the presence or absence of PROTAC 2.Progressive behavior impairment is the hallmark of ALS pathology . Other taluation . Herein,n system  . Furthermore, we utilized YFP control strain to confirm the degradation specificity of PROTAC 2. As shown in Additional file 2. Conclusively, PROTAC 2 only decreased the cytosolic aggregates in YFP-C-TDP-43 strain  as a CRBN binder, a benzothiazole-aniline (BTA) as a binder of C-TDP-43 protein in β-sheet structures, and a tetraethyleneglycol (TEG) linker to promote formation of the POM–TEG–BTA tertiary complex and thus enable degradation of C-TDP-43 aggregates via ubiquitin proteasome system. Compared to the positively charged thioflavin T (ThT), the electrically neutral BTA may have better cell permeability [S13) having aliphatic linkers (Supplementary Scheme S3) and a lenalidomide–BTA compound S18 that is connected by a triazole-containing linker (Supplementary Scheme S4). However, these compounds rendered low water solubility as the hydrophobicity of linkers increased, and their use as PROTACs was not further examined.PROTAC technology is known for inducing targeted-protein degradation and has been utilized in the field of cancer, immunity, and virus studies –17. Receeability  and higheability  to amylo1) significantly lost its TDP-43-degrading capability . Taken together, optimizing the length of linker is essential for the development of PROTACs in order to gain the potent efficacy and drug safety.During the PROTACs design and evaluation process, we found the length of linker was correlated with the degradation ability and cytotoxicity. Generally, the linker should be long enough to avoid steric hindrance between the target protein and E3 ligase, while the linker cannot be too long to cause futile transfer of ubiquitins . Zorba aity Fig. B. It wase.g. GSPT1) have just been identified [2 did not induce GSPT1 off-targeting degradation , unpredictable side effect of PROTAC may sometimes occur by occupying endogenous E3 ligase , 78. Anoentified ; howeverAlthough PROTACs appear to have many advantageous features for potential therapeutic uses, there are still many obstacles to be overcome, such as pharmacokinetics in the human body and blood–brain-barrier (BBB) penetration for treatment of CNS diseases. Lipinski’s rule of five (Ro5)  has been2 significant reduced C-TDP-43 aggregates, the beneficial effect on C-TDP-43 cytotoxicity in Neuro-2a and motor neuron defects in C. elegans were relatively minor. This disparity between degradation efficiency and beneficial results may arise from the loss of function of TDP-43 [2 treatment. Furthermore, while PROTAC 2 has been reported to degrade the misfolding protein aggregates, we provided new evidence to demonstrate PROTAC 2 could decrease both the amount and the compactness of cytosolic oligomers synchronously. As progressive transition of aggregation reflects the intrinsic energy states in aberrant amyloid protein [FRET of C-TDP-43 oligomeric intermediate in the aforementioned experiment suggested PROTAC 2 could also decrease the intrinsic energy state and interfere with the conformation of C-TDP-43 oligomers. The similar case has been reported in the other amyloid protein binding peptides (i.e. peptides against mutant Huntingtin protein). [It is also worthy to note that though PROTAC f TDP-43 . Increasf TDP-43 . Conclus protein , reductirotein). 2 is the first case which can effectively degrade C-TDP-43 oligomers in addition to its protein aggregates, which provides an alternatively therapeutic strategy against TDP-43 proteinopathy in ALS. To further examine the detailed therapeutic effects of PROTAC 2, the advanced studies of other animal models, including rodents with the mutant TDP-43 aggregates [As far as we know, PROTAC gregates , 89, wou2 (JMF4560) significantly reduced C-TDP-43 aggregates and alleviated C-TDP-43-induced toxicity through the proteasomal degradation (Fig. C. elegans, we also observed that PROTAC 2 was capable of reducing C-TDP-43 aggregates in the nervous system and exhibited beneficial effect on its motility (Fig. 2 could reduce the compactness of C-TDP-43 oligomeric intermediates and decrease their population. By demonstrating the efficiency of our newly-designed PROTACs on the degradation of C-TDP-43 aggregates and oligomers, we wish to develop these small molecules as new drugs against misfolding proteins in neurodegenerative diseases.Herein, we demonstrated that PROTAC 2 JMF450 signifi2 JMF450 signifiAdditional file 1. Figures S1–S9, synthetic schemes S1–S4, synthetic procedures and characterization of compounds, 1H and 13C NMR, absorption, and fluorescence spectra, and HPLC diagrams.Additional file 2. Video of YFP transgenic C. elegans during motility assay.Additional file 3. Video of YFP-C-TDP-43 transgenic C. elegans during motility assay.Additional file 4. Video of YFP-C-TDP-43 transgenic C. elegans upon PROTAC 2 treatment during motility assay.


Hospital-onset (HO) carbapenem-resistant Enterobacterales (CRE) infections are increasing; recently, the Centers for Disease Control and Prevention (CDC) reported a 35% increase during the COVID-19 pandemic. We evaluated the impact of HO CRE blood stream infections (BSI) on outcomes compared to community-onset (CO) CRE BSI.Patients prospectively enrolled in CRACKLE-2 from 56 hospitals in 10 countries between April 30, 2016 to November 30, 2019 with a qualifying CRE bloodstream culture were eligible. Infections were defined per CDC guidelines as CO when the culture was obtained immediately prior to admission or through the first three days of hospitalization, and HO when the culture was obtained on or after the fourth day of admission. Categorical variables were tested using a chi-square test; continuous variable distributions were compared using Wilcoxon rank sum and Kruskal Wallis tests, as appropriate. The primary outcome was desirability of outcome ranking (DOOR) 30 days after index culture. Difference in 30-day mortality was calculated with 95% confidence intervals (CI).Figure 1. The probability of a better DOOR outcome in a randomly selected patient with CO BSI compared to a patient with HO BSI is 60.6% (95% CI: 56.8-64.3%). Mortality at 30-days was 11.6% higher in HO BSI than CO BSI . A Kaplan-Meier curve of all-cause 30-day mortality is in Figure 2Among 891 patients with CRE BSI (Table 1), 65% of BSIs were hospital-onset (582/891). Compared to those with CO CRE, patients with HO CRE were younger , had lower Charlson comorbidity score , and more often had Pitt bacteremia sore ≥4, indicative of critical illness, . ICU admission prior to first culture was associated with HO BSI . Distribution of DOOR outcome at 30 days is shown in Desirability of outcome ranking at 30-days. Events evaluated included 1) deleterious effects, including absence of clinical response, prolonged hospitalization (>30 days following first positive culture), or readmission within 30 days; 2) any adverse events including renal failure or C. difficile infection; and 3) mortality at 30 days. Two subjects  missing 30-day DOOR due to missing disposition information are not included in the figure.Two subjects  are excluded from the figure due to missing mortality outcome information. One subject in the hospital onset group died on day 30 and is still considered at risk in the figure.HO CRE BSIs are associated with worse outcomes than CO CRE BSIs; this unique population warrants special attention. As CRE often contain mobile genetic elements that facilitate horizontal transfer, close monitoring of HO CRE rates and optimizing hospital infection prevention methods are critical to prevent added morbidity and mortality.Yohei Doi, MD, PHD, bioMerieux: Advisor/Consultant|FujiFilm: Advisor/Consultant|Gilead: Advisor/Consultant|Gilead: Honoraria|GSK: Advisor/Consultant|Meiji Seika Pharma: Advisor/Consultant|Moderna: Advisor/Consultant|Moderna: Honoraria|MSD: Advisor/Consultant|MSD: Honoraria|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Shionogi: Honoraria Michael J. Satlin, MD, AbbVie: IDMC member|Biomerieux: Grant/Research Support|Merck: Grant/Research Support|SNIPRBiome: Grant/Research Support Vance G. Fowler, MD, MHS, Amphliphi Biosciences, Integrated Biotherapeutics; C3J, Armata, Valanbio; Akagera, Aridis, Roche, Astra Zeneca: Advisor/Consultant|Genentech, Regeneron, Deep Blue, Basilea, Janssen;: Grant/Research Support|Infectious Diseases Society of America: Honoraria|MedImmune, Allergan, Pfizer, Advanced Liquid Logics, Theravance, Novartis, Merck; Medical Biosurfaces; Locus; Affinergy; Contrafect; Karius;: Grant/Research Support|Novartis, Debiopharm, Genentech, Achaogen, Affinium, Medicines Co., MedImmune, Bayer, Basilea, Affinergy, Janssen, Contrafect, Regeneron, Destiny,: Advisor/Consultant|Sepsis diagnostic: Patent pending|UpToDate: Royalties|Valanbio and ArcBio: Stock Options David van Duin, MD, PhD, Entasis: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Honoraria|Qpex: Advisor/Consultant|Roche: Advisor/Consultant|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Union: Advisor/Consultant|Utility: Advisor/Consultant


The tensile yield strength of epoxy/graphene composites could be either enhanced or weakened by GBs under a tensile load parallel to the graphene sheet. The underlying mechanisms may be attributed to multi-factor coupling, including the tensile strength of the reinforcements, the interfacial interaction energy, and the inflection degree of reinforcements. A balance exists among these effect factors, resulting in the diversity in the tensile yield strength of epoxy/graphene composites. The compressive yield strength for epoxy/graphene composites is higher than their counterpart in tension. The tensile/compressive yield strength for the same configuration presents diversity in different directions. Both an excellent interfacial interaction and the appropriate inflection degree of wrinkles for GB configurations restrict the translational and rotational movements of epoxy chains during volume expansion, which eventually improves the overall gT. Understanding the reinforcing mechanism for graphene with GBs from the atomistic level provides new physical insights to material design for epoxy-based composites containing defective reinforcements.This work presents a molecular dynamics (MD) simulation study on the effect of grain boundaries (GBs) on the mechanical properties of epoxy/graphene composites. Ten types of GB models were constructed and comparisons were made for epoxy/graphene composites containing graphene with GBs. The results showed that the tensile and compressive behaviors, the glass transition temperature ( Epoxy with a cross-linked network structure is the most promising high-performance thermosetting polymer for multifunctional composites due to its excellent mechanical properties, good chemical stability, and durability. These characteristics make epoxy widely used in aerospace, coating, adhesives, electronics, automotive, and biotechnology fields ,3,4,5,6.Extensive experimental and computational studies were carried out on epoxy/graphene systems, highlighting significant improvements in their mechanical and thermal properties with the incorporation of graphene into epoxy ,20,21,22Although a significant amount of research has been performed to study the effect of graphene as reinforcement on the mechanical and thermal properties of epoxy, the reports are mostly focused on the effect of pristine and functionalized graphene. In fact, due to the limitations of synthetic techniques, the crystal growth of large-size graphene during the chemical vapor deposition (CVD) method leads to the formation of geometrical defects such as vacancies, dislocations, and grain boundaries (GBs) ,27. The Although these research works have made progress on the effect of GBs on the mechanical properties of graphene, there is a lack of research related to the effect of GBs on the mechanical and thermal properties of polymer/graphene composites. Verma et al.  evaluategT) of bi-crystalline graphene-reinforced epoxy composites. The stress–strain response, yield strength, elastic moduli (including Young’s modulus and compressive modulus), interfacial interaction energy, and gT of epoxy composites reinforced by pristine graphene and graphene with GBs are also estimated. The mechanism associated with the GB parameters on the mechanical properties and the thermal stability properties is proposed as well. Therefore, this work provides a theoretical basis for the industrialization of nano-reinforced graphene composites.In this work, using the MD method, we investigated in detail the effect of 10 types of GBs on the tensile/compressive behaviors and the glass transition temperature (n = 0). The atomistic models for the DGEBA and TETA segments are shown in L, mL) and  of the left and right crystalline domains. The constructions of GBs along the ZZ and AM directions are shown in The polymer matrix was composed of di-glycidyl ether of bisphenol A (DGEBA) as the resin matrix and triethylenetetramine (TETA) as the hardener. To reduce the computational cost, the polymerization degree of the initial DGEBA prior to cross-linking was set to 0  package . The ato−1, followed by another 100 ps equilibration under an isothermal-isobaric (NPT) ensemble at 300 K and 1 atm. Then, a series of cross-linked reactions were performed using a cross-linking algorithm [During the analysis, models with cross-linking degrees of around 84% were considered. The initial uncross-linked models were minimized using the conjugate gradient method with an energy convergence criterion of 0.0001 kcal mollgorithm ,41. No algorithm  and 100%−1. The tensile and compressive deformation processes were performed under an NPT  ensemble with an integration time step of 1 fs. The atomic stress was calculated using the virial theorem [After the initial cross-linked models were established, the mechanical properties of the epoxy/graphene composites were investigated by uniaxial tension and compression deformation using MD simulations. Three independent samples for each individual epoxy/graphene model were utilized for statistical analysis to quantify the errors. The final value of the mechanical properties was the average of three independent results and the error bars were determined by standard deviation. Before deformation, the initial cross-linked structures were minimized with the help of the conjugate gradient algorithm. Subsequently, the systems were relaxed with multi-step equilibration processes with an isothermal-isochoric (NVT) ensemble at 300 K for 1 ns, NPT  ensemble for 1ns, and an additional NVT (T = 300 K) ensemble for 1ns. The integration time step during the equilibration process was set to 0.25 fs. The root–mean–square displacement (RMSD) was examined to confirm the full equilibrium state. After the equilibration process, a constant uniaxial strain was applied along the x, y, or z directions at a strain rate of 0.0005 ps theorem ,34,43: and perpendicular to the graphene sheets were performed for all epoxy/graphene composites to investigate the effect of GBs on the mechanical properties of epoxy/graphene composites. Because it can directly influence the GB normal strength for graphene sheets, the stress component perpendicular to GBs can better reflect the effect of GBs on the properties of graphene . Thus, tTo further investigate the mechanism of GBs affecting the tensile mechanical properties of epoxy/graphene composites, the effect of the misorientation angle, the tensile strength of graphene sheets, the interaction energy, and the inflection angle on the tensile yield strength of epoxy/graphene composites with a tensile direction parallel to the graphene sheet were analyzed. In order to further comprehend the factors affecting the yield strength of epoxy/graphene composites, the interaction energy between the epoxy matrix and graphene with GBs was calculated using the following expression ,51.(2)ΔAs shown in In a word, the tensile yield strength of epoxy/graphene composites is affected by multi-factors simultaneously, including the tensile strength of the reinforcement, the interaction energy, and the structure of the reinforcement. The physics behind the enhancement in the mechanical properties of composites reinforced with graphene is complicated; a balance exists among these factors leading to a great difference in the tensile yield strength of all the GBs configurations.To further investigate the effect of GBs on the mechanical properties of epoxy/graphene composites, uniaxial compression along parallel and perpendicular directions to the interface was performed for different configurations of epoxy/graphene composites. The composite compression responses for pristine graphene and graphene with GB-reinforced epoxy composites are plotted in A uniaxial compressive simulation for the ZZ and AM configurations was performed with a compressive load parallel to the interface and perpendicular to the GB lines, as illustrated in From gT was calculated for all models. Note that it is a key thermal property affecting the mechanical properties, chemorheology, and internal stresses of thermosetting polymers [To understand the effect of GBs on the thermal stability of epoxy/graphene composites, polymers ,54,55.gT of all epoxy/graphene composites was determined by measuring the density of the composites while the NPT equilibration temperature levels gradually decreased from 600 to 300 K in increments of 20 K. During the cooling process, the structures were equilibrated for 500 ps under NPT ensemble (P = 1 atm) at each temperature with a density tolerance of 0.02 g/cm3. After fitting the data with a straight line, the value of gT could be obtained by searching for the intersection point of the density–temperature curves. In addition, the epoxy composite goes through a transition from a rubbery state, which is identified with a large slope, to a glassy state, which is identified with a small slope, during the cooling process [The  process .gT calculated by density-temperature curves are tabulated in gT of pristine graphene-reinforced epoxy composite is about 422.8 K, which is in good agreement with the value of 418.0 K from previous results [gT of epoxy composites. The gT of Epoxy/ZZ 8.9 and Epoxy/ZZ 9.4 are shown to be 456.2 K and 462.1 K, which are about a 7.9% and 9.3% improvement compared with that of epoxy/ZZ GRP, respectively. From gT of the other AM configurations is higher than that of Epoxy/AM GRP. The gT of Epoxy/AM 13.2 and Epoxy/AM 15.2 are shown to be 455.9 K and 455.6 K, which are about 33.1 K and 32.8 K higher than that of Epoxy/AM GRP, respectively. results . AdditiogT in pristine graphene and graphene containing GB reinforced epoxy composites is mainly related to the excellent interfacial interaction and the inflection degree of the wrinkles in graphene sheets. The strong interfacial interaction between the epoxy matrix and reinforcement can help to restrict the translational and rotational movements of epoxy chains during volume expansion. Moreover, the wrinkle structure of graphene with GBs increases the surface roughness, which is also conducive to blocking the slippage of epoxy chains. It also allows the composites to be heated evenly, avoiding heat concentration in the materials. However, the heavy wrinkle structure weakens the interfacial interaction energy to a certain degree, which creates a complex balance in influencing the gT of the epoxy/graphene composites. The gT of Epoxy/AM 27.8 is lower than that of epoxy/AM GRP, which is mainly due to its relatively weak interfacial interaction  and the low inflection angle of the graphene sheet (about 15°). The higher gT of Epoxy/ZZ 9.4 is related to its relatively strong interfacial interaction  and the appropriate inflection angle of the graphene sheet (about 41°). The improvement in gT indicates that the epoxy/graphene composites need a higher temperature to change from a glassy state to a rubbery state and that the strength of epoxy/graphene composites can remain stable at a higher temperature.The improvement of gT of epoxy/graphene composites are significantly affected by GBs. When the tensile load is parallel to the graphene sheet and perpendicular to the GB lines, the existence of GBs can either enhance or weaken the tensile yield strength of epoxy/graphene composites. The Epoxy/ZZ 9.4 and Epoxy/AM 21.8 configurations present stronger tensile yield strengths, which are considered to be superlative configurations. Multiple factors affect the tensile yield strength of epoxy/graphene composites, including the tensile strength of the reinforcement, the interfacial interaction energy, and the inflection degree of reinforcement. There is a complex balance of these influencing factors which leads to the difference in the tensile yield strength of all the GB configurations. The compressive yield strength for all GB configurations is stronger than the tensile yield strength, and diversity exists for the tensile (compressive) yield strength due to different loading directions. Graphene-containing GB-reinforced epoxy composites obtain a higher gT compared with pristine graphene-reinforced epoxy. The enhancement mechanism can be attributed to the excellent interfacial interaction and appropriate inflection degree of wrinkles in the graphene sheets with GBs. This work will shed light, from the molecular level, on the material design for epoxy composites containing defective reinforcements for practical applications.In summary, the effect of pristine graphene and graphene with GBs on the mechanical properties of epoxy composites was investigated using MD simulations. The results show that the yield strength, elastic moduli (Young’s modulus and compressive modulus), and