Graphene is a 2D material with fruitful electrical properties, which can be efficiently prepared, tailored, and modified for a variety of applications, particularly in the field of optoelectronic devices thanks to its planar hexagonal lattice structure. To date, graphene has been prepared using a variety of bottom–up growth and top–down exfoliation techniques. To prepare high‐quality graphene with high yield, a variety of physical exfoliation methods, such as mechanical exfoliation, anode bonding exfoliation, and metal‐assisted exfoliation, have been developed. To adjust the properties of graphene, different tailoring processes have been emerged to precisely pattern graphene, such as gas etching and electron beam lithography. Due to the differences in reactivity and thermal stability of different regions, anisotropic tailoring of graphene can be achieved by using gases as the etchant. To meet practical requirements, further chemical functionalization at the edge and basal plane of graphene has been extensively utilized to modify its properties. The integration and application of graphene devices is facilitated by the combination of graphene preparation, tailoring, and modification. This review focuses on several important strategies for graphene preparation, tailoring, and modification that have recently been developed, providing a foundation for its potential applications. Graphene is a key material for a new generation of electronic devices with unique electrical properties. In this review, current advances in the preparation, tailoring, and modification of graphene to obtain high‐quality graphene with precisely controlled patterning and specific functional groups are summarized. Physicists and chemists have been drawn to graphene due to its unique properties, such as high mechanical strength, high electrical conductivity, and large surface area. These excellent properties support the use of graphene for various electrical and optical applications. Effective approaches to graphene preparation, tailoring, and modification are required to realize these applications.Graphene, which consists of 2D spstrength,1 high Bottom‐up methods depend upon chemical reactions of precursors and produce large‐area graphene with controlled single layer, which is suitable for building large‐area electronic devices. Top‐down methods, consisting of mechanical exfoliation, liquid‐phase exfoliation, and so on, are proposed based on the unique structure of graphite, where carbon atoms in the sp2 hybrid manner form stable covalent bonds and the adjacent layers are combined through weak van der Waals attraction. Graphene flakes can be peeled from bulk graphite once the van der Waals attraction weakens. Top‐down methods are simpler and more convenient than bottom‐up methods because they do not require complex condition control or expensive equipment. More importantly, top‐down methods are suitable for building high‐performance devices to study the intrinsic characteristics of graphene.Graphene can be produced by both physical and chemical methods. The preparation methods of graphene can be classified into two types: bottom‐up and top‐down methods.10 Bot electron beam lithography (EBL), plasma etching, and so on, have been making great advancement and the precision has been improved to the nanoscale, leading to the rapid progress of microelectronic integrated circuits. However, conventional etching methods cannot achieve atomic‐level precision, which plays a crucial role in the fabrication of devices with uniform edge‐configuration, especially in spintronic devices. Gas etching has been discovered to have the feature of anisotropic etching, which can achieve atomic‐level precision, and may provide opportunities for precise processing.It is necessary to tailor graphene into various shapes in order to process and integrate it into electronic devices. For various functional devices, graphene etching is the primary method for patterning desired shapes and controlling layer numbers. Traditional etching techniques in the semiconductor industry, such as photolithography,25 ele Many organic reactions are applicable to graphene due to the richness of carbon chemistry. Electrophilic substitution reactions, free radical reactions, cycloaddition reactions, and coupling reactions are four main approaches, which are classified according to the types of chemical reactions. In addition, mechanochemical methods mainly make use of ball milling to break the graphic C─C framework and introduce the target groups, which is an effective and controllable method. The modifications are divided into two types based on the reaction sites: edge and surface modifications. Ball‐milling methods and Friedel–Crafts acylation are the most common methods for edge modification. Edge modification has been considered as a reliable way to tune its electronic and mechanical properties without damaging its conjugated π‐electron system. Surface modification of graphene can more effectively adjust the band gap and introduce the band level, resulting in more applications in the nanoelectronics fields. In comparison to pristine graphene, the modified graphene has outstanding characteristics and can be used for a variety of applications.Modification is necessary to endow graphene with various properties and functionalities, including noncovalent and covalent modifications to further realize the functional applications. The noncovalent modification uses π–π interactions to modify graphene surface, while covalent modification uses chemical bonds to decorate both the plane and edge of graphene, becoming a more stable and reliable method.36 ManIn this review, we pay attention to the recent significant development of graphene preparation, tailoring, and modification, supplemented by device fabrication Figure . First, 2The exploration of the property and further application of graphene depends on the development of preparation methods, which can be roughly divided into bottom‐up and top‐down methods, represented by chemical vapor deposition (CVD) and mechanical exfoliation, respectively. High‐density defects and wrinkles in graphene produced by CVD reduce its performance in electronic devices. The mechanical exfoliation method can directly exfoliate bulk graphite layer by layer to produce graphene with high‐quality, which is essential for the discovery of new phenomena and the development of high‐performance devices. Many efforts, such as optimizing deposition conditions, have been made to produce high‐quality graphene via CVD, but there are still challenges in achieving the same high quality as mechanically exfoliated graphene. However, most exfoliation methods produce small, low‐yield graphene flakes, limiting device fabrication efficiency and application possibilities. Mechanical exfoliation can be tweaked to address the aforementioned drawbacks. In the following part, we mainly discuss the improvement of mechanical exfoliation Table  after a 2.1 Under the catalysis of Cu, a typical catalyst for graphene growth, carbon species decomposed from hydrocarbons  nucleate on Cu, and the nuclei further grow into large domains until the surface of Cu foil is completely covered. Large‐area monolayer graphene can be made because of the poor solubility of carbon in Cu. Graphene films are often polycrystalline because of the misalignment of the domains. Moreover, wrinkles arising from the mismatch of thermal expansion coefficient between graphene and Cu are commonly observed. Different from the self‐limiting mechanism on Cu foils, the growth of graphene on Ni is mainly based on the precipitation and segregation of dissolved C atoms because of its better solubility of C and higher catalytical reactivity, which often results in inhomogeneous and multilayer graphene. In combination with the advantages of Cu and Ni, the Cu/Ni alloy has been intensively studied for CVD growth of graphene due to its good and controllable solubility of C. As the Ni content in the alloy increases, single‐crystal bilayer, trilayer, and even multilayer graphene can be prepared. In addition, large‐area and adlayer‐free monolayer graphene can also be achieved on Cu–Ni (111) alloy foils. For the preparation of high‐quality graphene, many factors including substrates, carbon sources, and growth conditions must be carefully considered and controlled. CVD is also widely used to produce large‐scale graphene films to meet the requirements of graphene industrialization for display and sensing applications. Automated transfer processes, such as roll‐to‐roll, are applied to improve transfer efficiency and avoid quality degradation, which is superior to wet etch transfer used in laboratories.CVD has been widely applied to fabricate large‐area graphene films.53 Und Usually, poly(methyl methacrylate) (PMMA) can be served to support graphene as a carrier, and copper foil is etched away by chemical etchant (FeCl3). The PMMA/graphene stack is picked up by a target substrate after floating on the surface of the etchant solution. After PMMA is dissolved with acetone, the graphene remains on the substrate. However, the contacts between PMMA and graphene surfaces are evitable, and there are difficulties in the complete removal of PMMA. For instance, acetone cannot entirely dissolve PMMA, and a thin layer of residues may cause degradation of the carrier mobility in graphene.Graphene grown on Cu foils often needs to be transferred to a target substrate for the fabrication of electronic devices. Wet transfer is the most common transfer method.68 Usu2 and sapphire (Al2O3), is vital for achieving high‐performance graphene‐based devices. Common approaches of direct growth on SiO2/Si require relatively high temperature, typically over 1100°C, resulting in low‐quality graphene due to the instability of SiO2. With the assistance of enhanced plasma, the temperature of graphene growth on SiO2/Si can be effectively lowered. Recently, a sapphire‐based multi‐cycle plasma etching‐assisted CVD method successfully led to the growth of wafer‐scale single‐crystal monolayer graphene, which produces graphene via a sacrificial layer of Cu rather than growing directly on sapphire. After a long annealing process, the copper grains are transformed into a regular orientation through which C atoms diffuse into the interface between Cu (111) and Al2O3 (0001). SiC is also a promising insulated substrate for obtaining graphene. At high temperatures above 1600°C, Si atoms on the SiC surface are sublimated, while C atoms are remained to grow into high‐quality graphene.In order to prevent contaminations and wrinkles introduced during wet transfer, the growth of graphene on insulators and semiconductors, such as SiO of SiO2.73 Wit2.2Mechanical exfoliation, in contrast to stronger in‐plane covalent bonding, relies on the relatively weak interlayer van der Waals forces, resulting in exfoliated flakes with a wide range of thickness and lateral dimensions. Though mechanical exfoliation can avoid defects caused by chemical synthesis and contamination introduced by wet transfer, the thickness, sizes, shapes, and locations of the obtained graphene are usually random, limiting the large‐scale device fabrication. Because of the disadvantages mentioned above, mechanical exfoliation is still a long way from mass production and commercialization, but it remains the best way to obtain high‐quality graphene for fundamental studies.2.2.1 This exfoliation process is illustrated in Figure  First, a piece of graphite is placed on the Scotch tape. After folding the tape in half several times to fully exfoliate the bulk crystal, it is pressed with the proper force on a Si wafer. Finally, single‐layer graphene is obtained by peeling the tape away from the wafer at a slow speed. This method is widely used in laboratories thanks to its advantages of producing high‐quality graphene, and it has also emerged as a promising method for obtaining other 2D materials, especially transition metal dichalcogenides (TMDs).In 2004, Geim and Novoselov et al. first isolated graphene with Scotch tape from highly oriented pyrolytic graphite (HOPG).14 ThiThe mechanical exfoliation depends on two kinds of forces, lateral force and normal force. The lateral force is derived from graphite's ability to self‐lubricate, which aids the movement between adjacent layers. Few‐layer graphene can be obtained by overcoming the van der Waals force between adjacent layers via the normal force. During the process of exfoliation, uneven interactions can cause the breakdown of in‐plane covalent bonds, producing fragmented small‐area graphene.The standard exfoliation method involves repeatedly folding Scotch tape to obtain graphene with random number of layers; thus, graphene quality and exfoliation yield are affected by a number of factors, including the substrate, the tape, the experimenters’ operation, temperature, and humidity. Among these factors, the tape and substrate play a vital role in improving peeling efficiency. The modification of substrate and tape is mostly considered to reduce the influence of uncontrollable factors  and improve the exfoliation yield.2.2.2 The ambient adsorbates are removed via oxygen plasma, and followed by heat treatment to ensure uniform contact with the interface. The oxygen plasma cleaning and mild annealing are considered key steps in this method  is reported.79 Thel Figure . The sha2.2.4 Under a kilovolt voltage, Na2O in the glass substrate decomposes into O2− and Na+ ions at high temperatures. Na+ ions migrate across the substrate under the influence of an electric field due to their relatively high mobility, while O2− ions are static, making the surface negatively charged. Thus, a strong electrical field is created, allowing for close atomic contact between graphite and substrate. The graphite is then cleaved away with an adhesive tape, leaving a few layers of graphene adhered to the substrate. The graphene produced can be hundreds of microns in size. However, this method requires high voltage (1.2−1.7 kV) and temperature (200−400°C), which is unavailable in most laboratories. The strong electrostatic effect makes it hard to transfer to other substrates, which may limit its further applications.The basic idea of anode bonding is to enhance the interaction between the bottom layer of graphite and the substrate via forming a strong electrical field.80 Und2.2.5 named covalent‐like quasi‐bonding, is considered to meet the requirements for mechanical exfoliation, which provides uniform and strong interaction with 2D materials. Only a few metals can help with graphene exfoliation due to differences in binding energies between metals and graphene. Ni can be used to peel graphene off due to its high binding energy. While Au, widely used to peel off TMDs, is less effective for graphene exfoliation due to the low binding energy. Metal‐assisted exfoliation becomes an ideal way to transfer materials tightly bound to the growth substrate, such as graphene grown on the silicon carbide (SiC) surface, due to the strong interactions between metal and the 2D materials.It is difficult to produce large‐area single‐layer graphene with traditional exfoliation method due to uneven interactions between graphite, tape, and substrate. Recently, a noncovalent interaction (∼0.5 eV per unit cell) between some metals and 2D materials,82 nam Due to the strong bonding of graphene to the SiC surface, transferring graphene from SiC is still challenging. A method for separating graphene from SiC has been proposed , the graphene layer is transferred to a SiO2/Si substrate. The exfoliation efficiency depends on the binding energy  and the stress of the metal film. Under the same deposition conditions, Ni has the highest stress and the largest binding energy (γGr‐Ni) among other metals (γGr‐Au < γGr‐Cu < γGr‐Pd < γGr‐SiC < γGr‐Ni), which exceeds γGr‐SiC. When Ni is used, a transfer yield of more than 95% can be achieved, whereas Cu, Pd, and Au peel off graphene with difficulty. Due to the large difference between γGr‐SiC and γGr‐Gr, there are still difficulties in directly separating single‐layer graphene from SiC with a Ni layer.Single‐oriented graphene can be obtained with the decomposition of SiC (0001) surface but is mostly one or two layers.91 Dued Figure .[84] As shown in Figure Gr‐Gr < γGr‐Au < γGr‐Ni, leaving the monolayer graphene on the other side.A single‐layer precision selective exfoliation method is developed according to the difference in binding energy between graphene and metals.84 As  The random conventional exfoliation is transformed into a controllable and deterministic process by deposition of metal films using graphite as the material , which is widely used in traditional etching processes, can also be applied to graphene tailoring. The basic working mechanism of RIE can be described as follows. Under the alternating electric field provided by radio frequency, the gas is excited and ionized, generating highly reactive plasma. The graphene basal plane is bombarded and reacted by ions and free radicals in the plasma. Particularly, physical etching reduces the etch selectivity, resulting in isotropic etching. For the preparation of graphene nanoribbons (GNRs) with a well‐controlled edge configuration, which is important for opening the band gap of graphene, atomic‐level etching is required. However, the required accuracy is still beyond the reach of current state‐of‐the‐art nanolithography techniques.To fabricate functional electronic devices, a variety of techniques have been used to tailor graphene.94 Tra and remote plasma etching, selectively remove C atoms at the edge of graphene owing to the difference in reaction rates between C atoms in zigzag and armchair sites, resulting in anisotropic etching with atomic precision. Metal nanoparticles, such as Ni and Fe, can anisotropically etch graphene when migrating on the basal plane, resulting in a crisscross line‐like etching pattern. However, this kind of etching occurs on the surface of graphene, where metal nanoparticles take random movements, making the etching sites uncontrollable, which cannot be applied to graphene patterning. After a brief introduction to EBL, we will primarily focus on anisotropic etching methods with various gases. The comparison of various anisotropic etching methods, in terms of etchant, substrate, edge configuration, and functional group, are summarized in Table Pure chemical etching methods, such as reactive gas etching99 and3.1 Desired patterns can be drawn directly on the resist, then transferred by etching or evaporation to obtain patterned materials, based on the interaction between electrons and resist such as PMMA. For example, as a top‐down processing method, EBL in combination with oxygen plasma etching can obtain specifically patterned graphene.As a typical top‐down method for micro‐nano processing, EBL can achieve nanometer‐level resolution.103 De The graphene is cut through the window of PMMA by oxygen plasma etching after an indented window on PMMA is obtained by EBL  and armchair graphene nanoribbons (AGNRs) terminated with epoxy oxygen atoms, the epoxy‐terminated armchair edge is more stable than the zigzag edge, which explains the existence of the armchair edge  can the hydrogenation of C atoms at the edge and the breakage of the C─C bonds be satisfied at the same time. Therefore, the reaction rate takes a “volcano type” curve with temperature changes  medium can be achieved at ∼130°C using the “direct” Friedel–Crafts acylation. The reactive C═O+ is generated by protonation of a carbonyl group of the benzoic acid derivatives via PPA. With the benzoic acid or benzamide derivatives, any desired functional groups, such as small molecules or macromolecules, can be attached to the edges of graphene. The C═O+ ion attacks the edges/defects of graphene without destroying the basal plane to preserve the conductivity and integrity of graphene in a mild and controllable way. Various functional wedges are introduced to the edge of graphite, endowing graphene materials with specific functions.As a typical electrophilic aromatic substitution reaction, Friedel–Crafts acylation reaction allows the synthesis of acylated products from the reaction between arenes and electrophiles. The edge‐selective graphene modification in a mild polyphosphoric acid (PPA)/phosphorous pentoxide  is determined by thermogravimetric analysis (TGA) showing ∼36 wt%. The covalent attachment of ABA grafts at the graphene edges has been verified using AFM images. The Friedel‐Crafts acylation reaction can introduce linear and hyperbranched polymers onto the edges of graphitic layers. For example, the para‐poly (ether‐ketone) (pPEK) can be covalently grafted to graphite. A series of characterization can verify the success of modification. The resulting pPEK grafted graphite (LPEK‐g‐graphene) shows better tensile properties than pPEK films. Dendritic macromolecular wedges can also be directly grafted to the edges of graphite via the Friedel–Crafts acylation. The solubility of dendritic macromolecules is improved in comparison with that of linear analogs. In general, graphite is treated with AB2 monomer for hyperbranched poly(ether ketone) (HPEK). According to AFM images, the edge of graphene is higher than its basal plane due to the existence of HPEK. The high degree of exfoliation is estimated using wide‐angle X‐ray diffraction. The cyclic voltammetry experiments results show that the carboxylic acid groups exist. The above‐mentioned aromatic 4‐aminobenzoyl moieties on the edge of AB‐EFG can play a dual role in “C‐welding” and “N‐doping” to obtain N‐graphite films, which have high conductivity, good mechanical properties, and high optical transmittance. N‐doped graphene has tremendous potential applications for its superior performance in field‐effect transistors (FETs).Small molecules, linear and hyperbranched macromolecules, are attached to the edges of graphene, as illustrated in Figure 144 Th In comparison with the graphene modification mentioned above, Friedel–Crafts chlorination happens at the edges/defects of graphene as confirmed by X‐ray single‐crystal analysis, which benefited from the solubility of nanographene to obtain the crystal structure. A series of well‐defined nanographene  are chosen to reflect precise chlorination. Chlorination is achieved at ∼80°C in CCl4 with iodine monochloride and catalyzed by AlCl3. Matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry confirms the elemental composition of chlorinated products. The isotopic distribution pattern of the mass peak is in good agreement with the calculated pattern. The precise edge chlorination can be served as an efficient model for edge‐functionalized graphene materials, providing an efficient way to tune properties of nanographene such as solubility, band gap, and the position of the frontier molecular orbital.A Friedel‐Crafts chlorination reaction for edge chlorination of nanographene is reported as a vital step toward controlled functionalization, resulting in well‐defined chemical functionalized graphene materials.151 In3‒) is prepared by chlorosulfonation and finally reduced to form graphene thiol (G─SH)  of sulfur‐modified samples shows the standard signatures for both sulfonate and thiolate groups, and the S:C atomic ratio of G─SO3− and G─SH is analyzed to be ∼0.3:100. Chemical functionalization of edges and defect sites of basal plane are visible in fluorescent‐labeled graphene under super‐resolution microscopy. It is possible to create the “glycographene” by radical addition, demonstrating the synthetic feasibility of directly linking reaction groups. Edge localization is visualized by epifluorescence microscopy of a lectin‐glycographene bioconjugate with fluorescently tagged.Unlike the Friedel–Crafts acylation, carbonyl groups cannot be introduced by the electrophilic aromatic substitution, which could slow electron transfer between the basal plane and the functional groups. Sulfur is electrophilic to attack electron‐rich graphene, then graphene sulfonate (G─SO) Figure .38]383‒) In this reaction, generated electrophilic ions overcome 2D π–π stacking of graphite and are covalently introduced into the edge of graphite, which ultimately turns to the more stable sulfonated graphene. SEM images of graphite have a flake shape with smooth surfaces, while sulfonated graphene shows an aggregated morphology indicating the presence of H‐bonds among polar sulfonic acid molecules at the graphene edges. The transmission electron microscopy (TEM) images indicate no chemical damage to the basal plane. Graphite can have up to ∼22.5 wt% edge‐selective functionalization.Electrophilic sulfonation is also realized by edge‐selective functionalization with chlorosulfuric acid.152 In4.2.22 carbons to sp3 carbons.Chemical reactions, laser irradiation, and photochemistry can all produce free radicals. Using diazonium salts, benzoyl peroxide, and halogen, free radicals can be added to the basal plane, transforming sp Delocalized electrons are transferred from graphene to aryl diazonium cations, which produce aryl groups of graphene after releasing a molecule of N2. The successful attachment of C6H4─NO2 groups on the graphene sheet can be confirmed by the Fourier transform infrared spectroscopy (FTIR) and time‐of‐flight secondary ion mass spectrometry (TOF‐SIMS) characterizations. The modification of the graphene largely enhances the room temperature resistance and changes the transport properties and electronic structure of graphene. For instance, modified graphene possesses a ∼0.4 eV bandgap. Using diazonium functionalization of graphene, it is possible to modify graphene not protected by PMMA to realize patterning and embroidery. Regular multiply functionalized patterns form molecular graphene embroidery including concentric regions of covalent addend binding. Furthermore, diazonium reaction and thermal treatment to functionalize the spatial structure using EBL on PMMA have been used to achieve reversible covalent patterning of graphene  Figure . F can be further covalently modified on graphene by treating it with the F‐substituted benzyne species, as shown by XPS and FTIR characterizations. Therefore, aryne cycloaddition is a facile and mild approach to obtain aryne functionalized graphene with good dispersibility.The aryne cycloaddition of graphene can be realized under mild reaction conditions. The aryne‐modified graphene sheets are stable and dispersible in a variety of solvents Figure .[163 Various enediynes directly attack sp2 carbon atoms at the surface of graphene. The modified graphene is soluble in many kinds of organic solvents and has excellent electric conductivity. This unique self‐coupling method can also be used to make polymer‐grafted graphite.Bergman cyclization is a cycloaromatization of enediyne‐containing compounds carried out a diradical intermediate Figure .[164 The nitrene addition could not take place only at the sheet's edges, and the resulting structure is similar to graphene oxide. The other method of nitrene addition is photochemical or thermal activation of graphene with perfluorophenyl azide (PFPA), which is converted to the active singlet perfluorophenylnitrene and undergoes C═C addition reactions with the graphene to produce the aziridine adduct. The solubility of graphene can be greatly regulated by chemical functionalization, and the functionalized graphene products disperse well in the corresponding solvents.Graphene nanosheets can be functionalized through nitrene addition of azido‐phenylalanine to exfoliate graphite Figure .[165 Specifically, graphite and graphene are used as dienophiles, both the 9‐methylanthracene and the 2,3‐dimethoxy‐1,3‐butadiene are used as dienes for the D‐A reaction. The carbons in graphene manage to transform from sp2 hybridized to sp3 hybridized through the D‐A chemistry. Attenuated total reflectance FTIR spectroscopy can be used to confirm the generality of graphene D‐A reactions. A solvent‐free D‐A reaction has been carried out by heating a mixture of graphite and typical dienophiles, such as maleic anhydride (MA) and maleimide (MI). Various characterization techniques have confirmed the functionalization of graphite with dienophiles, implying that graphite can be functionalized and delaminated into a few layers of graphitic nanosheets efficiently.Through the Diels–Alder (D‐A) reaction, graphene can be used as diene or dienophile to modify the electronic properties under mild conditions Figure .[167 The addition dichlorocarbene, diarylcarbene, and fluorinated olefins is the focus of most experimental work. The functionalization is operated based on a conventional method which is used to produce the dichlorocarbene. In brief, the graphene is suspended in a mixed solution of chloroform (CHCl3), sodium hydroxide (NaOH), and phase transfer catalyst under reflux.Since carbene functionalization on carbon nanotubes has been studied extensively, it is possible that dichlorocarbene functionalization on graphene will occur naturally Figure .[1694.2.4Mechanochemical methods and microwave assisted methods can cause some small gas molecules to react on the edge of graphene. This method is simple, low‐cost, environmentally friendly, and scalable for mass‐producing graphene nanoplatelets (GnPs) with various functional groups.A variety of functional groups could be grafted to the edge of graphite when mixing the graphene with solids, liquids, or even chemical vapors in a ball‐mill crusher according to the availability of mechanochemical reactions driven by ball milling. During ball milling, the high‐speed rotation of the stainless‐steel balls generates sufficient kinetic energy to break the graphitic C─C framework, and then various functional groups can be introduced at the broken edges of graphite. Edge‐selective functionalized graphene nanoplatelets can be achieved through mechanochemical reactions with hydrogen, carbon dioxide, sulfur trioxide, or carbon dioxide/sulfur trioxide mixture. The gas is captured by mechanochemically produced active carbon species resulting in graphene with edge‐selective functionalization. Active carbon species are formed when the edges of graphite are broken, which are reactive to pick up halogens. The presence of Cl, Br, and I is successfully confirmed by energy dispersive X‐ray spectroscopy with element mapping. Furthermore, it is found that the edge‐halogens cause the edge expansion of XGnPs. Two‐step ball‐milling of graphene yields the graphene nanoplatelets (ISGnPs) with iodine/sulfonic acid functionalized at the edge. First, graphite can be edge functionalized by iodine under ball‐mining to produce IGnPs, followed by ball‐milling with sulfur trioxide to produce ISGnPs. Phosphorus is covalently attached to the edges of graphene nanoplatelets by ball‐milling  is obtained with high yield by ball milling. The generated ECG can be dispersed in a lot of polar solvents to be exfoliated into graphene nanosheets. Because of the hydration induced by ball‐milling, the high‐energy carboxylates turn into carboxylic acids (─COOH), and residual activated carbon species turn into hydroxyl (─OH) and hydroperoxyl (─OOH) by oxygen and moisture in the air. The ball milling process makes the surface area increase to large extents. Carbon disulfide (CS2) can also be introduced to the edges of GnPs with abundant ─C═S/─C─S bonds through mechanochemical reactions with CS2  have been synthesized by mechanochemical reactions in the presence of a halogen gas Figure .[170g Figure .[1722 Figure . The res When further exposed to oxygen and moisture in the air, hydrogen‐GnPs, sulfonic acid‐GnPs, carboxylic acid‐GnPs, or carboxylic acid/sulfonic acid‐GnPs can be generated. They show excellent performance in electrocatalyst and functional device applications.Mechanochemical reactions with hydrogen, sulfur trioxide, carbon dioxide, or a mixture of sulfur dioxide and carbon trioxide can efficiently produce the graphene nanoplatelets (GnPs) with various functional groups on the edge selectively.46 Whe In this case, microwave irradiation may cause the halogen to become slightly ionized, which aids the halogenation reaction. The Cl and Br atoms have been covalently functionalized on the graphene sheet with ∼21% and ∼4% atom percentage. The graphene halides (G─X) can be easily exfoliated into monolayer graphene in organic solvents. After high temperature annealing, the chlorine content is reduced, and the structure of chlorinated graphene (G─Cl) can be restored. As a result, after annealing, the conductivity of the G─Cl film increases significantly. The high solubility and low cost of G─Cl allow it to meet the requirements for large‐area and economical electronic devices such as FETs, photovoltaics, and light‐emitting diodes. For instance, the G─Cl itself can be used as a functional component to prepare FETs. G─X could also be an important versatile precursor and intermediate material to fabricate new composite materials.Chemical synthesis of highly modified graphite can also be accomplished using microwave‐sparks‐assisted halogenation reactions Figure .[175 Under microwave radiation, molecular collisions caused by relatively high‐frequency electromagnetic radiation produce active singlet perfluorophenyl nitrene, which can react with C═C bonds of graphite via cycloaddition. Therefore, graphene can react with PFPA when exposed to microwaves. Any amine‐containing molecules can be covalently attached to the PFPA functionalized graphene. For instance, it can conjugate with glycosyl amine to give carbohydrate‐presenting graphene.PFPA can also be used to functionalize graphene through a microwave‐assisted reaction.176 Un5Graphene has been used in a wide range of applications, since it can be effectively exfoliated, tailored, and modified. Whether in laboratory research or industrial applications, large‐scale, high‐quality, and layer‐controlled graphene with specific functionalities is a worthy goal to pursue.As two parallel roads for graphene preparation, CVD and mechanical exfoliation provide strong support for the availability of large‐area and high‐quality graphene based on their complementary advantages, holding great prospects for the realization of graphene‐based integrated circuits and the exploration of novel phenomenon, such as superconductivity discovered in magic‐angle graphene superlattices. Much effort should be made to improve the quality and size of graphene. In case of CVD, growth conditions are needed to be optimized to obtain high‐quality single‐crystal graphene on metal substrates. Gentle and clean transfer procedures are required to preserve the excellent transport properties of graphene. New methods are needed to be developed to achieve graphene growth directly on dielectric surfaces. Furthermore, mechanism studies of graphene exfoliation are needed to be explored, which is crucial for obtaining large‐area and high‐quality graphene.Etching is a key step to fabricate various structures of graphene, endowing graphene with excellent properties. Traditional organic synthesis is also seen as a complementary choice for producing specific pattern of graphene or other materials, but involves complicated condition control and product post‐processing. In contrast, top‐down etching can effectively control the morphology of graphene over large area in combination with EBL, and anisotropic etching provide scientists with an alternative to studying topological and spin properties of edge states. However, it should be noted that an in‐depth understanding of the etching mechanism and the establishment of the theoretical model are required to study the underlying effects of different etching conditions.Through covalent functionalization, molecular catalysts can be immobilized on the graphene surface to form stable photocatalytic materials. In graphene chemistry, most reactions on graphene originate from the organic reactions of benzene. However, it is not rigorous enough to regard graphene as benzene, so the reaction mechanism on graphene needs to be further studied in the future. It is also necessary to precisely control the modified positions of graphene according to specific requirements, such as opening the bandgap or implementing new mechanisms. Target molecules should be introduced to realize the specific functions of graphene‐based devices.On the basis of improved graphene preparation, tailoring, and modification methods, large‐area and high‐quality graphene with precisely controlled patterns and specific functional groups should be used to fabricate devices. These three parts will invite in‐depth research on the amazing structures and electronic devices of graphene, such as magic‐angle graphene and graphene‐based single‐molecule junctions, to satisfy the exploration of novel properties and applications of graphene. Through delicate design, the processes of preparation, tailoring, and modification should be further combined to fabricate next‐generation electronic devices.The authors declare no conflict of interest.


Defects on graphene over a micrometer in size were selectively blocked using polyvinyl alcohol through the formation of hydrogen bonding with defects. Because this hydrophilic PVA does not prefer to be located on the hydrophobic graphene surface, PVA selectively filled hydrophilic defects on graphene after the process of deposition through the solution. The mechanism of the selective deposition via hydrophilic–hydrophilic interactions was also supported by scanning tunneling microscopy and atomic force microscopy analysis of selective deposition of hydrophobic alkanes on hydrophobic graphene surface and observation of PVA initial growth at defect edges. Polymer–based graphene nanocomposites hold great potential for applications such as sensors, energy harvesting, and gas barrier films because of their superior mechanical, optical, and barrier properties ,3,4,5,6.Furthermore, graphene defects do not occur only in the synthesis process. Defects may also occur in the process of transferring graphene to the target substrate . AlthougA number of graphene healing methods have been reported. For example, since DFT calculations or molecular dynamics could study the interactions of graphene with a variety of molecules, these methods hold great potential to design graphene–defect–healing processes ,26,27. AHere, we report that graphene defects over micrometer could be selectively filled with hydrophilic polymers using hydrophilic–hydrophilic interactions. Although the graphene surface is hydrophobic, graphene defects are generally hydrophilic due to the existence of hydrophilic functional groups such as hydroxyl and carboxyl groups ,36,37. U36H74), Sigma–Aldrich, St. Louis, MO, USA), and heptane  were used without a further purification process. As a solvent, ethanol was purchased from Sigma–Aldrich. The height of PVA in defects on graphene was obtained using AFM  in non–contact mode.Polyvinyl alcohol  method ,23,38. T2 plasma using reactive ion etching (RIE) equipment in order to remove graphene off the back side by inverting the graphene coated with PMMA. To obtain only graphene from CVD–graphene, Cu foil was etched in an aqueous ammonium persulfate (APS) solution for 4 h. Graphene floating on the APS solution was transferred to deionized (DI) water using PET film and the remaining APS solution was rinsed three times. Before graphene was transferred to Si wafer, the Si wafer was cleaned in acetone and isopropyl alcohol (IPA) using sonication for 10 min, respectively. Then, graphene was transferred to the target substrate and dried at room temperature for 30 min in order to prevent water from being trapped between the graphene and the Si wafer substrate. After that, it was put oven for an hour, dried completely, and the PMMA used as a supporting material in acetone for an hour on hot plate at 40 °C was removed. Graphene on Si wafer was rinsed in IPA and extra solvent was blown using nitrogen.Graphene was transferred onto the Si wafer through the wet transfer method using polymethylmethacrylate (PMMA) as a supporting material ,39. The Holes were manufactured using photolithography ,41. A phHTC heptane solution of 1 mM was prepared by ultra–sonicating the HTC and heptane mixture for 1 h at room temperature. For the PVA solution, PVA was dissolved in ethanol with a concentration of 0.1 g/mL by stirring the solution for 30 min at 45 °C.For HTC deposition, the HTC solution in heptane (b.p 98 °C) was dropped on patterned graphene with holes, and the solvent was slowly vaporized at room temperature to obtain multilayer HTC on graphene. For the PVA deposition, the PVA solution in ethanol was dropped on prepared graphene with a hole. Then, the ethanol solvent was slowly vaporized at room temperature and quenched before fully vaporized by blowing the solution to investigate the initial growth of PVA. For the full deposition of PVA, after dropping PVA solution on patterned graphene, the ethanol solvent was slowly and completely vaporized at room temperature. Then, the sample was annealed on a hot plate at 210 °C for 10 min to prepare the selectively deposited PVA films in graphene defects where Tg and Tm of PVA are 74.3 °C and 222.7 °C, respectively.Samples for AFM measurements were created by making artificial defects on CVD–graphene transferred on Si wafer substrate and dropping the HTC or PVA solutions as shown in the experimental methods 2.2, 2.3, 2.4, 2.5, and 2.6 above. The AFM imaging process was conducted in non–contact mode using park system’s NX10 equipment. Images were scanned measuring 10 μm × 10 μm or 20 μm × 20 μm in size. The values of the scan rate for the pristine graphene sample, graphene coated with multilayer HTC, and PVA in defects on graphene were 0.35 Hz, 0.6 Hz, and 0.3 Hz, respectively. The values of Z servo gain for the pristine graphene sample, graphene coated with multilayer HTC, and PVA were 1, 1.5, and 1, respectively.A self–assembled HTC monolayer on CVD–graphene was imaged using STM . The STM tip was created by cutting the tip of a wire made of Pt/Ir  alloy. In order to investigate a self–assembled HTC monolayer at molecular level, HTC was dissolved in 1–phenyloctane for STM study. The HTC solution was replaced with CVD–graphene on copper substrate. The STM tip went into the solution and approached the graphene surface. The self–assembled structure of HTC was mainly scanned at 700~800 mV and 200~300 pA. All image processing was performed using SPIP software.2 surface. The lack of a strong van der Waals interaction between HTC and surface leads to HTC molecules not being nucleated on the amorphous SiO2 surface in the early nucleation stage. Therefore, it was expected that HTC would only be deposited on patterned graphene and not on the amorphous SiO2 surface. This hypothesis is experimentally demonstrated using patterned graphene. The graphene has periodically aligned 5 μm defects as shown in 2 to graphene before the deposition was measured as ~2 nm through the non–contact mode of AFM , were deposited on patterned graphene in order to confirm that hydrophobic molecules prefer to be deposited on only a hydrophobic graphene surface rather than hydrophilic graphene defects as shown in e of AFM b. After In order to prove the hypothesis that hydrophilic–hydrophilic interaction between PVA and defects allows selective deposition, the initial growth of PVA was investigated before completing deposition. As shown in Although various healing methods for graphene defects have been developed, defects over micrometer size are not efficiently healed. Upon considering the hydrophobic surface of graphene and the hydrophilic characteristics of graphene defects, it was demonstrated that hydrophilic polymer could selectively fill graphene defects over micrometer size through hydrogen bonding with functional groups at defect edges. This mechanism study for selective deposition of polymers in graphene defects would provide insights for further development of polymer–graphene composites.


Our work demonstrates that the removal of less than 10% of the low-complexity sequence at N- and C-termini generates amyloid fibrils with comparable macroscopic features but different local structural arrangement. It highlights that the assembly mechanism of TDP-43, in addition to the aggregation of the hydrophobic region, is also driven by complex interactions involving low-complexity aggregation-prone segments that are a potential source of structural polymorphism.Aberrant aggregation of the transactive response DNA-binding protein (TDP-43) is associated with several lethal neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. Cytoplasmic neuronal inclusions of TDP-43 are enriched in various fragments of the low-complexity C-terminal domain and are associated with different neurotoxicity. Here we dissect the structural basis of TDP-43 polymorphism using magic-angle spinning solid-state NMR spectroscopy in combination with electron microscopy and Fourier-transform infrared spectroscopy. We demonstrate that various low-complexity C-terminal fragments, namely TDP-13 (TDP-43 Misfolding and deposition of proteins into fibrillar aggregates are causative of many neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease, and many other cognitive disorders . These pTARDBP gene and widely expressed in neurons. The protein binds to DNA and RNA  essential for TDP-43 function, and disruption of these interactions contributes to TDP-43 dysfunction and pathology  1–81) , a nucleathology . Recentlynuclein .postmortem brain tissue of FTLD patients  adopt multiple structurally distinct molecular conformations  to TDP-35 (TDP-4390–414) and full-length TDP-43. We used a construct lacking the GaroS2 domain ) to demonstrate that although GaroS2 is critical for the formation of amyloid fibrils in the full-length construct, its removal still results in the formation of fibrillar aggregates with cross-β properties (in vitro but using a different aggregation procedure (in vitro aggregated TDP-43 (311–360) and TDP-43 (262–414), respectively. By comparing the NMR chemical shift signature of these various TDP-43 fibrillar polymorphs, as well as to those formed after seeding with proteins from bacterial inclusion bodies , TDP-11 (TDP-43 residues 300–399) and TDP-10 (TDP-43 residues 314–414).. The polymorphic capability of the TDP-43 CTD amyloid fibrils has significant relevance in TDP-43 pathology and understanding its structure and function can provide insights into the underlying mechanisms of neurodegenerative diseases and guide the development of effective therapeutical strategies. The three fragments used in our study comprise the hydrophobic segment (318–340) and the Q/N rich segment (341–367) but varied in sequences from the GaroS1 and GaroS2 subdomains. The nomenclature of different C-terminal segments is chosen to be consistent with Mompean et al. and Shenoy et al.  low-complexity constructs studied here have been selected to be shorter than the LCD (267–414) as defined by Eisenberg and coworkers . In TDP-E. coli and purified under denaturing conditions following the procedure reported previously  is a good indicator because the spectral dispersion in this region allows for unambiguous identification of most aromatic contributions. Phenylalanine and tyrosine signals are observed in the INEPT spectra  of the TDP-11 construct (Hα-Cα correlation peaks are indicated with dotted squares in 13C–13C spectroscopy with a short proton-driven spin diffusion (PDSD) mixing time of 50 ms to probe intra-residue correlations of rigid residues. The aliphatic region of the 13C–13C correlation spectra of CTF aggregates is displayed in 13C line width of ∼100 Hz , indicative of the presence of residues associated with a relatively well-ordered structural arrangement within the amyloid core. Comparable 13C line widths have been reported for TDP-43311–360 by Lu and coworkers  techniques. CP favors the detection of rigid residues in amyloid aggregates. Hence, we employed 2Doworkers , TDP-432g et al.  and for oworkers . However primarily arises from backbone carbonyl stretching vibrations and is directly related to secondary structure (−1) corresponds to CN stretching and NH bending. ATR-FTIR analysis also evidences a composite secondary structure in TDP-43 CTF fibrils (−1), β-sheet , turns  and random coil   spectroscopy. FT-IR is a powerful method to obtain a rapid analysis of the secondary structure of peptides and proteins in their aggregated state . We perftructure , while t fibrils ; Table 146 cm−1) . The throid core .13C–13C correlation experiments with a mixing time of 150 ms to favor the detection of sequential  connectivity  of the 2Dspectrum , suggestspectrum . These ispectrum . Unfortuspectrum .et al. have pointed out that the interactions of the Phe-Gly motifs are a main driving force for the TDP-43 prion domain fibril formation from droplets by using solution and solid-state NMR analysis . This observation is in line with our previous findings that the GaroS2 domain is involved in the TDP amyloid core . Recentlanalysis . Interesanalysis . Strikinanalysis . Althouganalysis  for TDP-analysis .in vitro conditions. The CTD of TDP-43 is composed of the hydrophobic domain (318–340) and the Q/N-rich domain (341–367), surrounded by two low-complexity domains (GaroS1 and GaroS2). By shortening the primary sequence of these two low-complexity domains, we generated three CTF constructs, and demonstrated that using in vitro conditions, already employed for full-length TDP-43 aggregation , TDP-11 (300–399) and TDP-10 (314–414). The presence of specific C-terminal fragments is one of the structural hallmarks of the pathology of TDP-43 proteinopathies. Interestingly, the length and the starting amino acid of the fragments seem to be controlled during the disease process, e.g., ∼18–35-kDa CTFs are found in the brains of ALS and FTLD patients , while tregation , triggerregation  by increregation . Here wef TDP-43 , indicatus study . Such obus study  and TDP- shared the same solid-state NMR spectral fingerprint; however, it was not the case for the CTF TDP-16 (267–414). Interestingly, in the present work, we show that shorter CTFs are more prone compared to the larger constructs TDP-43 and TDP-35, to structural polymorphism and to aggregate into distinct structures despite the absence of a few residues from the CTD. For TDP-11, we truncated a part of the GaroS2 domain (residues 400–414) that is not observed by solid-state NMR as the rigid component of the so-called “second core”  by Murray et al.  or fibrils and droplets  by digestion with NdeI and XhoI, followed by ligation using T4 DNA ligase (New England Biolabs).E. coli strain BL21 (DE3) pLysS transformed with the pET24-TDP vector. The expression and purifications were either in LB or M9 minimal media supplemented 15NH4Cl and 13C-glucose. The bacterial cultures were induced with 0.75 mM IPTG at OD600 0.8 and grown for 3 h 30min at 37°C. The cells were harvested by centrifugation  and frozen (−80°C) until purification. Frozen bacterial pellets were thawed and resuspended on ice in 20 ml of lysis buffer , sonicated , and centrifuged at 15,000 g for 1 h at 4°C. The pellet was then resuspended in 20 ml of lysis buffer supplemented with 2% Triton X-100, incubated at 37°C , and centrifuged . To remove the detergent, the pellet was washed with lysis buffer  and then centrifuged . The pellet thus obtained was resuspended in 10 ml extraction buffer  supplemented with dry guanidine hydrochloride until saturation and incubated overnight . The solution was sonicated and centrifuged . The protein was purified using a 5 ml Histrap HP column with a gradient of 10–500 mM imidazole in denaturing buffer . The fraction containing pure protein was pooled and dialyzed two times against 20 mM MES buffer pH 7.5 to remove urea and imidazole and was allowed to self-assemble for 1 week at room temperature. It was then extensively washed in water, centrifuged, and transferred to the ssNMR rotor. The sample quantity per rotor was ∼10 mg. Note that the limited solubility of CTD constructs has hampered further solution NMR analysis of soluble monomers.The TDP proteins were expressed and purified following the same protocol as described in our previous study . In shor1H Larmor frequency spectrometers (Bruker Biospin) at a MAS frequency of 11 kHz and 278K on a triple resonance HCN 3.2 mm MAS probe , using 90 kHz SPINAL-64 1H decoupling. DSS was used as an internal reference. The rigid part of the fibrils was probed using two-dimensional 13C–13C correlation spectra using a PDSD mixing scheme. A cross-polarization (CP) contact time of 1 ms was used in the initial transfer. Intra-residue correlations were probed using a PDSD mixing time of 50 ms for acquisition times of 20 ms (direct) and 7.5 ms (indirect), 288 scans for TDP-13, 480 scans for TDP-10 and 384 scans for TDP-11 and a recycle delay of 3 s led to a total experimental time of ∼5 days. Sequential correlations were probed using a PDSD mixing time of 150 ms for acquisition times of 20 ms (direct) and 6.3 ms (indirect), 792 scans and a recycle delay of 3 s leading to a total experimental time of ∼7 days. The 2D 1H-13C INEPT was recorded using acquisition times of 20 and 6 ms in direct and indirect dimensions, respectively, with 128 scans, for a total experimental time of ∼18 h. All two-dimensional spectra were processed using Bruker Topspin with a sine-bell shift of pi/4 and analyzed using CCPNMR  at the copper wavelength. The concentrated hydrated samples were mounted in a MicroLoops™ from Mitegen  on a goniometer head under the cold nitrogen flow. Each diffraction pattern corresponds to a 360° rotation along the phi axis with an exposure time of 720 s after subtraction of a ‘blank’ image of the same exposure time with only the loop on the goniometer head.TDP fibrils were diluted twice in 150 mM acetic acid buffer and applied to glow-discharged 300 mesh carbon-coated copper grids for 1 min, washed with water, stained with a 2% uranyl acetate (w/v) solution for 1 min, and dried in dark condition, and monitored in an FEI CM120 transmission electron microscope at an accelerating voltage of 120 kV under TEM low-dose mode at a magnification of 200,00×. TEM images were recorded using a Gatan USC1000 2 k × 2 k camera .−1 and a one-level zero filling. The hydrated TDP fibril samples were deposited (10 μl) on a germanium ATR crystal. Excess water was removed with a nitrogen gas flow, and the spectrum was recorded with 200 scans. The spectra were analyzed to determine the secondary structure elements of each protein with an algorithm based on a second-derivative function and a self-deconvolution procedure  to examine the number and wavenumber of individual bands within the spectral range of the amide I band .The ATR-FTIR spectroscopy was performed on a Nicolet 6700 FT-IR spectrometer  equipped with a liquid nitrogen-cooled mercury–cadmium–telluride detector , with a spectral resolution of 4 cm


Using this concentration in the seed amplification assay, ACI-5891.9 was able to neutralize the transactive response DNA-binding protein of 43 kDa pathogenic seeds derived from amyotrophic lateral sclerosis patient CSF. This translational work adds to the evidence of transmission of transactive response DNA-binding protein of 43 kDa pathology via CSF that could contribute to the non-contiguous pattern of clinical manifestations observed in amyotrophic lateral sclerosis and demonstrates the ability of a therapeutic monoclonal antibody to neutralize the toxic, extracellular seeding-competent transactive response DNA-binding protein of 43 kDa species in the CSF of apparently sporadic amyotrophic lateral sclerosis patients.In amyotrophic lateral sclerosis, a disease driven by abnormal transactive response DNA-binding protein of 43 kDa aggregation, CSF may contain pathological species of transactive response DNA-binding protein of 43 kDa contributing to the propagation of pathology and neuronal toxicity. These species, released in part by degenerating neurons, would act as a template for the aggregation of physiological protein contributing to the spread of pathology in the brain and spinal cord. In this study, a robust seed amplification assay was established to assess the presence of seeding-competent transactive response DNA-binding protein of 43 kDa species in CSF of apparently sporadic amyotrophic lateral sclerosis patients. These samples resulted in a significant acceleration of substrate aggregation differentiating the kinetics from healthy controls. In parallel, a second assay was developed to determine the level of target engagement that would be necessary to neutralize such species in human CSF by a therapeutic monoclonal antibody targeting transactive response DNA-binding protein of 43 kDa. For this, evaluation of the pharmacokinetic/pharmacodynamic effect for the monoclonal antibody, ACI-5891.9,  et al. report the detection of seeding-competent transactive response DNA-binding protein of 43 kDa (TDP-43) in CSF of amyotrophic lateral sclerosis patients using seed amplification assay. These seeding species can be neutralized by an antibody targeting the C-terminal of TDP-43 providing evidence for an immunotherapy approach to target amyotrophic lateral sclerosis.Audrain   Staging of TDP-43 pathology based on post-mortem immunohistochemical analysis of ALS brain and spinal cord has revealed the spatio-temporal spread of pathology from the initially affected region to other neuroanatomically connected brain regions.7 These data advocate for the presence of specific pathogenic forms of TDP-43 that can transmit from cell to cell and transform conformation of the native protein into pathogenic form in a prion-like mechanism. The cell-to-cell spreading of TDP-43 pathology in brain has been demonstrated in transgenic mouse models injected with pathogenic TDP-43 seeds derived from brains of patients with TDP-43 proteinopathies.8 More recently, spinal cord extracts from ALS patients were demonstrated to seed and spread TDP-43 pathology in ALS cerebral organoids.9 However, this mechanism by itself does not explain the non-continuous clinical manifestations frequently seen in ALS.10 To account for this, it has been hypothesized that CSF can be a vector for local and distal propagation with subsequent disease progression occurring independently either via axonal11 transport (anterograde and/or retrograde), tunneling nanotubes,12 extracellular vesicles13 and CSF transmission.14 In fact, the neurotoxicity conferred by CSF of ALS patients in vitro suggests the presence of one or more toxic factors in ALS CSF,15 supporting an implication in disease pathogenesis.16 In ALS, a primary TDP-43 proteinopathy, it is hypothesized that disease-associated misfolded TDP-43 forms resulting from neurodegeneration in the CNS might be present in patient’s CSF and thereby contribute to disease pathogenesis.10Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder with high unmet medical need due to the lack of disease-modifying therapies.in vitro12 and in vivo.17 Recently, an ultrasensitive assay based on amplification of seeds has emerged that enabled the detection of misfolded TDP-43 forms in CSF from confirmed genetic ALS patients that ‘seed’ or ‘induce’ aggregation of native TDP-43.18 Such a seed amplification assay (SAA) was originally conceptualized for antemortem detection of prions under the name of protein misfolding cyclic amplification and was based on the tendency of misfolded prion seeds to serve as template and induce native protein to shift to pathogenic conformation.19 Subsequently, the assay was successfully adapted to detect tau,20-22 α-synuclein23-25 and TDP-4318 seeds and has been reported under the name of real-time quaking-induced conversion. In this report, based on the recent consensus, such a technique is referred to as a SAA to reflect the proposal for a unifying terminology for this concept.26Several attempts have been made to establish the impact of ALS patient CSF exposure on TDP-43 aggregation and the associated downstream features 27 The published data clearly demonstrated, using multiple in vitro and in vivo models of ALS and frontotemporal dementia (FTD), that mAbs such as ACI-5891 with epitopes in the C-terminal region result in the reduction of pathology as well as confer neuroprotection from toxic patient brain-derived TDP-43 aggregates.27 Furthermore, the sequence of ACI-5891 was humanized to a therapeutic suitable format, ACI-5891.9, which involved insightful antibody engineering, producing a candidate with optimal potency and pharmacokinetics to maximize successful disease modification in upcoming clinical trials.28To study the seeding propensity of CSF obtained from ALS patients and establish the translational potential to neutralize TDP-43 seeds with immunotherapy, the C-terminal region targeting TDP-43 monoclonal antibody (mAb), ACI-5891, was assessed. Previously, we have established that this region of TDP-43 is key for efficacy.Thus, to study patient-derived extracellular seeding-competent TDP-43 and implications for clinical research, a robust SAA was established, employing a unique reaction substrate to both confirm the presence of seeding-competent TDP-43 species and determine the effect using a therapeutic mAb, ACI-5891.9. In parallel, a novel target engagement assay complemented with pharmacokinetic (PK) data from non-human primates (NHPs) was created to obtain the pivotal data to model the dose needed for target saturation in patients. Together, the data confirmed that CSF of apparently sporadic ALS patients contains species which can induce TDP-43 aggregation. Furthermore, when using the high-affinity mAb, ACI-5891.9, full inhibition of seed amplification was observed at clinically relevant concentrations. Hence, these data add to the growing body of literature to support that spreading of pathology in ALS occurs at least in part by species found in CSF. As such, treating patients with a passive immunotherapy approach by ACI-5891.9 should result in slowing and ameliorating devasting TDP-43-mediated neurodegenerative diseases such as ALS.Human CSF was provided by the Northeast Amyotrophic Lateral Sclerosis Biofluids Repository, Boston, USA. All samples were collected and used with written informed consent. Participants were not tested for all known ALS-causative genes, however, as these participants had no family history of ALS or FTD, they were classified as ‘apparently sporadic ALS’. Due to the small size of cohorts, the healthy controls (HCs) were not perfectly age matched, but majority of them overlapped with the ALS patients in age . CSF was352NNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGSSMDSKSSGWGM414-OH) was chemically synthesized at Pepscan and obtained with a purity >99% (UPLC/UV215). Lyophilized powder was first dissolved in pure hexafluoro-2-propanol (HFIP) for 5 days under agitation at RT and let dry under the hood overnight (200 µL of HFIP for 0.5 mg of lyophilized substrate). Films were then resuspended in ice-cold H2O, aliquoted and kept at −80°C. Concentration was assessed by measuring UV absorbance at 280 nm using NanoDrop Spectrophotometer (Thermo Fisher).The TDP-43 peptide used as assay substrate (H-Reactions were performed in 96-well black/clear bottom plates (Thermo Fisher #265301) in a total volume of 200 µL per well and in the presence of one glass bead of 3 mm diameter (Supelco #104015) in each well. The reaction master mix contained 40 mM Tris-HCl, 5 mM DTT, 0.002% SDS, 50 µM thioflavin T (ThT) and 10 µM substrate peptide  was arbitrarily compared at 12 h between the different conditions. Additionally, the difference in time to reach 3000 rfu between each sample incubated with ACI-5891.9 versus isotype control (ratio) was assessed.The morphology of assay substrate assemblies was assessed by transmission electron microscopy in a CM 100 Biotwin transmission electron microscope (Philips) following adsorption onto formvar carbon-coated 400 mesh grids and negative staining with 1% uranyl acetate. The images were recorded using a bottom-mount TVIPS F416 camera (4K × 4K).Escherichia coli as a fusion protein with N-terminal His-SUMOstar tag. Protein expression was induced with 0.1 mM IPTG at 18°C for 16 h in Luria–Bertani medium (Sigma) supplemented with kanamycin. Following cell lysis, the protein was purified from the soluble fraction using affinity chromatography. Subsequently, the His-SUMOstar tag was removed using the SUMOstar Protease I (Tebu-bio). Finally, the protein was applied to size exclusion chromatography using 16/600 Superdex 75 column to obtain high-purity soluble protein in buffer .Human TDP-43 was expressed in d-galactosidase . Plates were washed between each incubation using a magnetic microplate washer (Quanterix Corporation) and read with a SR-X SIMOA® instrument (Quanterix Corporation). Assay optimizations were accomplished by adjusting the concentrations of mAbs either conjugated to the paramagnetic beads or after biotinylation. The molar ratio of biotin and linker used for mAb–biotin conjugations and the concentrations of SBG were also adjusted. Paramagnetic beads conjugated to the capture antibody were diluted in bead diluent (Quanterix Corporation), and human CSF samples were diluted in the general detector and sample diluent (Quanterix Corporation). SBG was diluted in SBG diluent (Quanterix Corporation). The lower limit of quantification was determined as the mean blank for a readout of average enzyme per bead (AEB) + 2.5 times the standard deviation. The lower limit of detection was set based on the signal at the lowest concentration of the calibrator (0.023 AEB). For calibrator, full length recombinant TDP-43 with a polyglycine tag in N-terminal was used (described above). The raw values in AEB rather than the extrapolated concentrations for CSF were used as the levels of TDP-43 signal was between lower limit of detection and lower limit of quantification.The Single Molecule Array  technology was used for the development of high-sensitivity custom immunoassays using two proprietary murine IgG2a mAbs binding TDP-43: ACI-5891 and ACI-5965. ACI-5891 was conjugated to paramagnetic beads (Quanterix Corporation) as a capture reagent, and ACI-5965 was biotinylated (Thermo Fisher) to be used as a detection antibody. Assays were performed using a three-step protocol with all incubation steps performed at 25°C on a microplate shaker set at 800 rpm (Quanterix Corporation). The incubation times were 30 min with the antibody-conjugated capture beads, 10 min with the biotinylated detection antibody, and 10 min with streptavidin-β-The assay described above was used to quantify free (unbound) TDP-43 in human CSF. Different concentrations (from 0 to 5000 ng/mL) of ACI-5891.9 (ACI-5891 humanized variant on human IgG1 backbone) were spiked into three different human CSF samples prior to use in the assay. This resulted in concentration-dependent saturation of the epitope by ACI-5891.9, preventing capture of TDP-43 by the ACI-5891-coated magnetic beads and allowing assessment of target engagement by measuring free TDP-43. A four-parameter logistic regression model was used to plot each curve for each human CSF sample independently.28 Murine complementarity-determining regions (CDRs) of ACI-5891 VH and VL were grafted onto selected frameworks VH1-69-2/VK2-28. Back-mutations were introduced at critical framework residues known to influence CDR conformation and based on the molecular model of ACI-5891 Fv domains to generate ACI-5891.9.28 For recombinant antibody productions, CHO cells were transiently transfected with equimolar quantities of heavy- and light-chain vectors. Antibodies were purified from supernatants by protein A chromatography . The identity and purity of the purified antibodies were confirmed using native and reducing SDS-PAGE. Concentrations of antibodies were measured using the NanoDrop Spectrophotometer (Thermo Fisher).The Fv domain of ACI-5891 was discovered by hybridoma technology, from mice immunized using the SupraAntigen® vaccine technology.in vivo study was approved by the national ethical guidelines and performed in an animal facility accredited by the provincial animal management office. NHPs ≥ 2 years old, weighing ≥ 2 kg, were purchased from Hainan Jingang Laboratory Animal Co. Ltd. ACI-5891.9 was administered by intravenous bolus injection single dose at 40 mg/kg (n = 4). Serum was collected from each animal at the following time points: pre-dose (−24), 0.05, 1, 2, 4, 8, 16, 24, 48, 168, 336, 504, 672, 840, 1008, 1176 and 1344 h (Day 56).The Serum PK time points were selected to accurately capture the initial antibody concentration at the start of the study, the distribution phase during the first 48 h and the typical inflexion point of the PK profile . Following this, weekly time points were collected for 8 weeks to characterize the elimination phase. PK parameters were estimated by non-linear mixed effect modelling using Monolix  by a two-compartmental model with linear elimination from central compartment. PK parameters are reported as typical population PK parameters, and the terminal half-life was derived from it.CSF was collected at the following time points: pre-dose (−24), 48 and 1344 h (Day 56), except for the NHP#1 for which both the pre-dose and 48 h time points were not collected as per protocol. The pre-dose time point allowed baseline quantification of TDP-43. The time point at 48 h post-dose allowed assessment of antibody penetration in the CNS when serum concentration was expected to be high, and the terminal time point was used to assess antibody concentration at the end of the study when antibody serum concentration had declined. Animals were checked daily for body condition, natural behaviour and appearance. Haematology and blood chemistry were performed pre-dose and at 168 h post-dose.y2 weighting.The total concentration of human antibody in NHP serum was determined by enzyme-linked immunosorbent assay (ELISA). A biotinylated mouse polyclonal anti-human kappa chain antibody (Southern Biotech) diluted to 1.5 µg/mL in phosphate buffered saline (PBS) with 0.05% Tween 20 was captured onto a streptavidin-coated microplate (Microcoat Biotechnologie GmbH) for 1 h at RT. Plates were washed four times with PBS with 0.05% Tween 20 and blocked with 100 µL of PBS with 1% bovine serum albumin (BSA) and 0.05% Tween 20 (dilution buffer) for 1 h at RT. Standard curves prepared from a 2-fold serial dilution starting at 50 ng/mL and test samples diluted in 1:100 dilution buffer supplemented with 1% cynomolgus monkey serum (Neo-Biotech) were loaded to the plates, incubated for 90 min at RT and washed as previously described. Bound human antibodies were detected with an anti-human kappa chain antibody conjugated to horseradish peroxidase (Southern Biotech) diluted to 4.0 ng/mL in dilution buffer. Plates were incubated for 30 min at RT and washed as previously described. Finally, TMB substrate (SeraCare) was added to each well and incubated for 10–15 min. Reactions were stopped by adding 100 µL of Stop Solution (Bethyl) to each well. Absorbance was read at 450 nm with a reference wavelength at 690 nm using a BioTek microplate reader. Concentrations of all serum samples were back-calculated against a reference standard using a non-linear four-parameter regression fit including a 1/2 (Jackson), at 20 nM (3 µg/mL) in PBS with 1% BSA and 0.05% Tween 20 for 1 h was performed at 37°C. Fifty microlitres per well of the TMB substrate (SeraCare) for 15 min at RT in the dark was used for the detection, and the reaction was stopped by adding 50 µL per well of H2SO4. Absorbance was read at 450 nm using a Tecan plate reader. Concentrations of CSF samples were also back-calculated using a reference standard using a non-linear four-parameter logistic model. The sensitivity of the assay was 0.93 ng/mL.The total concentration of human antibody in NHP CSF was also determined by ELISA. Briefly, coating of 96-well plates was done with an anti-human IgG at 10 nM (=1.5 µg/mL) in carbonate–bicarbonate buffer  using 50 µL/well at 4°C overnight. The washing steps were done using a plate washer and 200 µL per well of PBS/Tween 20 (0.05%). Blocking used 5% BSA in PBS/Tween 20 (0.05%) at RT for 30 min. Fifty microlitres per well of the sample  were incubated at 37°C for 1.5 h. CSF was diluted to 1/100. Incubation with the detection antibody, anti-human IgG F(ab’)A similar assay design to that described for CSF was used to assess target engagement in the serum of NHPs previously treated with ACI-5891.9 during the PK study. However, capture and detection antibodies were inverted. The RNA recognition motif (RRM) binding TDP-43 mAb ACI-5965 was coupled to acceptor beads to capture total TDP-43, while detection was performed using a biotinylated ACI-5891. Additionally, serum samples were diluted (1/10) in a diluent  specifically formulated for optimum assay performance in serum. The concentration of free TDP-43 was extrapolated in picograms per millilitre or as a fold change compared to the pre-dose serum for each animal.n values) and statistical tests are also indicated in ﬁgure legends. For data in t-test (two-tailed) was used whereas for data in post hoc analysis was used. The normal distribution of all data has been checked. P-values are reported as asterisks based on the following values: ****P < 0.0001, ***P < 0.001 and *P < 0.05.Graphs represent the mean of all samples in each group ± SEM or SD. Sample sizes  with characteristics suitable for SAA was identified. Following an initial lag phase, the substrate alone showed a concentration-dependent aggregation as monitored by ThT fluorescence, confirming the formation of β-sheet-containing fibrils . FollowiFollowing the assay qualification, CSF from 20 apparently sporadic ALS patients and 12 HC  technology was set up. A mouse anti-human C-terminal targeting, TDP-43 mAb, ACI-5891, was coupled to acceptor beads allowing capture of total TDP-43 in human CSF. However, when ACI-5891.9, a humanized version of ACI-5891, was pre-incubated in human CSF sample, immune complexes with TDP-43 were formed impeding their capture in the assay due to the unavailability of the ACI-5891 epitope (50) of 129.2 ng/mL was calculated for ACI-5891.9 confirming its high binding affinity to TDP-43 (90 (1100 ng/mL), these data demonstrated that a mAb concentration of approximately ≍1100 ng/mL was required to saturate all forms of TDP-43 in human CSF  has been shown to represent the amyloid core of pathological TDP-43 aggregates in the brain of ALS patients,46 regions adjacent to this core have been demonstrated to adopt β-sheet conformation.47 This was further demonstrated in this study by the ThT-positive fibrillar structure adopted by the assay substrate (amino acids 350–414).To maximize the chances of capture, neutralization and clearance of all seeding-potent forms of TDP-43 including the C-terminal fragments found to be enriched in TDP-43 aggregates found in patients,in vitro and in vivo for use of an immunotherapy approach with ACI-5891.9 can be used in support of clinical rational and dosing regimens for trials in patients with apparently sporadic ALS. Furthermore, the SAA and target engagement assays can be used to help stratify and follow efficacy of disease-modifying therapies in patients.In conclusion, in addition to providing the field with an optimized SAA, this study provides evidence for extracellular seeding-competent disease-associated TDP-43 species in the CSF of patients with apparently sporadic ALS. The extensive work to determine saturating conditions both fcad306_Supplementary_DataClick here for additional data file.


Here, we reported a fast preparation method of self-propagating high-temperature synthesis to realize in situ compositing of BiCuSeO and Cu2Se to optimize the service stability. Additionally, using the interface design by introducing graphene in these composites, the carrier mobility could be obviously enhanced, and the strong phonon scatterings could lead to lower lattice thermal conductivity. Ultimately, the Cu2Se-BiCuSeO-graphene composites presented excellent thermoelectric properties with a ZTmax value of ~2.82 at 1000 K and a ZTave value of ~1.73 from 473 K to 1000 K. This work provides a facile and effective strategy to largely improve the performance of Cu2Se-based thermoelectric materials, which could be further adopted in other thermoelectric systems.Thermoelectric materials can realize direct conversion between heat and electricity, showing excellent potential for waste heat recovery. Cu 2Se-based materials. They obtain a maximum ZT value of ~2.82 at 1000 K on Cu2Se-BiCuSeO-graphene composites.Here, the authors devise a synthesis strategy to optimize the stability and thermoelectric performance of Cu A good thermoelectric performance is needed for further application, which is generally determined by the dimensionless figure of merit, ZT = S2σT/κ. The parameters S, σ, κ, and T stand for the Seebeck coefficient, electrical conductivity, thermal conductivity, and absolute temperature, respectively. Thus, achieving an outstanding ZT value relies on a large power factor (PF = S2σ) and a low κ, where the κ is mainly consisted of carrier thermal conductivity (κc) and lattice thermal conductivity (κl)6. However, the strongly coupled correlation among S, σ, and κc has always been a big challenge for improving ZT values8. To overcome this problem, various strategies have been proposed, such as band structure engineering11, mobility optimization14, lattice thermal conductivity suppression17, and so on. To date, polycrystalline SnSe recorded the highest ZT value of ~3.1 at 783 K18. The maximum ZT (ZTmax) values over 2.0 have also been reported in Cu2Se20, PbTe22, GeTe24, and single-crystal SnSe26 thermoelectric materials.Waste heat will be a potential and huge energy resource if it can be properly utilized. Based on carrier and phonon transport, thermoelectric technology with the capacity of direct energy conversion between heat and electricity could be an attractive alternative for waste heat recovery, which has been widely investigated in the past decades2Se-based thermoelectric materials with high ZT values are highly promising and notable in mass production and commercial applications29. The reason for the high thermoelectric performance of Cu2Se could mainly be the highly mobile Cu+ ions, which cause strong phonon scattering and further lead to intrinsically low κl30. Meanwhile, the superionic nature has detrimental effects on the stability of Cu2Se and then damages its thermoelectric performance under some certain application conditions31. Thus, improving the stability and remaining a high ZT value is a challenge for Cu2Se-based materials. Inhibiting the long-range migration of Cu+ ions by introducing ion-blocking interfaces has been proposed as an effective strategy to improve the stability of Cu2Se34. It is proposed that Cu2Se/BiCuSeO interfaces could stabilize Cu2Se by blocking the long-range migration of Cu+ ions and obstructing the reduction reaction of Cu+ into Cu metal precipitates34.Due to the elemental abundance, non-toxicity, and wide working temperature range, Cu2Se (lower than 20 cm2 V−1 s−1) and BiCuSeO (lower than 10 cm2 V−1 s−1) hinders the further improvement of thermoelectric performance, especially the average ZT (ZTave) values39. Thus, optimizing carrier mobility is urgent for achieving higher ZT values of Cu2Se-based materials. Recently, carbon materials have been attractive in optimizing thermoelectric performance since it has excellent electrical properties42, which can introduce extra electrically conductive paths and are expected for improving the carrier mobility of composites. In addition, previous work has shown that introducing carbon structures can also decrease lattice thermal conductivity because of the interfacial phonon scattering and strong phonon reflection at phase interfaces41.However, the relatively low carrier mobility of Cu0.88Pb0.06Ca0.06CuSeO (BPCCSO)35 than that of pristine BiCuSeO and the robust level of carrier mobility in graphene41, the Cu2Se-BPCCSO-graphene composites were designed and prepared by a fast method of combining self-propagating high-temperature synthesis (SHS) with spark plasma sintering (SPS). We found that compositing with BPCCSO and graphene synergistically optimized electrical and thermal properties. Excellent electrical performance with the highest average PF (PFave) value of 14.49 μW cm−1 K−2 from 473 K to 1000 K was obtained, which could be mainly attributed to the optimized weighted mobility. Meanwhile, the multiple interfaces brought strong phonon scatterings to achieve a relatively low lattice thermal conductivity value of 0.11 W m−1 K−1 at 1000 K. Ultimately, attractively high ZT values including a ZTmax value of ~2.82 at 1000 K and a ZTave value of ~1.73 from 473 K to 1000 K were realized, which exhibited good potential for future practical applications.Considering the higher thermoelectric properties of Bi2Se-BPCCSO composites and explored the optimum compositions for further investigation. For comparison, Cu2Se and BPCCSO were also prepared by the same method in the meantime. All the samples were highly dense with a relative density of over 95%, as shown in Supplementary Table 2Se-BPCCSO composites in the whole temperature range was much higher than that of Cu2Se . For instance, the carrier concentration increased from 2.22×1020 cm−3 of Cu2Se to 2.80×1020 cm−3 of 0.9Cu2Se-0.1BPCCSO  is shown in Supplementary Fig.ρ) can be seen in Supplementary Table Cp) was estimated according to the Neumann–Kopp rule based on the measured Cp of Cu2Se in this work . The temperature-dependent κl and Lorentz constant (L) are presented in Supplementary Fig. 2Se and BPCCSO, the Cu2Se-BPCCSO composites had lower lattice thermal conductivity due to the interfacial phonon scatterings.By using the SHS-SPS method, we first prepared Cu2Se Fig. , which w2Se Fig. , Cu2Se-Bues Fig. . FurtherZT values were calculated and shown in Fig. 2Se-0.1BPCCSO sample possessed the highest ZTmax value of ~2.14 at 973 K. To test the service stability of such composites, we re-measured the 0.9Cu2Se-0.1BPCCSO sample and conducted heating and cooling cycles test. As shown in Supplementary Fig. ZT values all presented excellent stability with only small changes under experimental uncertainty. Furthermore, it is proposed that electrical conductivity has a positive correlation with critical voltage and can be a simple indicator to rapidly predict the stability of Cu2Se-based materials, where the higher electrical conductivity is, the better stability will be43. Therefore, the Cu2Se-BPCCSO composites should have better stability than Cu2Se. In addition, with the compositing of BPCCSO, the bending strength of Cu2Se-BPCCSO composites largely improved from ~45 MPa to ~70 MPa  can be analyzed via the X-ray diffraction (XRD) patterns, as shown in Supplementary Fig. 2Se-0.1BPCCSO-x wt% graphene composites  were almost the same and mainly consisted of two phases, Cu2Se and BPCCSO, indicating the high efficiency of the facile and fast preparation method for fabricating such complex composites. Owing to the low content of graphene, it was difficult to detect its phase by XRD. Besides the main phases, two minor peaks of Bi2O3 around 27.5° and 32.7° were observed in composites as well, which formed different scales of precipitates embedded in the matrix to strengthen phonon scatterings, bringing a further decrease in lattice thermal conductivity. The existence of Bi2O3 may be ascribed to the evaporation of Bi elements, and a small amount of Bi2O3 was unreacted as impurity phases.The phase structure of 0.9Cu2Se-0.1BPCCSO-x wt% graphene composites  is presented in Supplementary Fig. 2Se, which was confirmed by energy dispersive spectroscopy (EDS)  plane of BPCCSO was parallel to the (−206) plane of Cu2Se. Such in situ formed interfaces can effectively scatter phonons without impeding carrier transport, which is beneficial to decoupling carrier-phonon interactions. In addition, graphene with different sizes has been found in composites, as shown in Supplementary Fig. The fracture microstructure of 0.9Cuses Fig. , which wses Fig. . Parts o2Se-0.1BPCCSO-x wt% graphene composites  are presented in Fig. 2Se-0.1BPCCSO composite, the addition of graphene  further increased the electrical conductivity of the Cu2Se-BPCCSO-graphene composites, where the maximum value of 1538 S cm−1 was 36% higher than that of the 0.9Cu2Se-0.1BPCCSO composite without graphene and the value was approximately twice than that of Cu2Se. The results could be attributed to that the appropriate content of graphene could introduce extra electrically conductive paths and effectively improve the mobility of composites. Here, the Hall mobility reached 39.63 cm2 V−1 s−1, which was 57% higher than that of the 0.9Cu2Se-0.1BPCCSO composite  is shown in Fig. 2Se-0.1BPCCSO-x wt% graphene composites  were significantly improved within the entire temperature range, reaching 16.97 μW cm−1 K−2 at 1000 K of 0.9Cu2Se-0.1BPCCSO-0.035 wt% graphene, which was much higher than that of the Cu2Se-BPCCSO composite and Cu2Se sample. At the same time, the average PF values in the mid-to-high temperature range significantly increased. The average PF value of 0.9Cu2Se-0.1BPCCSO-0.035 wt% graphene reached 14.49 μW cm−1 K−2 in the temperature range from 473 K to 1000 K  composites possessed much higher weighted mobility than others, which was consistent with the Hall mobility and PF values, proving their good electrical transport properties.To get a better understanding of inherent electrical transport properties, the weighted mobility of the composites was calculated based on the measured Seebeck coefficient and electrical conductivity, as shown in Fig. 2Se-based thermoelectric materials are well known for their relatively low thermal conductivity due to their superionic nature. The temperature-dependent thermal conductivity of 0.9Cu2Se-0.1BPCCSO-x wt% graphene composites  is shown in Fig. x = 0.02 to x = 0.04, the thermal conductivity first increased and then decreased. Compared to 0.9Cu2Se-0.1BPCCSO, although 0.9Cu2Se-0.1BPCCSO-0.035 wt% graphene showed higher electrical conductivity, they had close thermal conductivity, which may be due to the much lower lattice thermal conductivity. Therefore, by subtracting carrier thermal conductivity, we obtained the lattice thermal conductivity of 0.9Cu2Se-0.1BPCCSO-x wt% graphene composites . The temperature-dependent lattice thermal conductivity and Lorentz constant (L) are presented in Supplementary Fig. 2Se-0.1BPCCSO composite sample possessed a relatively low lattice thermal conductivity, it is noted that the lattice thermal conductivity of the 0.9Cu2Se-0.1BPCCSO-x wt% graphene  composite samples was even lower. With the increasing content of graphene, the lattice thermal conductivity increased, which may be ascribed to the aggregation of graphene that reduced the interfacial phonon scattering effects. As revealed by the STEM results, due to the increase of multiple interfaces among BPCCSO, Cu2Se, and graphene, more phonon scattering sources strengthened the phonon scattering and improved the interfacial thermal resistance.In addition to the excellent electrical transport properties, Cuvl) and shear sound velocity (vs) were obtained. And the average sound velocity (va), phonon mean free path (lph), and elastic properties including Young’s modulus (E), Poisson ratio (νp), and Grüneisen parameter (γ) were calculated and listed in Supplementary Table va and lph values of 0.9Cu2Se-0.1BPCCSO-x wt% graphene  were also plotted in Fig. −1 to 1428 m s−1, which meant the softening of the phonons. Meanwhile, the declined phonon mean free path from 0.34 nm to 0.20 nm also proved the increase of phonon scattering.Furthermore, by measuring sound velocity, the longitudinal sound velocity  experiments were performed to have measured the generalized phonon density of states (GDOS) for Cuδ) was introduced, which is defined as ω) is the normalized intersection height of GDOS at a frequency ω51. The δ of Cu2Se-BPCCSO-nano graphite was 0.376. As a comparison, the δ of Cu2Se-nanographite, and BPCCSO-nano graphite were also calculated and the values were 0.437 and 0.514, respectively. The poor overlap provided a good explanation for the ultralow lattice thermal conductivity of Cu2Se-BPCCSO-graphene composites. Moreover, the peaks shifted to lower energy when measured at 600 K  were calculated and demonstrated in Fig. ZT values of the Cu2Se-BPCCSO-graphene composites were significantly improved. The ZT values of 0.9Cu2Se-0.1BPCCSO-0.035 wt% graphene reached 2.82 at 1000 K. Compared to Cu2Se and 0.9Cu2Se-0.1BPCCSO, the ZTmax value increased by ~50% and ~32%. Furthermore, the average ZT (ZTave) values of 0.9Cu2Se-0.1BPCCSO-0.035 wt% graphene in the temperature range from 473 K to 1000 K was over 1.7, which was superior to most Cu2Se-based materials with good thermoelectric performance45, as shown in Fig. ZTmax and ZTave values, the Cu2Se-BPCCSO-graphene composites in this work could meet the needs of thermoelectric devices.The temperature-dependent 2Se-BPCCSO-graphene composites were successfully prepared by a fast preparation method of combining self-propagating high-temperature synthesis (SHS) with spark plasma sintering (SPS). The Cu2Se-BPCCSO-graphene composites presented excellent thermoelectric properties with a ZTmax > 2.8 at 1000 K and ZTave > 1.7 in the mid-to-high temperature range. The underlying mechanisms of carrier and phonon transport were deeply investigated. On one hand, the electrical performance was largely improved with the highest PFmax value of 16.97 μW cm−1 K−2 at 1000 K and PFave value of 14.49 μW cm−1 K−2 from 473 K to 1000 K, which could be attributed to the optimized carrier concentration and Hall mobility by compositing with BPCCSO and graphene. On the other hand, multiple scattering sources like interfaces between BPCCSO, Cu2Se, and graphene revealed by STEM strongly scattered phonons, resulting in extremely low lattice thermal conductivity. The weak interface thermal transport in such composites was further proved by inelastic neutron scattering (INS) techniques. This work gives a paradigm to improve the thermoelectric performance of Cu2Se-like superionic semiconductors, which could be further adopted in other thermoelectric systems.In this study, Cu2Se-0.1Bi0.88Pb0.06Ca0.06CuSeO (BPCCSO)-x wt% graphene  composites were fabricated by a fast preparation method of combining self-propagating high-temperature synthesis (SHS) with spark plasma sintering (SPS). Stoichiometric amounts of Bi , Bi2O3 , Cu , Se , PbO , and CaO  powders were thoroughly mixed by hand grinding, then cold pressed into pellets and underwent the SHS process. For each sample, the weight of the pellet was about 10 g. The pellet was put in an alumina crucible. The SHS process started by heating the bottom of the crucible to the ignition temperature with an alcohol lamp. Once the reaction began from the lower melting point component, we put the lid on the crucible and moved the alcohol lamp away. The combustion wave was persisted by the energy released from the initial reaction and spread to the whole pellet in few seconds. The obtained bulks were ground into fine powders and fully mixed with graphene  by hand grinding for more than 30 min, and then sintered by an SPS furnace at 973 K under a uniaxial pressure of 40 MPa for 3 min in vacuum.The 0.9Cuλ = 0.15406 nm) at the scanning rate of 1° min−1. The samples for XRD measurement were fine powders crushed and ground from the sintered bulks. The microstructure of different samples was observed by field-emission scanning electron microscopy  using backscattering electron imaging (BEI) mode at 15 kV. The crystallography, compositions, and interfacial structure were further investigated by a double CS-corrected scanning transmission electron microscopy  equipped with probe and image correctors simultaneously. The probe convergence angle and high angle annular dark-field (HAADF) acceptance angles were 25 mrad and 48-200 mrad, respectively. The TEM specimens were prepared by ion milling with an Ar gas source, and the energy was 2 kV for perforation and 0.5 kV for the removal of the amorphous layer.The identification of phase purity and crystal structure for the composites was analyzed by X-ray diffraction  with a Cu Kα radiation  as a function of energy transfer at the neutron-energy-gain side. The GDOS accounts for the temperature dependence of the scattering function, which is related to the thermal population of excitations.The INS experiments were conducted on the time-of-flight cold neutron spectrometer, Pelican, at the Australian Nuclear Science and Technology Organisation (ANSTO). The five powder samples are Cuσ) and Seebeck coefficient (S) were measured by a commercial thermoelectric measurement system  from room temperature to 1000 K under the protective atmosphere of helium gas. The sample size was about 3 × 3 × 11 mm3. The carrier concentration (n) and Hall mobility (μΗ) were determined from room temperature Hall coefficient (RH) measurements by using the van der Pauw method on a homemade system equipped with a maximum 5 T superconducting magnet . The sample size was about 10 × 10 × 0.5 mm3. Thermal conductivity (κ) was calculated from the equation, κ = DρCp, where ρ is the density of the specimen, Cp is the specific heat capacity, and D is the thermal diffusivity, respectively. The ρ was measured by the Archimedes method. The Cp was measured by differential scanning calorimetry on a DSC  under an argon atmosphere and estimated according to the Neumann–Kopp rule in this work. And the D was measured by a laser flash method  from room temperature to 1000 K under a continuous argon flow. The longitudinal and shear sound velocities were obtained at room temperature via an ultrasonic pulse-echo method using an Olympus 5072 PR pulser/receiver. The sample size used for thermal diffusivity and sound velocity measurements was about 10 × 10 × 1.5 mm3. The electrical and thermal properties of all the samples were measured in the same direction (perpendicular to the direction of pressure). The uncertainties of the measured Seebeck coefficient and electrical conductivity are approximately 3% and 5%, respectively. The uncertainty of thermal conductivity measurement is about 10%. As a result, the uncertainty of ZT is estimated to be about 20%.The values of electrical conductivity (Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary