Meanwhile, 3D-GS-CBF can be stable in the solution even after 6 months, addressing the instability issues of conventional graphene networks.A multiscale modeling including non-equilibrium molecular dynamics simulations and heat conduction model is applied to interpret experimental results. 3D-GS-CBF aqueous medium can largely improve the solar vapor evaporation rate (by 1.5 times) that are even comparable to the interfacial heating system; meanwhile, its cooling performance is also superior to commercial coolant in thermal management applications.Highly thermo-conductive aqueous medium is a crucial premise to demonstrate high-performance thermal-related applications. Graphene has the diamond comparable thermal conductivity, while the intrinsic two-dimensional reality will result in strong anisotropic thermal conductivity and wrinkles or even crumples that significantly sacrifices its inherent properties in practical applications. One strategy to overcome this is to use three-dimensional (3D) architecture of graphene. Herein, 3D graphene structure with covalent-bonding nanofins (3D-GS-CBF) is proposed, which is then used as the filler to demonstrate effective aqueous medium. The thermal conductivity and thermal conductivity enhancement efficiency of 3D-GS-CBF (0.26 vol%) aqueous medium can be as high as 2.61 W mThe online version of this article (10.1007/s40820-020-00478-2) contains supplementary material, which is available to authorized users. The −1 K−1 , while tn issues . The rean issues . 3D grapn issues , while tn issues , 34, hav−1 K−1, which yields a record high TCEE of ~ 1300%. Meanwhile, it exhibits a long-term stability (> 6 months) in the solutions, resolving the instability issues of conventional non-covalent graphene structures. We further implement multiscale modeling technique (including non-equilibrium molecular dynamics simulations and heat conduction equations) to study the underlying mechanisms. Two examples of the solar thermal conversion and LED thermal management processes are then used to prove the potential thermal application of the 3D-GS-Fin aqueous medium.In this paper, we suggest an efficient aqueous medium based on the 3D graphene continuous structure with covalent-bonding nanofins (3D-GS-CBF) on the skeleton. In the 3D-GS-CBF, apart from the graphene skeleton which conducts heat, the nanofins covalently grown on the top of the skeleton can exchange energy with the liquid medium as well. As a result, the heat transfer at the graphene–liquid medium interfaces can be improved largely Fig. b. With a4 (5 mL min−1) and H2 (5 mL min−1) was introduced into the quartz tube. The pressure of chamber was maintained at 30 Pa during the growth procedure. After deposition for 1 h, the system was cooled to room temperature. The as-synthesized graphene-Ni foam was immersed in polymethyl methacrylate (PMMA) solution (4 wt% in ethyl lactate) for seconds and then dried at 80 °C for 2 h. The PMMA-coated graphene-Ni foam was immersed into a 3 M HCl solution at 80 °C overnight to dissolve the Ni template. After removing the PMMA with hot acetone solution at 50 °C, the hierarchical and free-standing 3D-GS-CBF was obtained. The dried sample was then functional group modified by 500 ppm moist ozone flow (1 L min−1) for 5 min. As for the preparation of 3D-GS, the commercial Ni foam with the same size was heated to 1000 °C in a quartz tube under a mixture of Ar (500 mL min−1) and H2 (200 mL min−1) for 5 min. Thereafter, CH4 of 10 mL min−1 was introduced into the reaction tube at ambient pressure. After feeding the reaction-gas mixture flow for 5 min, the sample was rapidly cooled to room temperature at a cooling rate of ∼100 °C min−1 under the mixed H2 (200 mL min−1) and Ar (500 mL min−1) flow. The etching process was consistent with those described in the previous work.The commercial Ni foam (1.6 mm in thickness) was cut into pieces with the lateral size needed. Then, it was placed into a cylindrical quartz tube . The quartz tube was first evacuated to a low pressure of 3 Pa and then heated up to 700 °C. An inductively coupled plasma (ICP) source with a radiofrequency power of 250 W was employed. A mixture of CHα of aqueous medium was measured by a laser flash analysis apparatus (NETZSCH LFA467 Nanoflash). Then the thermal conductivity k could be calculated as k = ραc. The thermal conductivity enhancement (TCE) is defined as TCE = (k −kwater)/kwater × 100%, where kwater denotes thermal conductivity of water. The thermal conductivity enhancement efficiency (TCEE) is defined as TCEE = TCE/ϕ, where ϕ presents the fraction of filler in water.The morphology and structure of sample were inspected by scanning electron microscope  and transmission electron microscope . Raman spectra were confirmed by Raman spectrometer (LabRAM HR Evolution) with a 532-nm laser beam. X-ray photoelectron spectroscopy (XPS) measurement was taken through VG ESCALAB MARK II spectrometer. The contact angles of samples were tested by a computerized contact angle analyzer , and images of droplet impact were captured with high-speed camera (REDLAKE MotionXtra HG-100 K). The thermal diffusivity 2, which was measured by light intensity meter (PD130). A quartz container with an inner diameter of 26 and 6 mm in depth was put at the solar thermal position. A thermocouple (TT-T-36) was fixed at the bottom of the container to measure the temperature change, and an electric balance  was used to record the mass change for calculating the evaporation rate. The solar vapor generation rate can be calculated as:−2 h−1), m and t are the evaporation amount of the stabilization state, respectively, A is the area of evaporation setup. Afterward, solar vapor generation efficiency can be defined as:I is the power density of the light (W m−2).Simulated solar light was generated by a xenon lamp (PLS-SXE300D). The light intensity at test spot was kept at 1000 W/mThe experimental setup consisted of a pump, a tank and a DC power supply (UTP1306S). The test section consisted of a water block, a high-power LED light and thermocouples. The water block was assembled by a poly-(methyl methacrylate) (PMMA) cover and an aluminum plate with an interspace of 40 × 40 × 2.5 mm. High-power LED light  was attached on Al plate by a layer of thermal interfacial material. Three thermocouples were applied to measure the temperature of the LEDs, inlet, and outlet water, respectively. The operating current of LEDs was recorded using a Keithley 2602 source meter.VA and VR denote the attractive and repulsive pair terms, respectively; bij term presents the reactive empirical bond order between carbon atoms. In the extension from REBO to AIREBO potential, non-bonded interactions and dihedral-angle interactions term are added, i.e.,The inter-atomic interactions of carbon atoms in graphene sheets were characterized by the adaptive intermolecular reactive empirical bond order (AIREBO) potential, which can accurately describe the carbon nanostructure system. The REBO function is defined mathematically as:qi, rij, εi and σi represent the charge of ith atom, the distance between ith and jth atom, the minimum energy and zero energy separation distance, respectively. The inter-atomic LJ parameters between different species were calculated based on Lorentz–Berthelot mixing rules  potentials were employed to characterize the interactions between graphene and water molecules. Based on the XPS results, hydroxyl groups were also applied on the graphene surface to accurately describe the interfacial interactions. In these models, the interactions between atomic sites can be expressed as:mentclass2pt{minim–6). Periodic boundary conditions are applied in all directions. During the MD simulation, the system is firstly relaxed for 5 ns in the NVT ensemble, followed by NPT ensemble for 2 ns (at 1 atm and 300 K). Subsequently, an extra 2 ns in NVE ensemble is carried out to confirm the equilibrium state of system. To get steady temperature gradient, a heat source (350 K) and a heat sink (250 K) are set on the left and right graphene atoms, respectively. When the steady temperature profiles are established, the interfacial thermal resistance can be calculated as Rb = AΔT/J, where J is the heat flux, ΔT is the temperature drop, and A is the surface area of interface.The non-equilibrium molecular dynamics (NEMD) simulations were performed by Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) . A cutofz-direction. The transient temperature difference is conducted within 8 × 10–6 s before the system approach to the stable equilibrium state. In this FEM calculation, interfacial thermal resistance Rb is obtained from MD simulations, while other parameters are empirical parameters , 3D graphene structure (3D-GS) and 3D graphene structure with covalent-bonding nanofins (3D-GS-CBF). GN is synthetized via the modified Hummer’s method, while 3D-GS and 3D-GS-CBF are prepared through plasma-enhanced chemical vapor deposition (PECVD). GN is dispersedly distributed without framework, exhibiting a petal-liked nanosheet morphology and soft-layered structure with the lateral size of several micrometers Fig. S1). The discrete GN distribution in aqueous solutions is lack of continuous heat transfer pathways, resulting in strong phonon scattering between the graphene nanoplates  is about ~ 1.21, also indicating that there are numerous graphene nanofins with edges in the 3D-GS-CBF .3D-GS-CBF shows a similar 3D porous skeleton structure with 3D-GS Fig. c, f. Meak of GN aqueous medium is 0.66 W m−1 K−1 with the GN volume fraction of 0.1 vol%  once the same volume fraction of 3D-GS-CBF is added into the water, demonstrating its potential as efficient heat dissipation materials. In addition, we also study the effect of volume fraction of the filler, i.e., 3D-GS-CBF. With improving the volume fraction of 3D-GS-CBF in the aqueous medium to 0.26 vol%, the apparent thermal conductivity k will increase to a record high value of 2.61 W m−1 K−1 with a thermal conductivity enhancement (TCE) of 355%  and 2.82 W m−1 K−1 (at 80 °C), demonstrating the good thermal stability for practical long-term work. On the other hand, our 3D-GS-CBF sample shows that the structure can be stable in the solution even after 6 months, addressing the stability issues  of conventional non-covalent graphene networks as aqueous medium fillers  [To demonstrate the merits of our structure design, we also compare the thermal transport property of 3D-GS-CBF aqueous medium with other existing aqueous mediums with various fillers. Our aqueous medium exhibits an excellent thermal conductivity with only 0.26 vol% of 3D-GS-CBF, which is comparable to that of boron nitride (BN) nanosheets filling aqueous medium with over 20 vol% of BN fillers  , 37–41, Rb = 6.7 × 10–9 K m2 W−1 [To investigate the underlying mechanisms of the thermal transport process, we implement the multiscale modeling approach, i.e., non-equilibrium molecular dynamics (NEMD) simulations and heat conduction equations, to capture the detailed microscopic information. The interfacial thermal conductance between graphene and water is firstly calculated via NEMD simulations and is then used in the heat conduction models. Based on the temperature distribution Fig. a, the inK m2 W−1 , 43), muBased on the NEMD results, heat diffusion simulations are carried out to study the temperature evolution and distribution of the graphene structures. All the parameters used in the heat conduction equations are listed in Table −2 h−1 , while those of Au and GN cases are merely 0.50 and 0.74 kg m−2 h−1, respectively. As presented in Fig. 3O4@CNT [2 [−2 h−1 and efficiency of 50~90%) with the optimized light adsorption capability , rational thermal management  to reduce heat loss and ingenious water pathway design, etc., paving a new way of enhancing the inherent heat transfer efficiency of aqueous mediums to achieve high-performance volumetric solar-driven vapor generation system.Before closing, we apply our samples for two practical applications: solar thermal conversion and thermal management of LEDs. The experimental setup for solar thermal conversion application is presented in Fig. −1 Fig. S, while t 3O4@CNT , 46.8% o4@CNT [2 ). More iThe second practical application example is the thermal management of LEDs. Here, we study the cooling process of LEDs via pure water, 3D-GS-CBF and commercial coolant . The corresponding experimental setup is presented in Fig. −1 K−1) with an ultralow loading of 0.26 vol%, yielding a record high TCEE of 1300%, which is almost orders of magnitude larger than those in the state-of-the-art studies (< 200%). The multiscale simulations suggest that the large amount of nanofins on the top of graphene skeleton will increase the surface ratio largely and then increase heat exchange at the graphene–water interfaces significantly. As a result, in comparison with GN counterpart, the excellent thermal transport properties of 3D-GS-CBF aqueous medium can largely enhance the evaporation rate by 1.5-fold (up to 1.13 kg m−2 h−1) and its corresponding efficiency by 1.7-fold (up to 70.8%) in the solar thermal conversion applications. Moreover, the 3D-GS-CBF aqueous medium can also reduce the operation current by 18.3%, demonstrating a superior thermal management performance over commercial coolant. This work suggests a new venue of fabricating 3D continuous, covalent graphene networks to prepare high thermally conductive aqueous medium for high-performance advanced thermal applications. As a prime example, improving the thermal property can potentially increase the temperature rising rate of aqueous medium and thus boost the reaction kinetics of photocatalytic reactions, which deserves to be investigated in future work.In conclusion, 3D graphene continuous structure with covalent-bonding nanofins is fabricated through PECVD method to realize high thermally conductive aqueous mediums. Our results show that 3D-GS-CBF can enhance the thermal transport properties of its corresponding aqueous medium by ~ 4.4-fold (2.61 W mSupplementary file1 (PDF 320 kb)Below is the link to the electronic supplementary material.


Graphene oxide exhibits high hydrophilicity and dispersibility. Thus, it is difficult to be separated from aqueous solutions. Therefore, functionalization with magnetic nanoparticles is performed in order to prepare a magnetic GO nanocomposite that combines the sufficient adsorption capacity of graphene oxide and the convenience of magnetic separation. Moreover, the magnetic material can be further functionalized with different groups to prevent aggregation and extends its potential application. Until today, a plethora of magnetic GO hybrid materials have been synthesized and successfully employed for the magnetic solid-phase extraction of organic compounds from environmental, agricultural, biological, and food samples. The developed GO nanocomposites exhibit satisfactory stability in aqueous solutions, as well as sufficient surface area. Thus, they are considered as an alternative to conventional sorbents by enriching the analytical toolbox for the analysis of trace organic compounds.Graphene oxide (GO) is a chemical compound with a form similar to graphene that consists of one-atom-thick two-dimensional layers of sp Solid-phase extraction (SPE) and liquid-liquid extraction (LLE) are two widely used and well-established techniques for the extraction of organic compounds. However, these conventional techniques tend to have many fundamental drawbacks such as complicated and time-consuming steps, requirement for a large amount of organic solvents and sample, as well as difficulties in automation ,2,3,4. RMagnetic solid-phase extraction (MSPE) is a form of dispersive solid-phase extraction in which a magnetic sorbent is added into an aqueous sample in order to adsorb the target analytes. The sorbent is easily separated by applying an external magnetic field . Subsequ18) [Because of the evolution of technology and nanotechnology, novel extraction sorbents with improved chemical and physical properties have been synthesized and successfully used for magnetic solid-phase extraction of target analytes. Moreover, with the use of these materials, high extraction efficiency, good reproducibility in combination with low detection and quantification limits can be achieved ,2. Typic18) , activat18) , carbon-18) , graphen18) , graphen18) , metal-o18) , covalen18)  and zeolGraphene oxide is the oxidized form of graphene that can be easily prepared from natural graphite powder with Hummer’s method after reaction with an anhydrous mixture of sulfuric acid, sodium nitrate and potassium permanganate ,22,23,242-bonded carbon and the material is rich in oxygen-containing groups including hydroxyl, carboxyl and epoxy groups, which assist the interaction between the sorbent and organic molecules through strong π-π stacking, hydrophobic interaction and hydrogen bonding [In analytical chemistry, GO has been successfully employed for the sample preparation of a wide variety of samples including biological, food and environmental matrices ,27,28. G bonding ,30,31.3O4 [Graphene oxide is an ultra-light material that poses high dispersibility in aqueous solutions as well as high hydrophilicity which makes its separation from this kind of solutions difficult. In order to improve the separation, GO can form magnetic nanocomposites with magnetite through electrostatic interaction between the negatively charged graphene oxide sheets and the positively charged surface of Fe3O4 . The mag3O4 ,34.Many reviews have been published regarding the applications of graphene and graphene-based sorbents in the field of the sample preparation ,37,38,393O4 has been employed for the extraction of a wide variety of organic compounds from various samples. The surface of magnetic graphene oxide is rich in hydroxyl and carboxyl groups, which assist the interaction between the sorbent and the target analytes through strong π-π stacking, hydrophobic interaction as well as hydrogen bonding [Due to its high surface area and its superparamagnetic properties GO/Fe bonding ,29,30,312+  and of Fe3+  are added, the mixture is heated and ammonium hydroxide is slowly added and the magnetite nanoparticles are formed [The one-step co-precipitation approach is the most common synthetic route for the preparation of magnetic GO. In this approach, graphene oxide is dispersed in water. Subsequently, salts of Fee formed .3+ salt  and sodium acetate, under vigorously stirring for 30 min at room temperature [3+ to Fe3O4.Another common synthetic procedure is the solvothermal approach, in which GO is added in a dispersion of a Feperature ,40. The 3O4 under ultrasonic irradiation, stirring or mechanical shaking. In this case, Fe3O4 nanoparticles are dispersed in nitric acid solution in an ultrasonic bath for 30 min in order to generate a positively charged surface. Graphene oxide is dispersed in deionized water in order to generate a negatively charged surface. Subsequently, the dispersions of Fe3O4 and GO are mixed, the pH of the mixture is adjusted to a desired value and the mixture is subjected to vigorous magnetic stirring, mechanical shaking or ultrasonic radiation for a certain time span  [3O4 material can be prepared after hydroxylation of Fe3O4 nanoparticles by mixing with GO and dimethyl sulfoxide. After ultrasonic treatment of the mixture for a certain time at room temperature, the desired material is obtained [Magnetic GO can be prepared by mixing GO and Feg., 5 h) . Finallyobtained .3O4. 3O4 for the MSPE of organic compounds from biological, environmental and food samples.Organic molecules of various chemical groups have been extracted with GO/Fe3O4 material was found to be reusable in most cases.Regarding the analysis of biological samples, graphene oxide has been employed for the MSPE of tamsulosin hydrochloride , pseudoe3O4 magnetic nanocomposite has been employed for the extraction of 2,4,4′-trichlorobiphenyl (PCB 28) [3O4.The GO/Fe(PCB 28)  and atra(PCB 28)  from wat(PCB 28) , polycyc(PCB 28) , 2,4,6-t(PCB 28) , malachi(PCB 28) , the cyt(PCB 28)  as well (PCB 28)  have bee3O4 sorbent prior to their determination by HPLC–MS/MS [3O4 prior to its spectrophotometric and optimized angle mode-mismatched thermal lens spectrometric determination (OAMTLS) [Sulfonamides have been extracted from milk samples by GO/FeLC–MS/MS  or capilLC–MS/MS . Sulfadi(OAMTLS) .3O4 for the extraction of organic compounds from food samples include the extraction of patulin from apple juice [Other applications of GO/Fele juice , flavorsle juice , lignansle juice , flavonole juice , azo dyele juice .3O4 nanomaterial include acetone, n-hexane, acetonitrile, methanol etc., however, only a small quantity of these solvents was required. In most cases, the GO/Fe3O4 sorbent was found to be reusable after a regeneration and a washing step. The as-prepared nanomaterial has some significant benefits including high surface area, superparamagnetic properties and strong magnetism. The synthetic procedures that were reported were simple, mild, rapid an economic and the MSPE method provided the benefits of limited organic waste, no requirement for time-consuming centrifugation, column-passing or filtration steps.The organic solvents that were mainly used for the elution of the adsorbed analytes from the GO/Fe3O4 nanoparticles and the graphene sheets, the magnetic RGO sorbent shows distinguished properties including good dispersity, high surface area, high adsorption efficiency and good super-paramagnetism [Reduced graphene oxide (RGO) is a nanomaterial obtained by chemical reduction of graphene oxide that contains less oxygen groups and has properties closer to those of graphene ,58. RGO agnetism .3O4 sorbents. The co-precipitation approach is a multi-step procedure in which GO/Fe3O4 previously prepared by precipitating Fe2+ and Fe3+ in the presence of GO is reduced with the addition of hydrazine hydrate [3O4 can be also prepared through the hydrothermal method, which is similar to the solvothermal but instead of organic solvents that are used in the solvothermal method, water is used as a solvent in the hydrothermal approach. For this purpose, a salt of Fe3+ and sodium hydroxide is added to an aqueous solution of graphene oxide and the mixture is heated in an autoclave for a certain time span [3O4 prepared by solvothermal (a), hydrothermal (b) and co-precipitation (c) methods.There are different synthetic procedures for the fabrication of RGO/Fe hydrate ,65. For  hydrate ,66. The ime span ,67. Figu3O4 prior to their determination by HPLC-DAD [3O4 has been also used for the MSPE of bisphenol A from water samples prior to its determination by HPLC-UV. The MSPE technique was coupled with dispersive liquid–liquid microextraction (DLLME) in order to utilize the benefits of both sample preparation techniques. The magnetic sorbent was separated conveniently and rapidly from the sample matrix and it was found to be reusable for at least 12 repeated cycles [Sudan dyes have been extracted from tomato sauce and chili-containing foods with RGO/FeHPLC-DAD . The devd cycles .3O4@Au sorbent was used for the MSPE of organochlorine pesticides from seawater samples prior to their determination with GC-MS [In 2016, Mehdinia et al. developed a microwave-assisted synthesis of reduced graphene oxide decorated with magnetite and gold nanoparticles. Gold nanoparticles offer the benefits of chemical stability, biocompatibility, satisfactory magnetic properties and possibility for chemical modification. The RGO/Feth GC-MS .A magnetic polyethyleneimine functionalized reduced graphene oxide nanocomposite was synthesized and used for the MSPE of polar non-steroidal anti-inflammatory drugs from water samples  and pola3O4-carbon nanotubes composite via a simple and green one-pot solvothermal approach. For this purpose, iron (III) chloride hexahydrate, sodium acetate, GO and CNTs were added in a Teflon-liner and the suspension was heated at 200 °C for 10 h. It was found that the Fe3O4 nanoparticles were covered with RGO and CNTs, while CNTs were inserted between the RGO sheets in order to prevent the aggregation of the RGO sheets. The novel sorbent was used for the MSPE of sulfonamides from milk prior to their determination with HPLC-UV [3O4 nanoparticles. After incorporation into alginate hydrogel microspheres, the developed sorbent was successfully applied for the MSPE of phthalates from bottled drinks [In 2018, Feng et al. synthesized RGO/Fe HPLC-UV . Pinsritd drinks .3O4 sorbent, Mahpishanian and Sereshti reported the one-step green synthesis of β-cyclodextrin/iron oxide-RGO nanocomposite. The sorbent was used for the MSPE organochlorine pesticides residue from honey samples prior to their determination by gas chromatography-electron capture detection (GC-ECD). In this case, functionalization with β-cyclodextrin was chosen, due to the high potential supramolecular recognition capability of β-cyclodextrin [In order to increase the selectivity of the conventional RGO/Feodextrin .Akbarzade et al. synthesized zero valent Fe-RGO quantum dots and used them for the MSPE of organophosphorus pesticides from real water and fruit juice samples prior to their determination with GC-MS. The quantum dots were prepared with a hydrothermal cutting method and sodium borohydride was used for the reduction. The novel sorbent provided short extraction time, simple operation as well as high enhancement factor . The main disadvantage of graphene oxide-based adsorbents is the important π–π stacking interactions between graphene oxide nanosheets, which are responsible for serious aggregation and restacking of the nanosheets, resulting in a potential block of the active adsorption sites of the sorbent and a decrease of its specific surface area. In order to overcome this problem, functionalization of the sorbent with different molecules that can enter between the GO nanosheet and prevent them from aggregation and restacking can take place .In order to overcome this limitation, Yilmaz et al. developed a magnetic nanodiamond/graphene oxide hybrid and used it for the MSPE of sildenafil from alleged herbal aphrodisiacs by HPLC-DAD system. Functionalization with nanodiamond successfully prevented the aggregation and restacking of GO nanosheets .Functionalization of graphene oxide-based adsorbents can also take place to enhance the extraction efficiency of the material by introducing compatible chemical molecules with high surface area and abundant functional groups in the structure of GO.3O4 sorbent exhibited higher extraction efficiency compared to the conventional GO/Fe3O4 nanocomposite.Polyamidoamine (PAMAM) dendrimer has been used to develop amino-terminated hyper-branched PAMAM polymer grafted magnetic graphene oxide nanosheets for the MSPE of selective serotonin reuptake inhibitors from plasma samples . Due to 3O4 due to the high density of surface functional groups such as amines, amides and carboxylic groups of SEP [Functionalization with soluble eggshell membrane protein (SEP) was found to increase the stability and adsorption performance as well as accuracy and recoveries of GO/Fes of SEP .3O4 nanocomposite and the sorbent was used for the MSPE of sulfonamides from tap and river water samples [3O4.Porphyrin has been also used for the functionalization of GO/Fe samples . Due to 3O4 in order to develop a sorbent for the MSPE of chlorophenols from environmental water prior to their determination by HPLC-MS/MS [A co-polymer of divinylbenzene (DVB) and glycidylmethacrylate (GMA) was used for the functionalization of GO/FeLC-MS/MS . Due to 3O4 nanoparticles [Polystyrene (PS)  and polyarticles .3O4 [3O4, providing satisfactory extraction efficiency. The functionalized nanocomposite was used for the MSPE of chlorpheniramine [Polythionine was also employed for the functionalization of magnetic graphene oxide through an oxidative polymerization reaction of thionine on the surface of GO/Fe3O4 ,85. Thisniramine  and dulo3O4 was applied for extraction of PAHs from vegetable oils. Due to the super-amphiphilicity of phytic acid, the dispersibility of the conventional GO/Fe3O4 sorbent increased. [In order to enhance the dispersibility of magnetic GO in hydrophobic media, functionalization with phytic acid has been reported. Phytic acid-stabilized GO/Fecreased. .3O4 can also be performed for the enhancement of its selectivity towards the target analytes. In 2015, Abdolmohammad-Zadeh and Talleb synthesized a β-cyclodextrin (β-CD) grafted GO/Fe3O4 nano-hybrid and used it for the MSPE of gemfibrozil from human serum and pharmaceutical waste-water samples followed by determination using spectrofluorometry. This chemical compound can selectively bind with various organic, inorganic and biological guest molecules into its cavity to form stable host–guest inclusion complexes by a series of forces such as hydrophobic and van der Waals interactions. Therefore, due to the surface modification of graphene oxide with β-cyclodextrin, selective separation of the target analyte from complex sample matrices was achieved [Functionalization of GO/Feachieved .3O4 are triethylenetetramine [3O4 nanocomposites. Other examples of chemical molecules that have been used for the functionalization of GO/Feetramine , silica etramine , sporopoetramine  and phenetramine . The sup3O4 and its application in the MSPE procedure.Moreover, Molecularly imprinted polymers (MIPs) are highly selective, tailor-made synthetic polymeric materials that exhibit high adsorption capacity. Moreover, they can be easily prepared with economic synthetic procedures. Therefore, MIPs have been applied for the extraction and preconcentration of trace analytes in diverse fields, including natural, agricultural, and food products and environmental samples ,93,94. T3O4 nanoparticles were grafted with acrylic acid. Subsequently, the MIPs-GO/Fe3O4 sorbent was prepared from 17β-E2 (template molecule), acrylamide , ethylene glycol dimethacrylate (cross-linker), and 2,2′-azobis(isobutyronitrile) (initiator) in acetonitrile dispersion of the functionalized magnetic GO material. With the use of developed sorbent, specific recognition, as well as highly effective removal of 17β-E2 from complicated matrices, were achieved [Ning et al. developed a molecularly imprinted polymer on magnetic GO and used it for the extraction of 17β-estradiol from milk powder samples. For this purpose, GO/Feachieved . Barati achieved .Metal-organic frameworks are mixed organic-inorganic supramolecular materials that became popular in 1995, when Yaghi and Li reported the synthesis of a MOF with large rectangular channels . These mThe combination of graphene oxide and metal organic frameworks enhances the merits of sorbent including its reusability, its pore volume, its dispersion capability, its extraction capacity, its mechanical strength as well as its surface area ,103,104.3O4 and MOFs for the rapid separation and highly selective adsorption of triazole pesticides [Liu et al. developed a sorbent based on magnetic graphene oxide functionalized MOF-199 with the aim to combine the benefits of GO/Festicides . The sorA high-affinity graphene oxide-encapsulated magnetic zirconium-MOF was developed for the MSPE of photosensitizers hematoporphyrin and hematoporphyrin monomethyl ether from human urine prior to their determination by ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) . The nov3O4 nanoparticles with core-shell structured through covalent bonding and subsequently the GO surfaces were modified with Cu-MOFs. The silica shells prevented the oxidation and agglomeration of Fe3O4 nanoparticles and served as a platform to integrate the Fe3O4 particles and GO nanosheets through covalent bonds. Finally, the functionalization with the Cu-MOFs enhanced the extraction efficiency by providing more active sites for adsorption because of the high porosity and tunability of MOFs. 3O4@SiO2-GO-MOFs (b) as well as the TEM Cu-MOFs (c) and Fe3O4@SiO2-GO-MOFs (d).Wang et al. synthesized magnetic Cu-MOFs embedded within graphene oxide nanocomposites and used it for the MSPE of benzenoid-containing insecticides prior to their determination by HPLC-UV . For thi3O4/polytyramine (GO/MOF-74/Fe3O4/PTy) nanoporous composite and used it for the magnetic dispersive micro solid-phase extraction (MD-μ-SPE) of prokinetic drugs prior to their determination with HPLC–UV [Pourbahman et al. synthesized a graphene oxide/metal–organic framework-74/Fe HPLC–UV . The sur HPLC–UV  and the  HPLC–UV . The fir HPLC–UV . The com HPLC–UV . HoweverMicroporous organic polymers (MOPs) represent a class of amorphous porous materials, composed of fully covalently bound organic building blocks. MOPs have lately gained much research interest, due to the combined superiority of porous materials and functional polymers ,109,110.3O4 nanopaticles were modified by triazine-based polymeric prepared from melamine and terephthaldehyde. Subsequently, the functionalized Fe3O4 nanoparticles were mixed with GO in THF and the mixture was sonicated. The novel sorbent showed well-defined porosity, high surface area, good chemical stability and tunable surface chemistry. Due to the different functional groups of the nanocomposite , simultaneous extraction of basic and acidic pesticides was achieved [By combining MOPs and magnetic GO it is possible to develop nanocomposites that combine the extraordinary properties of both MOPs and GO. Shahrebabak et al. synthesized a triazine-based polymeric network modified magnetic nanoparticles/GO nanocomposite and used it for the MSPE of basic and acidic pesticides from food  and water samples by HPLC-UV. For the fabrication of the material, Feachieved .Ionic liquids (ILs) and deep eutectic solvents (DESs) are an alternative to environmentally harmful ordinary organic solvents. ILs are generally composed of bulky, non-symmetrical organic cations  and different inorganic or organic anions ,118,119.3O4@SiO2 magnetite microspheres functionalized with amino-groups (Fe3O4 @SiO2-NH2) and 1-carboxymethyl-3-methylimidazolium chloride were used to functionalize graphene oxide. The novel sorbent exhibited great adsorption capacity and was successfully used to extract both polar and non-polar chlorophenols from tap, river and well water. The sorbent was found to be reusable for at least six times.In 2016, Cai et al. synthesized a planar graphene oxide-based magnetic ionic liquid nanomaterial for extraction of chlorophenols from environmental water samples coupled with HPLC-MS/MS . For thi3O4 nanoparticles were treated with 1-hexadecyl-3-methylimidazoliumbromide in a phosphate buffer solution (pH 7.0) and the mixture was subjected to ultrasonic mixing 5 min. Due to the π-π stacking, the hydrophobic and the electrostatic interactions between the mixed hemimicelles and analytes, the modified material exhibited high surface area and excellent adsorption capacity. Moreover, after the extraction and desorption cycle, it is possible to regenerate the GO/Fe3O4 with water and methanol. A decrease in the sorbent amount was observed after six cycles, however no significant loss of the sorption capacity occurred.Wu et al. developed a mixed hemimicelles MSPE method for the extraction of cephalosporins from biological samples based on ionic liquid-coated magnetic graphene oxide nanoparticles prior to their determination by HPLC-UV . For the3O4 nanocomposite was prepared and used for the MSPE of PAHs from vegetable oil prior to their determination by GC–MS [A three-dimensional ionic liquid functionalized magnetic GO/Feby GC–MS . The ion3O4, in order to develop a MSPE sorbent for the preconcentration of methadone in biological and water samples followed by GC–FID and GC–MS [3O4 in a round-bottomed flask under stirring at room temperature for 1 h. The highest extraction recovery for methadone was observed with the TNO hydrogen-bond donors at a molar ratio of 1:2. of ChCl:TNO. The developed methods exhibited high preconcentration factor and satisfactory extraction recovery, however no reusability data were reported.Lamei et al. evaluated different DESs, based on choline chloride (ChCl) for the modification of GO/Fend GC–MS . For thi3O4 nanocomposite contains a large amount of hydroxyl and carboxyl groups, which assist the interaction between the sorbent and organic molecules through π-π stacking, hydrophobic interaction as well as hydrogen bonding. In most MSPE methods, the GO/Fe3O4 sorbent was found to be reusable after the desorption step, indicating satisfactory stability of the materials. Moreover, the synthetic procedures for the preparation of magnetic graphene oxide are simple, mild, rapid and economic.Due to the sufficient surface area and its superparamagnetic properties, magnetic graphene oxide has successfully been employed for the extraction of a wide variety of organic compounds from environmental, food, biological and agricultural samples. The surface of magnetic GO/FeThe main limitation of graphene oxide-based adsorbents is aggregation and restacking of the nanosheets, which may lead to blocking of the active adsorption sites of the sorbent, as well as to the reduction of its specific surface area. A wide variety of functional groups have been employed to overcome this problem. Enhancement of the dispersibility of magnetic graphene oxide in hydrophobic media can be also achieved through functionalization with appropriate chemical groups. Functionalization of magnetic graphene oxide has been also employed in order to enhance the extraction efficiency of the nanocomposite by introducing compatible chemical molecules with high surface area. Moreover, by using functional groups that can selectively bind with various organic, inorganic and biological guest molecules such as β-cyclodextrin, the selectivity of the extraction towards the target analyte can be significantly improved.Microporous organic polymers, molecular imprinted polymers, metal-organic frameworks, and zeolitic imidazole frameworks have been used in order to create nanocomposites with extraordinary properties. The resulted materials combined the advantages of magnetic graphene oxide and the chosen material and exhibited sufficient surface area, great extraction efficiency, ease in handling and separation and good stability.Finally, modification of magnetic graphene oxide with ionic liquid and deep eutectic solvents can significantly enhance the extraction efficiency of the sorbent. In this case, the modification process is simple, rapid, and environmentally friendly since no toxic organic solvents are required. Moreover, magnetic procedures are energy and time saving, and they require only small quantities of solvents during extraction and elution steps; therefore, they play a key role in green analytical chemistry.


TARDBP, encoding the RNA-binding protein TDP-43, are one cause of ALS, and TDP-43 mislocalization in MNs is a key pathological feature of >95% of ALS cases. While numerous studies support altered RNA regulation by TDP-43 as a major cause of disease, specific changes within MNs that trigger disease onset remain unclear. Here, we combined translating ribosome affinity purification (TRAP) with RNA sequencing to identify molecular changes in spinal MNs of TDP-43–driven ALS at motor symptom onset. By comparing the MN translatome of hTDP-43A315T mice to littermate controls and to mice expressing wild type hTDP-43, we identified hundreds of mRNAs that were selectively up- or downregulated in MNs. We validated the deregulated candidates Tex26, Syngr4, and Plekhb1 mRNAs in an independent TRAP experiment. Moreover, by quantitative immunostaining of spinal cord MNs, we found corresponding protein level changes for SYNGR4 and PLEKHB1. We also observed these changes in spinal MNs of an independent ALS mouse model caused by a different patient mutant allele of TDP-43, suggesting that they are general features of TDP-43-driven ALS. Thus, we identified SYNGR4 and PLEKHB1 to be deregulated in MNs at motor symptom onset in TDP-43-driven ALS models. This spatial and temporal pattern suggests that these proteins could be functionally important for driving the transition to the symptomatic phase of the disease.Amyotrophic lateral sclerosis (ALS) is an incurable neurological disease with progressive loss of motor neuron (MN) function in the brain and spinal cord. Mutations in  Amyotrophic lateral sclerosis  is a progressive, adult-onset neurodegenerative disorder that mainly affects lower and upper motor neurons (MNs) in the spinal cord and brain, respectively . DiseaseSOD1), and for more than a decade, this was the only ALS gene that was known  gene, which encodes the TDP-43 protein, in fALS and sALS cases (*605078). Meanwhile, many other fALS genes have been identified, including repeat expansions in C9ORF72 which are the most abundant genetic cause of ALS  is a major component of protein aggregates frequently observed in postmortem brain and spinal cord of ALS patients, with the striking exception of ALS caused by mutations in SOD1 ,17. Soone of ALS ,22. Manye of ALS ,24. Even alleles ,25.Despite its name, TDP-43 is mainly characterized as an RNA-binding protein. It interacts with thousands of RNAs by preferentially binding to long UG repeats or UG-enriched RNA sequences  and reguSyngr4 and Plekhb1, neither of which has previously been implicated in neurodegenerative diseases. Importantly, we show by quantitative analysis of spinal cord immunohistochemistry that the corresponding proteins synaptogyrin-4 (SYNGR4) and pleckstrin homology domain-containing family B member 1 (PLEKHB1) are respectively increased or decreased within MNs specifically during the transition from the asymptomatic to early symptomatic stage. Moreover, we observed similar changes in the levels of these proteins in a second TDP-43-dependant ALS mouse model driven by another TDP-43 patient mutation (Q331K). Collectively, our work identifies MN-specific regulatory targets of mutant TDP-43 in disease. The pattern of deregulation of these targets specifically at the transition to overt symptoms makes them promising candidates to drive the pathology within diseased MNs.Here, we reveal new proteins whose deregulation specifically within lower MNs correlates with the appearance of motor symptoms in mouse models of ALS driven by patient mutant variants of TDP-43. This was enabled by combining behavioral phenotyping across the disease time course with genome-wide gene expression analysis in MNs. Focusing on late asymptomatic and early symptomatic timepoints, we applied translating ribosome affinity purification (TRAP) to spinal MNs with consecutive RNA sequencing. Inclusion of animals that overexpress WT hTDP-43, but that do not develop symptoms, allowed us to filter out hundreds of changes in MN gene expression that do not correlate with disease. Two genes that passed this filter were Chat bacTRAP . In contrast, females show no phenotypes with any test at early time points and a clear transition from pre-symptomatic to early disease in the 9–12-week time window based on both neurological score and rotarod performance . As shown in WThTDP-43. This line expresses WT human TDP-43 under the control of the same prion promoter as the A315ThTDP-43 line, but reportedly does not develop any symptoms of ALS when hemizygous for the transgene (i.e. when only one chromosome is transgenic)  . Using nt tested . Thus, CChat bacTRAP; A315ThTDP-43 mice become symptomatic, we next performed genome-wide MN-TRAP at corresponding time points  of the motor neuronal marker Chat mRNA, and de-enrichment of non-motor neuronal mRNA Gfap in the presence and absence of the TDP-43 transgenes  Chat bacTRAP; WThTDP-43 mice. For each sample, a fraction of the spinal cord lysate was retained as an ‘input control’ (IC) and analyzed in parallel to evaluate the sensitivity and specificity of MN-TRAP. We also tested ALS-like motor symptoms for all animals used in TRAP assays by applying the neurological score and body weight tests. As expected from our previous behavioral characterization, all 14-week old Chat bacTRAP; A315ThTDP-43 female mice used for MN translatome analyses had difficulties in extension of the hind limbs, but showed no differences in body weight when compared to the Chat bacTRAP or Chat bacTRAP; WThTDP-43 control mice  and glial markers (Gfap and Aldh1l1) across genotypes . In all samples, we observed robust enrichment of the motor neuronal marker mRNAs, enotypes , indicatgenotype . Similargenotype . In compgenotype  or time genotype  appear sChat bacTRAP; A315ThTDP-43 mice (Chat bacTRAP controls and identified 2067 deregulated genes (802 up and 1265 down). A second comparison to Chat bacTRAP; WThTDP-43 revealed 1061 genes that were deregulated (168 up and 893 down). The intersection of these two comparisons revealed a set of 327 genes that were either consistently upregulated or downregulated in the mutant line relative to both controls  terms  terms , of whicO) terms . ExampleChat bacTRAP; A315ThTDP-43 mice to symptomatic mice revealed 52 downregulated genes in common  of r SYNGR4  or PLEKHr SYNGR4 . The dis 9 weeks .A315ThTDP-43 mice and controls  is preferentially expressed in mammalian brain and sensory systems. In retina, it was shown to be an integral membrane protein found in the outer segment of both cones and rods . Membranry cells ,59. The ry cells . Finallyry cells .Plekhb1 KO mice displayed no obvious phenotype when examined up to 1 year of age .Mouse lines used in this study are indicated in For behavioral experiments, each tested group was initially composed of 8–12 animals of the same genotype and sex. Behavioral tests were conducted under the guidelines from ‘working with ALS mice: Guidelines for preclinical testing & colony management’, provided by the Jackson laboratory . Four teStandard criteria were used to evaluate neurological score . Mice wepost hoc test was used if a significant difference was found from the analysis of variance. Data is presented as mean ± SEM and *, ** and *** indicates a P-value of P < 0.05, P < 0.01 and P < 0.001, respectively.Data analysis and statistics was done with Excel and GraphPad Prism 6 software, respectively. A two-way repeated measurement ANOVA (two-way RM ANOVA) test was used to calculate the significance among genotypes within the same time point and among genotypes at different time points. The Bonferroni’s Ketanest®) (12 mg/mL) and Xylazine (®Rompun) (1.6 mg/mL) and after deep sleep induction were perfused with 4% paraformaldehyde (PFA). The hindlimbs were disconnected above the hip and the skin was carefully removed. Post-fixation of the hindlimbs was performed in 4% PFA solution for 1 h at room temperature (RT). The samples were washed with 1× PBS and all residual connective tissue was removed. The deep lumbrical muscles of the hindlimbs were dissected out and the NMJs were labelled using an established immunohistochemistry protocol in order to visualize pre-synaptic 2H3/SV2 proteins (medium weight neurofilament/SV protein) and post-synaptic acetylcholine receptors (AChRs) (Mice were anesthetized with a mixture of Ketamine ( (AChRs) . MusclesThe TRAP methodology was adapted from the original protocol from ‘Cell-Type-Specific mRNA Purification by Translating Ribosome Affinity Purification (TRAP)’ .m HEPES, 150 mm KCl, 5 mm MgCl2, 1% NP-40, 0.5 mm DTT, 100 ug/mL cycloheximide, protease inhibitors, and RNase inhibitors) and 50 ug of 19C8 and 19F7 antibodies  were added. The mixture was incubated at RT for 1 h on a rotator. The beads were washed with 0.15 M KCl buffer 3×, after which were re-suspended in 200 uL of 0.15 M KCl buffer. The IPs were stored on ice until the spinal cords were extracted and lysed.Streptavidin MyOne T1 Dynabeads (Invitrogen) were incubated with 120 uL of Biotinylated Protein L (Fisher) for 35 min at RT on a rotator. The coated beads were washed 5× with 3% bovine serum albumin IgG and protease-free solution (Gibco) in PBS. The beads were re-suspended in 1 mL 0.15 M KCl buffer ). The tissue was then transferred to a glass homogenizer in lysis buffer . The homogenate was centrifuged at 4°C for 10 min at 2000 g to remove cellular debris. The supernatant was collected and kept on ice. NP-40 (Thermo Scientific) was added to a final concentration of 1% (v/v), followed by addition of dihexanoylphosphatidylcholine . The mixture was centrifuged at 4°C for 10 min at 20 000g. 10% of the supernatant was collected separately as an IC. The remaining 90% was incubated overnight at 4°C on a rotator with the 200 uL of the previously prepared beads. On the second day, the beads were washed 4× with 0.35 M KCl buffer . After the final wash, the supernatant was discarded from the beads and 800 μL trizol (Life Technologies) was added to both IPs and ICs.Animals were anesthetized with a mixture of 80% of COg. The upper phase was transferred to a fresh tube, and an equal amount of 100% EtOH was added. The RNA was purified using the PureLink RNA kit (Ambion). The purified RNA was treated with DNase I (Roche) to digest DNA, for 15 min at 37°C. The RNA was concentrated by an overnight sodium acetate precipitation (5 M sodium acetate (Sigma), 100% EtOH (Roth), GlycoBlue (Ambion) at −80°C. On the third day, the samples were removed from the −80°C freezer and directly centrifuged at 13 000g for 15 min at 4°C. The samples were washed twice with 70% EtOH with a spinning in between at 13 000g for 15 min at 4°C. The pellets were left to dry at RT for 15 min prior to being re-suspended in RNAse-free water (Ambion).The samples were incubated with 200 μL chloroform and shaken vigorously by hand for 15 s followed by 15 min centrifugation at 4°C, 12 000® II Reverse Transcriptase (Life Technologies) according to the manufacturer’s recommendation. Residual RNA was digested with RNase H (NEB) for 20 min at 37°C followed by enzyme inactivation by incubation for 10 min at 70°C. qPCR primers were either selected from the mouse primer depot website (www.mouseprimerdepot.nci.nih.gov)  . The cDNbcl2fastq2  and sequencing quality control was performed using FastQC software (www.bioinformatics.babraham.ac.uk/projects/fastqc/). Sequencing reads were aligned to the Ensembl reference genome of Mus musculus . Scatter plots of individual genes were generated from normalized expression values without VST using the R package ggplot2 .Samples were de-multiplexed with mbl.org,  using thhat bacTRAPA315T; hTDP-43 versus Chat bacTRAP and (2) Chat bacTRAPA315T; hTDP-43 versus Chat bacTRAPWT; hTDP-43. Candidates were required to be either consistently upregulated or downregulated in both comparisons (1 and 2). Candidate identifications was carried out for both the motor neuronal translatome (TRAP) and whole spinal cord samples (input control). With identified candidates from TRAP samples, a gene set enrichment analysis of biological process GO terms was performed using the online tool ToppGene (toppgene.cchmc.org). The top 100 significantly regulated GO terms were used to construct an enrichment map (tidygraph (version 1.1.2.9999) and ggraph (version 2.0.0.9000) were used.To identify potential disease-driving candidates, we compared symptomatic mutant animals versus two control lines at 14 weeks of age, e.g. (1) Cment map  that cluKetanest®) (12 mg/mL) and Xylazine (®Rompun) (1.6 mg/mL) and after deep sleep induction they were perfused with 4% PFA (Roth) in PBS. Post-fixation of the spinal cord was done in 4% PFA solution for 2 h at RT, after which it was put in 30% sucrose solution in PBS until complete sank of the tissue. The tissue was then cut in three parts, corresponding to the cervical, thoracic and lumbar regions of the spinal cord and embedded in Tissue-Tek (Sakura) prior storage at −80°C.The mice were anesthetized with a mixture of Ketamine  in 0.1% triton in PBS at RT for 45 min. The primary antibodies against EGFP , NeuN , PLEKHB1 , SYNGR4 , synaptophysin  and ChAT  were diluted in 0.1% triton in PBS solution and left overnight at 4°C. On the next day, the slices were washed 3× with PBS and incubated with secondary antibodies , Alexa 555 ), in 1× PBS for 3 h at RT. After the incubation period, the slices were washed 3× with PBS. The slides were incubated with DAPI  in PBS for 10 min and embedded in roti mounting medium before sealing of the slide. The slices were stored at −20°C. Images were acquired from a Zeiss LSM 700 microscope and processed by ImageJ Fiji. A total of 5–6 images per experiment were taken, and a total of 4–5 age-matched animals per genotype were used.t-test. Data are presented as mean ± SEM, and *, ** and *** indicates a P-value of P < 0.05, P < 0.01 and P < 0.001, respectively.Statistical analyses were performed using GraphPad Prism 6 software and a paired R.F.M. planned and performed most experiments and analyzed the data; J.B.E. performed bioinformatic data analysis and interpreted results; K.K. helped to perform experiments; R.A.J. and T.H.G. planned, performed, analyzed and interpreted N.M.J. imaging experiments; T.L. and G.S.R. supervised RNA-seq library generation and performed bioinformatic data analysis; M.A.F. helped plan experiments and interpret results; K.E.D. conceived and supervised the project, planned experiments, helped to analyze data and interpreted results. K.E.D., R.F.M. and J.B.E. wrote the manuscript. All authors gave input on the manuscript and approved the final version.Marques_et_al_Supplementary_material-PDF_ddaa140Click here for additional data file.Supp_Table_3_Differential_gene_expression_14w_ddaa140Click here for additional data file.Supp_Table_4_Gene_set_enrichment_analysis_14w_ddaa140Click here for additional data file.Supp_Table_5_Differential_gene_expression_9w_ddaa140Click here for additional data file.Supp_Table_6_Gene_set_enrichment_analysis_9w_ddaa140Click here for additional data file.Supp_Table_7_Overlap_differential_gene_expression_between_9w_14w_ddaa140Click here for additional data file.CLEAN-Marques_et_al_Supplementary_material_ddaa140Click here for additional data file.HIGHLIGHTED-Marques_et_al_Supplementary_material_ddaa140Click here for additional data file.