III complexes with neutral bisguanidine ligand units. Further one‐electron oxidation is ligand‐centred in the case of Co–bisguanidine complexes with fully alkylated guanidino groups, giving CoIII complexes with radical monocationic bisguanidine ligands. On the other hand, the hydrogen‐bond strengthening upon oxidation of the Co–bisguanidine complex with partially alkylated guanidino groups initiates metal reduction (CoIII→CoII) and two‐electron oxidation of the guanidine ligand, providing the first example for the stimulation of redox‐induced electron transfer by interligand hydrogen bonding.Octahedrally coordinated cobalt(II) complexes with a redox‐active bisguanidine ligand and acac co‐ligands were synthesized and their redox chemistry analysed in detail. The N−H functions in a bisguanidine ligand with partially alkylated guanidino groups form N−H⋅⋅⋅O hydrogen bonds with the acac co‐ligands, thereby massively influencing the redox chemistry. For all complexes, the first one‐electron oxidation is metal‐centred, leading to Co III complex with a neutral redox‐active bisguanidine ligand, leading to metal reduction (CoIII→CoII) and two‐electron oxidation of the bisguanidine ligand. In the absence of interligand hydrogen bonding, oxidation leads to a CoIII complex with a radical monocationic guanidine ligand.The strengthening of interligand hydrogen bonding triggers a redox‐induced intramolecular electron transfer (RIET) upon oxidation of a Co III complex preferred at low temperature into a high‐spin CoII complex favoured at higher temperature.III into a higher‐energetic CoII complex by light at low temperature (similar to the light‐induced excited spin state trapping (LIESST) effect discovered by Gütlich et al.III complex by light (similar to reverse‐LIESST).‐benzodioxole (L1), 5,6‐bis‐2,2‐dimethyl‐‐benzodioxole (L2), and 5,6‐bis‐2,2‐dimethyl‐‐benzodioxole (L3) sketched in Figure II complexes with one of the bisguanidines L1–L3 as ligand and two acetylacetonato (acac) co‐ligands led to stable complexes that were fully characterized. We present the first example for the initiation of RIET by hydrogen‐bond enforcement, enriching the arsenal of IET stimulants by a biomimetic, innovative alternative.Herein, we report the first IET in a cobalt complex with a redox‐active bisguanidine ligand, being triggered in an unprecedented way by interligand hydrogen bonding. We used the three bisguanidines 5,6‐bis2(L1)], [Co(acac)2(L2)], and [Co(acac)2(L3)] 2(L3)] (see Lewis structure in Scheme +) salt Fc(PF6), and two electron oxidation either also with Fc(PF6) or with the stronger oxidant NO(SbF6). The complexes were isolated and fully characterized in all three redox states. The first one‐electron oxidation is metal‐centred (CoII→CoIII) for all complexes. Further one‐electron oxidation of the complexes with L1 or L2 is ligand‐centred, giving CoIII complexes with radical monocationic ligand L1.+ or L2.+. On the other hand, oxidation of the monocationic complex [Co(acac)2(L3)]+ to the dication [Co(acac)2(L3)]2+ is coupled with IET (RIET) leading to cobalt reduction (CoIII→CoII). RIET is triggered by the increase of the interligand N−H⋅⋅⋅O hydrogen‐bond strength upon two‐electron oxidation of the ligand. Hence detailed analysis clearly shows that the dication [Co(acac)2(L3)]2+ is a high‐spin CoII complex with dicationic guanidine ligand. In principle, a further one‐electron oxidation is imaginable, leading to a tricationic CoIII complex with dicationic guanidine ligand. Indeed, cyclic voltammetry measurements indicate the possibility of reversible three‐electron oxidation (see below). However, it was not possible to isolate these ultimately oxidized complexes in pure form in experiments that relied on NO(SbF6) as oxidizing reagent. According to the experiments, partial degradation leads to cleavage of the bond between the metal and the dicationic guanidine in significant amount. In the following, the electronic structures of the neutral, as well as the singly and doubly oxidized complexes will be evaluated in detail. The discussion concentrates on the comparison between complexes of the ligands L1 and L3. The experimental results obtained for the complexes with L2 showed similar behaviour as the complexes of L1.The three neutral Coo(acac)2L], [Co(aco(acac)2L],  2(L1)]+/ [Co(acac)2(L1)]0, E1/2=−0.03 V for [Co(acac)2(L1)]2+/ [Co(acac)2(L1)]+, and E1/2=0.41 V for [Co(acac)2(L1)]3+/ [Co(acac)2(L1)]2+. Also, three redox processes are found for [Co(acac)2(L2)], at E1/2=−0.54 V for the redox couple [Co(acac)2(L2)]+/[Co(acac)2(L2)]0, E1/2=−0.21 V for [Co(acac)2(L2)]2+/[Co(acac)2(L2)]+, and E1/2=0.32 V for [Co(acac)2(L2)]3+/[Co(acac)2(L2)]2+. The potential for the first one‐electron oxidation is almost equal for [Co(acac)2(L1)] (E1/2=−0.56 V) and [Co(acac)2(L2)] (E1/2=−0.54 V), and significantly lower than the potentials required to oxidize the free ligands L1 (E1/2=−0.25 V for L1.+/L10) or L2 (E1/2=−0.46 V for L2.+/L20). These results clearly indicate that the first one‐electron oxidation is metal‐centred (CoII→CoIII), as expressed by the Lewis structures in Scheme −1) a small feature is visible on the high‐potential side of the first oxidation wave for all three complexes, vanishing at slower scan rates. The disappearance of this feature at slow scan rates and the similar behaviour of all three compounds argue against the presence of a compound with different composition in the solutions. The change from high‐spin CoII to low‐spin CoIII brings about massive structural and electronic changes, and therefore it might be possible that the presence of an intermediate complex (e.g. a CoIII complex with different spin multiplicity that relaxes to the low‐spin CoIII ground state) causes the additional weak features at high scan rates.In the cyclic voltammogram recorded for 2+/[Co(acac)2(L1)]+ and −0.21 V for [Co(acac)2(L2)]2+/[Co(acac)2(L2)]+, are both higher than the potentials for one‐electron oxidation of the free ligands. Such an increase could easily be explained by the bonding to the Lewis‐acidic metal. Moreover, the difference in the E1/2 values of 0.18 V is almost equal to the difference of 0.21 V for the free ligands. On these grounds, the second one‐electron oxidation is assigned to a ligand‐centred oxidation process, leading to CoIII complexes with radical monocationic guanidine ligands  are clearly potentially separated (ΔE1/2=0.44 V for L1 and 0.53 V for L2), comparing with ΔE1/2=0.14 and 0.08 V for the free ligands L1 and L2, respectively.The s Scheme . The thi2(L3)] is also included in Figure E1/2=−0.53 V (Eox=−0.46 V), E1/2=−0.19 V (Eox=−0.09 V), and E1/2=+0.11 V (Eox=+0.17 V), assigned to the couples [Co(acac)2(L3)]+/[Co(acac)2(L3)]0, [Co(acac)2(L3)]2+/[Co(acac)2(L3)]+, and [Co(acac)2(L3)]3+/[Co(acac)2(L3)]2+, respectively. The potential of the first one‐electron process is close to those measured for the analogue complexes with L1 and L2, and lower than the potential measured for the free ligand L3; therefore it is assigned to a metal‐centred oxidation (CoII→CoIII).The cyclic voltammogram of 2+/[Co(acac)2(L3)]+ is large (0.22 V), possibly due to the significant structural changes induced by the RIET process. From the synthesis of stable salts of the monocation [Co(acac)2L3]+ and dication [Co(acac)2L3]2+ by chemical oxidation of the neutral compound (see below) an inherent instability of the redox states could be excluded. The CV measurements suggest the third one‐electron redox process, leading to a CoIII complex with dicationic guanidine ligand, to be reversible. However, it was not possible to synthesize salts of these complexes in pure form.The second and third one‐electron redox events then belong to ligand‐centred oxidation. We will see that the second redox event is accompanied by a RIET process 2(L1)]. For [Co(acac)2(L2)], g⊥=7.53 and g∥=2.00 add up to an effective g value is 3.84. Values of g⊥=6.66 and g∥=2.68 are obtained for [Co(acac)2(L3)], resulting in an effective g value of 4.00. Hyperfine coupling to the nuclear spin of the 59Co nucleus (I=7/2) should produce splitting into eight lines. Indeed, eight lines are visible for [Co(acac)2(L2)] ], the others being buried. The coupling constants for [Co(acac)2(L1)] (98 G) and for [Co(acac)2(L2)] (95 G) are very similar. For [Co(acac)2(L3)], eight sharp lines show, but also additional lines due to coupling with other nuclei. With 72 G, the A value is slightly smaller, presumably due to the structural peculiarities produced by the two hydrogen bonds (see structure below).2(L1)](PF6) in CH2Cl2, indicating that [Co(acac)2(L1)]+ is a diamagnetic CoIII complex with neutral ligand in the whole temperature region 6–300 K. Complementary with these results, signals appeared in the 1H NMR spectrum of the compound . The EPR spectrum of [Co(acac)2(L1)](PF6)2 in a frozen CH2Cl2 solution at 6 K shows a sharp signal due to a ligand‐centred radical with a g value near 2 (1.997). At 293 K, a clear hyperfine structure due to isotropic hyperfine coupling with the 59Co nucleus is visible, resulting in line splitting with a hyperfine‐coupling constant A=97 G ](PF6)2 . Hence, [Co(acac)2(L1)]2+ and [Co(acac)2(L2)]2+ are CoIII complexes with radical monocationic ligands, in line with the results from cyclic voltammetry and the Lewis structures in Scheme No EPR signal was detected for [Co(acac)G Figure . Similar2L](PF6) in2(L3)](PF6)2, confirming the absence of an organic radical and the formulation as a CoII complex with dicationic ligand unit L32+. In similarity to the neutral complexes, the absence of clear signals could be rationalized by signal broadening due to fast relaxation at room temperature. Interestingly, a weak and broad signal that might be assignable to an organic radical appeared in the 6 K EPR spectrum, together with weak, broad signals in the region 550–3000 G . In principal, we could not completely exclude that these signals arise from the presence of a minor impurity, but their absence in the room temperature spectra seems to be not in accordance with this explanation. Another possibility is a change in the electronic structure of the [Co(acac)2(L3)]2+ complex at very low temperature, that is also motivated by the calculated small energy difference between two redox isomers (see discussion below).On the other hand, no sharp signal appears in the room‐temperature EPR spectrum of +, [Co(acac)2(L2)]+, and [Co(acac)2(L3)]+ are also free of strong bands, arguing for metal‐centred oxidation (CoII→CoIII), in line with the results from cyclic voltammetry and EPR spectroscopy. The spectra recorded for the dications [Co(acac)2(L1)]2+ and [Co(acac)2(L2)]2+ obtained upon two‐electron oxidation contain a band at 364 nm and a broad absorption in the visible region with an absorption maximum at ca. 554 nm for [Co(acac)2(L1)]2+ and 503 nm for [Co(acac)2(L2)]2+, indicating the presence of a CoIII complex with radical monocationic ligand. On the other hand, bands at 295 and 450 nm in the spectrum of [Co(acac)2(L3)]2+ in CH2Cl2  in the visible region, being responsible for the red colour of the compounds. The visible regions in the spectra of the monocations [Co(acac)2L]+, [Co2(L1)] is displayed in Figure 2(L2)] is shown in the SI . In both compounds, the bisguanidine ligand binds with its two imino N atoms to the octahedrally coordinated CoII atom. The Co−N bond lengths measure 2.135(2)/2.149(2) Å for [Co(acac)2(L1)] and 2.085(2)/2.245(2) for [Co(acac)2(L2)]. Due to combined σ‐ and π‐contributions to the metal−guanidine bonding,2(L1)] and [Co(acac)2(L2)], respectively.2(L1)] to the monocation [Co(acac)2(L1)]+ and the dication [Co(acac)2(L1)]2+  to a low‐spin CoIII complex upon oxidation. The decrease of the Co−N bond lengths (by 0.183/0.189 Å) and Co−O bond lengths (by 0.163/0.187 Å) after two‐electron oxidation add up to an average bond length decrease of 0.180 Å, being similar to the value reported for VT between CoII and CoIII complexes with two oxolene ligand units.II complexes with reduced, neutral L1, measuring in average 1.321 Å, are elongated to an average value of 1.382 Å upon two‐electron oxidation. The N−C bonds connecting the guanidino groups to the C6 ring decrease from 1.408 Å before to 1.362 Å after two‐electron oxidation. In summary, the crystal structures fully support the Lewis structures in Scheme The Co−N and Co−O bond lengths decrease significantly from neutral [Co(acac)2L] to the 2L] to the 2(L3)], and [Co(acac)2(L3)](PF6)2. The Co–N distances slightly increase, from average 2.150 Å before to 2.175 Å after oxidation, and the Co–O distances slightly decrease, from average 2.083 Å before to 2.043 Å after oxidation, but these values remain in the region typical for CoII (Table 2(L3)]2+ is best described as a CoII complex with dicationic ligand unit L32+. To obtain structural information about the hydrogen atoms within the hydrogen bonds, the positions of the relevant hydrogens in [Co(acac)2(L3)]2+ and [Co(acac)2(L3)]2+ were taken from difference Fourier maps and freely refined. The experimental NH⋅⋅⋅O distances decrease from 2.22(6)/2.15(6) Å in [Co(acac)2(L3)] to 2.020(2) Å in [Co(acac)2(L3)]2+. The more reliable distances between the N and O atoms within the N−H⋅⋅⋅O hydrogen bonds decrease from 3.033(9)/2.966(8) Å in [Co(acac)2(L3)] to 2.762(2) Å in [Co(acac)2(L3)]2+ (see SI), a value that is quite small in comparison to previously reported interligand N−H⋅⋅⋅O hydrogen bonds.(PF6)2 and [Co(acac)2(L3)](PF6)2 to confirm the differences in the electronic structure ](PF6)2, the χ T value measures 0.181 cm3 K mol−1 at 300 K and slightly increases to 0.276 cm3 K mol−1 at 40 K, being close to the theoretical value of 0.375 cm3 K mol−1 obtained from the Curie law for a compound with one unpaired electron (S=1/2). Hence, the data are in line with the presence of low‐spin CoIII and L1.+.e Figure . For [Coχ T value of ca. 2.5 cm3 K mol−1 was found for [Co(acac)2(L3)](PF6)2 at 300 K. This large value fully supports the formulation as a high‐spin CoII complex (S=3/2) with dicationic ligand, L32+ and an unquenched orbital contribution. Due to significant spin‐orbit coupling (4T1g ground term in hO symmetry), it is higher than the spin‐only value for high‐spin CoII (S=3/2) of 1.876 cm3 K mol−1 predicted by the Curie law. The value decreases almost linearly with decreasing temperature until ca. 50 K  EPR spectrum of a frozen CH2Cl2 solution , and by the calculated small energy difference between two redox isomers (see below). However, it is not possible to reach a final conclusion on this point.A much higher omplexes.52, 53Quantum‐chemical calculations. Finally, B3LYP/def2‐TZVP calculations were carried out, the solvent effect being included by single‐point calculations with the conductor‐like screening model (COSMO). The calculated structures are in pleasing agreement with the experimentally derived structures in the solid state (see SI for details). In Figure 2(L1)]2+ and [Co(acac)2(L3)]2+. The calculations for the quartet state of [Co(acac)2(L1)]2+ did not converge to a CoII complex with dicationic ligand L12+, but instead to an intermediate‐spin CoIII complex with radical monocationic ligand L1.+  is preferred by ΔE=85 kJ mol−1 at ϵr=37.5 with respect to this intermediate‐spin CoIII redox isomer.II redox isomer of [Co(acac)2(L3)]2+ (S=3/2) with dicationic ligand, L32+, the N−H⋅⋅⋅O hydrogen bond lengths measure 1.824 Å, being significantly shorter than in the calculated CoIII redox isomer with radical monocationic L3.+ unit (2.061 Å). Additionally, the calculated distances between the N and the O atom in the N−H⋅⋅⋅O hydrogen bonds in the neutral and the dicationic complexes of L3 are similar to those found in the corresponding crystal structures. Hence experimental values of 3.033/2.966 and 2.762 Å were found for [Co(acac)2(L3)] and [Co(acac)2(L3)]2+, respectively, being close to the calculated values of 2.905/2.835 and 2.788 Å (see SI). The decrease of the hydrogen‐bond lengths signals a stronger interligand interaction, leading to a stabilization of the CoII redox isomer by the two strong interligand hydrogen bonds. Consequently, the calculations predict the otherwise highly unfavourable quartet spin state (being high‐energy states for complexes of L1 and L2) to be even slightly preferred over the doublet state (by 2 kJ mol−1) at ϵr=37.5 (CH3CN solution); without solvent effect the doublet state (CoIII redox isomer) is slightly preferred, but by not more than 10 kJ mol−1.In the calculated Co2(L1)]2+ in its S=3/2 state with free L3 to give [Co(acac)2(L3)]2+ in the S=3/2 ground state and free L1, a ΔG value (at 298 K) of −76 kJ mol−1 was obtained, underlining the massive stabilization of the S=3/2 state (CoII redox isomer) with respect to the S=1/2 state (CoIII redox isomer) for the complex with L3 by hydrogen‐bonding.The Gibbs free energy for a hypothetical ligand exchange reaction was calculated to further highlight the impact of hydrogen bonding Figure . For theIII complex with the neutral guanidine ligand leads to metal reduction and two‐electron oxidation of the guanidine ligand, resulting in a CoII complex with dicationic guanidine ligand unit. The results of this study demonstrate the possibility to alter the outcome of redox processes and to initiate intramolecular electron transfer (IET) by introducing intramolecular hydrogen‐bond interactions. Thereby, they pave the way to a sophisticated control of redox and IET processes. Nature extensively uses the weakening or strengthening of hydrogen bonding up to the point of proton transfer to trigger electron transfer,The first mononuclear, octahedrally coordinated cobalt complexes with redox‐active bisguanidine ligands were synthesized and their redox chemistry was studied. Three different redox‐active bisguanidines were used in this study, two of them with fully alkylated and one with partially alkylated guanidino groups. Interligand N−H⋅⋅⋅O hydrogen bonds between two guanidino N−H functions and oxygen atoms of the acetylacetonate (acac) co‐ligands are established in the Co–bisguanidine complex with partially alkylated guanidino groups. The increase of the hydrogen‐bond strength upon oxidation of this guanidine ligand triggers a redox‐induced electron transfer (RIET) that is absent in the complexes with the guanidine ligands having fully alkylated guanidino groups. Hence, one‐electron oxidation of the monocationic CoThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


A solvent-free thermoplastic forming processing of graphene materials is invented by polymer intercalation from graphene oxide precursor.The correlation between interlayer spacing and thermoplastic forming capability of polymer-intercalated graphene oxide solid is uncovered.The multi-scale forming of graphene materials from Gaussian curved shapes to surface relief patterns with size precision down to 360 nm is realized.The online version contains supplementary material available at 10.1007/s40820-021-00755-8. 5 S m−1) and thermal conductivity (745.65 W m−1 K−1) after removal of polymers. The thermoplastic strategy greatly extends the forming capability of GO materials and other layered materials and promises versatile structural designs for more broad applications.The processing capability is vital for the wide applications of materials to forge structures as-demand. Graphene-based macroscopic materials have shown excellent mechanical and functional properties. However, different from usual polymers and metals, graphene solids exhibit limited deformability and processibility for precise forming. Here, we present a precise thermoplastic forming of graphene materials by polymer intercalation from graphene oxide (GO) precursor. The intercalated polymer enables the thermoplasticity of GO solids by thermally activated motion of polymer chains. We detect a critical minimum containing of intercalated polymer that can expand the interlayer spacing exceeding 1.4 nm to activate thermoplasticity, which becomes the criteria for thermal plastic forming of GO solids. By thermoplastic forming, the flat GO-composite films are forged to Gaussian curved shapes and imprinted to have surface relief patterns with size precision down to 360 nm. The plastic-formed structures maintain the structural integration with outstanding electrical (3.07 × 10The online version contains supplementary material available at 10.1007/s40820-021-00755-8. Graphene macroscopic materials have exhibited attractive properties and promised wide applications in structural components, thermal management, sensors and electronic devices –4. To saTd) [Thermoplastic processing of graphene is forbidden by its extremely high glass-transition point up to 6300 K, which exceeds the decomposition temperature of graphene (Td) . To achiTd) –28. In tTd) , 29. By Td) . The strTd) . HoweverTg) starts to appear and the rigid polymer-intercalated GO solid (Pi-GOS) is able to transform into plasticity when the interlayer spacing exceeds 1.4 nm, indicating a thermal forming criterion of containing above 45%. In the thermoplastic range, we forge flat films into stereoscopic structures with different Gaussian curvatures. We develop a thermoplastic imprinting method to design multiscale surface patterns and periodic arrays in a broad size precision from 360 nm to 200 μm. The thermoplastic imprinting allows the modulation of properties, including Janus material for actuators and responsive film surface with voltage-dependent wetting. Despite of the introduction of polymer plasticizer, the thermoplastic forming materials remain similar electrical (3.07 × 105 S m−1) and thermal conductivity (745.65 W m−1 K−1) as raw films after thermal annealing. The solvent-free thermoplastic forming strategy extends polymer-like forming techniques to graphene and other layered solids and meets complex structural designs in broader applications including humidity sensor, thermal management and functional surfaces.Here, we demonstrate the solvent-free thermoplastic forming of graphene oxide solids by polymer intercalation, achieving the size precision from macro- to sub-micrometer. The thermal transition of intercalated polymer endows the bulk composite film with plasticity. We reveal that the glass-transition temperature (−1) was acquired from Hangzhou Gaoxi Technology Co. Ltd (www.gaoxitech.com). Poly   was received from ACROS Organics Co. Ltd. Polyethylene glycol  and polyvinylpyrrolidone  were purchased from Rhawn.Aqueous graphene oxide dispersion  80 wt.% GO with 20 wt.% PVA; (2) 67 wt.% GO with 33 wt.% PVA; (3) 50 wt.% GO with 50 wt.% PVA; (4) 33 wt.% GO with 67 wt.% PVA and (5) 20 wt.% GO with 80 wt.% PVA. Pi-GOSs were prepared by casting the GO/PVA complex solutions on a polyethylene terephthalate (PET) substrate and the drying process was maintained at the ambient temperature.PVA was dispersed into deionized water and the mixture was heated to 60 °C with magnetic stirring for 12 h to prepare solution with a concentration of 1%. Then, PVA solutions were mixed with GO dispersions which is diluted into 5 mg mL3PO4, 50 wt.%) at 60 °C for 60 min to remove AAO film. To clean off the residual aqueous solution on the surface of disc, the demolded sample was washed by ethanol and n-hexane successively.For macro- and micro-scale forming, the Pi-GOS was placed on or between the metallic moulds with designed structures, and then pressed at the forming temperature of 95 °C for varied time. After cooling to room temperature, the formed solid was easily separated from die. For the nanoimprinting process, we chose AAO membrane as template with diameter for 13 mm and hole size for 360 nm. After molding processing, the whole disc was reduced by hydriodic acid at 95 °C for 12 h and immersed in phosphoric acid solution . The measurements were carried out at a frequency of 1 Hz and a temperature ramp of 5 °C min−1. 3D-profile images were performed using an optical profilometer (Wyko NT9100). The indentation test was carried out by an Instron 2344 equipped with a load cell. The shells were placed on a plat and stiff base pressed by an indentor at the constant speed of 10 μm s−1. Scanning electron microscope (SEM) images were taken on Hitachi S4800 and SU8010 field-emission SEM system. Transmission electron microscope (TEM) images of nanotubes were taken on a HT-7700 HR-TEM. Contact angle measurements were conducted by a video-based, contact angle measuring device (OCA 20).The molding processing was carried out on a press (GT-7014-A50C). X-ray diffraction (XRD) profiles were collected on a X`Pert Pro  diffractometer using monochromatic Cu 17 Kα1 radiation (λ = 1.5406 Å) at 40 kV. The glass-transition temperature was investigated by differential scanning calorimetry (DSC) using a TA Q20 instrument. Mechanical tests were taken on TA Q800 at a loading rate of 1 mm minTg < Td) and good affinity to GO, so the variety is constrained. To enable the thermoplastic forming of GO, we introduced polyvinyl alcohol (PVA) into GO solution and prepared homogenously mixed solid papers as raw material for thermoplastic forming . The illustration of polymer intercalating process is shown in Fig. S3. The polymeric plasticizers can extend to other species with appropriate Tg, like Polyethylene glycol  and polyvinylpyrrolidone  . The film is thermally compressed against template and specific patterns with high precision are formed by the solids creeping into the hole of mold , the polymer segments in the interlamination begin able to move, thus endowing the plasticity of whole material. By thermoplastic processing on Pi-GOS, structures with large scale spanning from 4 to 390 nm were fabricated, including origami with stereo-structures, multiple patterns and nanopillar arrays.The strong interlayer interactions of graphene oxide solid hamper the free motion of graphene oxide layers. Given this, thermoplastic polymer was intercalated between GO layers for the reduction of interlayer van der Waals force, as well as the introduction of thermal response –36. The g Figs. S and S2. d) with the increasing weight ratio of PVA to GO (α) and the interlayer spacing remains stable after long-term storage . The peak shift in XRD patterns indicates the expansion of interlayer spacing  to 25 (E25) °C and d, we have detected a negative correlation curve with changing fit slope  as raw material for following forming.DMA tests revealed a similar transition in storage modulus of Pi-GOS as the interlayer spacing increases. For the Pi-GOS with  modulus , 40. Whed weakens the van der Waals interaction between GO sheets by intercalated polymer, so that the chain segments of PVA are able to move above Tg [d at room temperature , the failure strain is enhanced when temperature increasing to 95 °C, which implies a prominent assistance provided by the motion of polymer chains and the impact becomes prominent when the contents of PVA gradually increased. While d is narrowed down to 1.2 nm or below, for instance 0.9 nm, the elongation shows a diminution due to the frozen PVA chains. Meanwhile, the downward trend of Young’s modulus caused by both temperature and interlayer space can be interpreted as decreasing percentage of “bricks” (GO) and the motion of “mortar” (PVA segments). A microscopic contrast of activated sheet sliding under tension at room temperature and imprinting temperature is observed through fracture morphology analysis by SEM  of the spherical shell, defined as the slope of the force–displacement curves, is higher than that of cylinder shell , following the deduction as Kcylinder ~ Kspherical shell (t/R) 1/2 <  < Kspherical shell, where t is the thickness and R is the principal radius of curvature (t/R <  < 1) [The materials with curved geometry usually possess enhanced mechanical stability compared to that of original flat sheets . However <  < 1) –45. The  <  < 1) , 46.Fig.We used thermo-imprinting to fabricate micro-scale structures on the surface of Pi-GOS and the process can be double-imprinting. As shown in Fig. H) of imprinting patterns. While the imprinted shape is settled as circle with constant diameter (D), the aspect ratio (H/D) denotes the shaping capability. For an immobile interlayer space (1.4 nm) of Pi-GOS, the imprinting pressure (t) follow a simplified classic Norton–Bailey’s creep power law [Bi  are constants related to diameter, material properties and processing temperature .We further analyzed the influence factors in the height  membrane with 360 nm apertures as template  to Semi-Cassie model (hydrophilic) . Those complex deformations modify mechanical property and responsiveness of Pi-GOS and the thermoplastic forming materials remain good electrical as well as thermal conductivity after thermal annealing. The solvent-free thermoplastic forming approach provides new forming technology of GO materials and other layered materials, and further broadens the applications in versatile devices.Supplementary file1 (PDF 8783 kb)Below is the link to the electronic supplementary material.


Due to the different binding forces of the transferred graphene surfaces, there may have been pollutants present. PMMA residues, graphene laminations and other defects that occurred in the wet transfer process were analyzed by X-ray photoelectron spectroscopy and Raman spectroscopy. These defects influenced the square resistance of the produced graphene films, and of these defects, PMMA residue was the most influential; square resistance increased with increasing PMMA residue.Graphene has been widely used due to its excellent electrical, mechanical and chemical properties. Defects produced during its transfer process will seriously affect the performance of graphene devices. In this paper, single-layer graphene was transferred onto glass and silicon dioxide (SiO Since the discovery of single-atom layers of graphene in 2004 ,2, graph3, H2O, NO2) on the graphene surface also change the local carrier density, resulting in step-like changes in resistance [3 content increases when placed in the air for a long time [The PMMA-assisted transfer method is the most popular way to transfer graphene to a target substrate ,23,24. Psistance ,30. The ong time ,32. The ong time .2 substrates by the wet transfer method, and the resistance of each sample is tested by a four-probe square resistance tester. Then, the surface roughness of the SiO2 substrate and graphene are measured by AFM to observe impurities in the produced graphene. The surface hydrophilicity is measured by a surface tension instrument to determine the binding force of the surface, characterizing the graphene’s surface quality. By X-ray energy spectrum analysis, the adsorbed elements of the representative samples on the slides and SiO2 substrates are determined. Additionally, Raman spectroscopy is used to analyze the surface spectra of two groups of samples with different representative resistances to study the layer distribution on the graphene surface after transfer. The experiment explores the influence of defects produced during the wet transfer on the obtained graphene’s electrical properties, laying a foundation for the subsequent improvement of these electrical properties.In this paper, single-layer graphene is transferred to glass slides and SiO3 solution was prepared. Due to its ultra-thin characteristic, graphene will quickly adsorb impurities, such as molecules and particles, in an aqueous solution. So, a dust-free cloth was used to filter the impurities on the solution surface. The residual FeCl3 contaminants in the graphene film were neutralized by 10–20%-diluted hydrochloric acid for 1 h and then further cleaned with deionized water. The substrate was washed with deionized water and acetone successively, and deionized water was finally used to reduce ion pollution. Then, the graphene/PMMA was transferred to the substrate and let stand to naturally dry. Finally, the PMMA was removed by acetone to complete the transfer process of graphene film. The specific steps of the operation are shown in The graphene film used in the experiment was prepared by the CVD method and deposited on copper. The PMMA-assisted wet transfer method was adopted. Firstly, 4 g of PMMA powder was weighed and placed in a brown, wide-necked bottle. Then, 100 mL of benzaldehyde solution was poured into the bottle, and the bottle was heated in a water bath at 50 °C and magnetically stirred for 6 h. The PMMA was prepared by spin coating onto the graphene surface by a homogenizer and was heated and cured at 180 °C for 3 min. Next, 1 mol/L of FeCl2 substrates. On different substrates (SiO2 and glass), three groups of samples, named “No.1”, “No.2” and “No.3”, were prepared. Using an HPS2661 four-probe resistance measuring instrument (produced by Helpass Electronic Technologies), each sample was measured five times and its square resistance was indicated by an error bar. The test results of the different substrates are shown in 2 substrate, the minimum square resistance was 5.66 kΩ/□ and the maximum square resistance was 13.07 kΩ/□. It can be concluded that the square resistance of the graphene transferred to the SiO2 substrate was lower than that transferred to the glass substrate. In addition, the square resistances of various samples on like substrates were different.Graphene was transferred to glass slides and SiO2 is different because of the impurities and defects produced during the transfer process, though surface roughness may also be one of the influencing factors. In order to clarify the reasons for the differences in square resistance, surface roughness, number of layers and impurities were analyzed by AFM, a surface tension instrument, XPS and Raman spectrum analysis.The square resistance of graphene on a slide and on SiO2 substrate by AFM with an Innova-IRIS atomic force microscope . Bare SiO2 was used as a blank control group in the experiment. As shown in 2 substrate. In the No.3 sample on the SiO2 substrate particles were evenly distributed and its flatness was average. There were some large particles in the No.2 sample on the SiO2 substrate. For the No.1 sample, the surface was more uneven. The surface roughness was obtained with the NanoScope analysis software. The surface roughness, expressed as Ra),of the bare SiO2 substrate was 1.23 nm, the Ra of the No.3 sample on the SiO2 was 4.65 nm and the Ra of the No.2 and No.1 samples on SiO2 were 6.71 nm and 7.86 nm, respectively. From 2 substrate. The surface roughness of the sample with the lowest square resistance . As shown in 2 substrate was 90°, the dip angle of the No.1, No.2 and No.3 samples on the SiO2 substrate were 68°, 65° and 63°, respectively , No.2 (9.53 kΩ/□) and No.3 (5.66 kΩ/□) samples transferred to the SiOectively . While tectively .(1)γSV=2 and slide substrates were different because the surface defects reduced the binding forces. The defect content of each sample was also different, as were the contact angles. Next, XPS was used to determine the kinds of residues or impurities on the graphene surfaces.The contact angles of the samples on the SiO+ X-ray photoelectron spectroscopy (Thermo Fisher). The contents of the elements C, N and O on the surfaces of the experimental samples after transfer were measured by XPS analysis, and information of the various elements’ peaks was obtained. The C1s peak is the most significant peak for the analysis of doping substances. Therefore, the C1s peak was processed by XPS peak separation. The chemical bond composition of C in the C1s peak was analyzed as the baseline for introducing impurity species during the graphene transfer process. Different substrate samples are selected for analysis, as shown in To further study the influence of graphene surface impurities on the electrical properties of the obtained graphene, it was necessary to analyze the graphene surface in the X-ray energy spectrum, for which we used an ESCALAB Xi2 substrate had a similar trend. On the same substrate, samples of greater square resistance had more C=C content. No matter the substrate, only PMMA molecules contained a large amount of C=C. PMMA, as an impurity, when doped with graphene, opens the graphene’s bandgap [As shown in  bandgap ,27,31,342O and doped graphene, in turn resulting in a smaller binding force and larger contact angle. Therefore, PMMA is the substance with the greatest influence on the square resistance because it can be doped with graphene and thereby open the graphene bandgap.Combined with the contact angle test results, it was found that the greater the square resistance of the same group of samples, the greater the contact angle. It was speculated that the substance on the surface of the sample affects the surface binding energy of deionized water. XPS analysis results showed that a large amount of C=C remained on the substrate surfaces, and only PMMA contained C=C during the experiment. Compared with the water contact angle test and XPS results , it was −1) is the characteristic peak of the sp2 structure, reflecting the symmetry and crystallinity of graphene, which is generated by the double-degenerate iTO and iLO optical phonon interactions in the center of the Brillouin zone, with E2g symmetry. The 2D peak  is generated by two inter-valley inelastic scatterings of iTO optical phonons near point K, which is used to characterize the stacking mode of the carbon atoms in graphene. In addition, the D peak and D’ peak reflect defects. If the laser is focused on a graphene samples containing defects or its grain boundary, a defect peak D, at about 1350 cm−1, and a peak D’, at about 1620 cm−1, appear. The number of graphene layers can be determined by the ratio of I2D/IG. If I2D/IG is two, graphene is of a single layer.; if I2D/IG is one, it is bilayered. In practice, the ratio of I2D/IG is generally not exactly equal to two. In such case, we can judge the number of graphene layers by its Raman feature. As there is often a multi-layer core at the center of each flake, when it is hard to judge graphene as single- or double- layered, a blue shift in the 2D peak of graphene can be used as the basis for evaluating double layers [2D/IG was less than one, indicating that the graphene stacking phenomenon was serious, and the number of graphene layers increased to various degrees after the transfer; thus, the square resistance significantly improved [A Renishaw inVia Qontor laser confocal Raman spectrum system with a 532-nm wavelength was used in the experiment. The Raman spectrum of graphene has two characteristic peaks: G and 2D peaks. The G peak (usually around 1580 cme layers . A samplimproved .2 substrate and its Raman spectra. According to the Raman spectra of the three tested positions, I2D/IG was about two, indicating that the graphene was monolayered and not destroyed after transfer. The number of graphene layers was maintained with good uniformity. However, it can be seen from the Raman spectrum of the first position that there was a defect peak D, indicating that a small number of defects appeared on the surface.1, P2 and P3, on the slide substrate, respectively. There were defect D peaks in all of them. The square resistance of graphene on the slide was more significant, overall, than on the SiO2 substrate, because the square resistance of graphene is proportional to the number of layers [According to f layers .2O and graphene; it can nonetheless widen graphene’s bandgap, leading to more significant square resistance. Therefore, PMMA affected the square resistance of the samples seriously. Additionally, there were stacks in the samples, observed by their Raman spectra, but they were not the main factor in the square resistances of the transferred graphene samples. This study provided a theoretical and experimental basis for graphene as a better electrical medium.In conclusion, the defects that occurred in the wet transfer process greatly influenced the electrical properties of the produced graphene. Defects, such as PMMA residue, delamination and cracking during the wet transfer process, were discovered with various instruments. AFM imaging found that, on the same substrate, samples’ roughness and square resistance increased. The samples with higher square resistance had larger water contact angles and more PMMA content. As PMMA residue can cause a smaller contact angle due to a competitive relationship with H


N-doped graphene samples with different N species contents were prepared by a two-step synthesis method and evaluated as electrocatalysts for the nitrate reduction reaction (NORR) for the first time. In an acidic solution with a saturated calomel electrode as reference, the pyridinic-N dominant sample (NGR2) had an onset of 0.932 V and a half-wave potential of 0.833 V, showing the superior activity towards the NORR compared to the pyrrolic-N dominant N-doped graphene  and the pure graphene . N doping could significantly boost the NORR performance of N-doped graphene, especially the contribution of pyridinic-N. Density functional theory calculation revealed the pyridinic-N facilitated the desorption of NO, which was kinetically involved in the process of the NORR. The findings of this work would be valuable for the development of metal-free NORR electrocatalysts. The global nitrogen cycle has been disrupted by human activities and industries, resulting in an increased nitrate concentration in the hydrosphere ,2. SinceThe electrochemical reduction of nitrate has attracted huge interest thanks to its controllable process and relatively low costs . HoweverCarbon nanomaterial is widely used as electrocatalysts due to their chemical stability and good conductivity ,40,41,422 electroreduction [N doping can significantly enhance the electrochemical activity of graphene for oxygen reduction , oxygen eduction , due to eduction ,53, it wHerein, we synthesized N-doped graphene containing different N species and evaluated their NORR activity. For the first time, an N-doped carbon material was adopted as the NORR electrocatalyst and the contribution of active N species was investigated. We demonstrated that pyridinic-N played the dominant role in the enhanced NORR activity and density functional theory (DFT) calculations were further conducted to reveal the mechanism.3, HCl, H2O2, and H2SO4 were obtained from Chongqing Chuandong Co., Ltd. . Dupont Nafion PFSA Polymer Dispersion D-520 was obtained from Dupont. All solid chemicals and 20% commercial Pt/C were purchased from Aladin Ltd. . HNOFirstly, graphene oxide (GO) was prepared by a modified Hummer’s method . Then, G2 adsorption–desorption isotherms were obtained at 77 K with a Quadrasorb 2MP instrument. Scanning electron microscopy  and transmission electron microscopy  were used to characterize the morphology of catalysts. Energy dispersive X-ray spectrometer (EDS) elemental mapping was recorded on an Iridium Ultra Premium EDS System . X-ray diffraction (XRD) patterns were carried out on a Shimadzu LabX XRD-6000 to investigate the crystal structure. X-ray photoelectron spectra (XPS) were obtained with an ESCALAB 250Xi spectrometer using a nonmonochromatized A1 Kα X-ray source (1486.6 eV) for elemental composition analyses, together with a Renishaw Micro-Raman system 2000 with He-Ne laser excitation for Raman spectroscopy. N−2 for all the samples. Linear sweep voltammetry was carried out in a 5 M HNO3 solution with a scan rate of 10 mV s−1. Chronoamperometry was conducted in a 5 M H2SO4 solution at 0.8 V in the absence and presence of 2 mM HNO3. The nitrate removal experiment was conducted in a two-chamber reactor divided by a Nafion 117 membrane. The working electrode was prepared with a carbon cloth as the substrate with an area of 4 cm2. The volume of the solution was 20 mL. The concentrations of nitrate and nitrite were determined by the standard colorimetric method according to the Chinese Environment Standards using a UV-2450 spectrophotometer  and Pt wire were chosen as reference electrode and counter electrode, respectively. All the potentials mentioned in this work are versus SCE. For the electrode preparation, the prepared catalyst powders were mixed with a Nafion solution and loaded on glassy carbon with a mass loading of 0.2 mg cmotometer .ads) of NO was defined as:NO/surf, Esurf, and ENO (g) are the energy of NO adsorbed on the surface, the energy of clean surface, and the energy of isolated NO molecules in a cubic periodic box with a side length of 20.00 Å and a 1 × 1 × 1 Monkhorst–Pack k-point grid for Brillouin zone sampling, respectively.The Vienna Ab initio Package (VASP) ,55 was eTo accurately investigate the effects of different N species on the NORR performance, NGR1 and NGR2 with similar elemental contents  were pre2 adsorption–desorption isotherms, as shown in 2 g−1, respectively. It is known that the BET surface area significantly affects electrochemical activity. The difference in BET surface areas between NGR1 and NGR2 was less than 10%, which is helpful to rule out the influence of the BET surface area on the NORR activity.X-ray diffraction (XRD) patterns of NGR1, NGR2, and GR a exhibitXPS was employed to reveal the elemental composition of samples. As shown in −1 corresponded to the D band, which was caused by the disorder in graphitic structure of carbon materials, and the G band peak, located at 1580 cm−1, arose from the sp2 hybridized graphitic carbon atoms. Generally, the D/G peak intensity ratio (ID/IG) was highly correlated with the quantity of structural defects, which may reveal the degree of structural defect. As shown in D/IG values for NGR1, NGR2, and GR were 0.989, 0.993, and 0.983, respectively, indicating the three samples had a similar degree of structural defect. Therefore, the effect of N species, i.e., pyrrolic-N and pyridinic-N, on the electrochemical performance could be accessible. Since it was reported that graphene defects could be the active sites for the NORR, it was important to clarify the degree of structural defect of the samples before evaluating the effect of N doping . Raman s3 solution with a scan rate of 10 mV s−1 to investigate the NORR activity of the samples. Each sample was tested in a fresh solution to exclude the possible by-products produced during the survey. As shown in −2) and a half-wave potential of 0.506 V, indicating that GR was electrochemical active towards the NORR. However, the activity of GR was still lower than that of commercial 20 wt % Pt/C (Pt/C) since Pt/C had a higher onset potential (0.83 V) and half-wave potential (0.642 V). When NGR1 was adopted, the onset potential and the half-wave potential positively shifted to 0.85 V and 0.732 V, respectively, which were 20 mV and 90 mV more positive, respectively, than that of Pt/C, demonstrating the higher activity of NGR1 compared to Pt/C. Since NGR1 and GR had the same degree of structural defect revealed by Raman measurements, the significantly enhanced activity of NGR1 could be attributed to the N doping. Linear sweep voltammetries (LSVs) were measured in a 5 M HNOTo further investigate the effect of different N species on the NORR performance, the activity of pyridinic-N-dominated NGR2 was also tested. The onset potential of NGR2 further increased to 0.932 V, which was 83 mV more positive than that of NGR1. Additionally, the half-wave potential of NGR2 was 101 mV more positive than that of NGR1. Although the catalytic activity of pyridinic- or pyrrolic-N sites alone could not be distinguished based on our experimental results, the boosted electrochemical performance of NGR2 compared to NGR1 implied that pyridinic-N played an important role in the enhanced NORR activity of N-doped graphene since the major difference between NGR1 and NGR2 was the contents of pyridinic-N and pyrrolic-N.+ to enhance the activity, since the C atoms located at defects had different density states. The N doping could change the charge densities of surrounding C atoms due to the larger electronegativity of N (3.04) compared to C (2.55), therefore we speculated that the mechanism for the NORR on N-doped graphene might be the same.The mechanism of the NORR on N-doped graphene was further discussed. Kamiya et al.  reported+, as shown in Equation (4). To demonstrate the critical role of nitrite for the reduction of nitrate in the autocatalytic reaction, a chronoamperometric measurement was employed here with NGR2 in a 5 M H2SO4 solution in the absence or presence of 2 mM nitrate at 0.8 V, which was more positive than the onset potential of GR. As shown in 2, NGR2 with NaNO2 presented a reduction current corresponding to the electrochemical reduction of nitrite. Remarkably, NGR2 with both 2 mM nitrate and 1 mM NaNO2 exhibited a larger current than that of NGR2 with only 1 mM NaNO2. The larger current could be ascribed to the sum of the reduction currents of nitrite and nitrate, indicating that the nitrate could be reduced in the presence of nitrite. The results demonstrated that nitrite was the important reactive intermediate in the NORR.According to this mechanism, the nitrite was not only the product, but also a key intermediate, since the nitrite could be reduced into NO2SO4. As shown in The nitrate removal experiment with NGR2 as the working electrode was conducted in the presence and absence of nitrite to identify the electrochemical reduction of nitrate, as well as provide more evidence supporting the importance of nitrite in this process. A two-chamber reactor was employed to exclude the influence of the counter electrode. The initial concentration of nitrate was 10 mM and the electrolyte was 5 M H+ onto the edge of graphene rather than the basal plane of graphene has been reported by Kamiya et al. [+, the adsorption of NO+ on the edge of graphene was extremely strong. It is well known that N doping results in the presence of defects on the graphene. Thus, it was reasonable to speculate that Equation (2) was not the rate-determining step. Based on the proposed mechanism, another important process was related to the desorption of NO according to Equation (3). NO was expected to be desorbed from the active sites so that it could react with HNO3 to produce HNO2 and the active sites were therefore regenerated for the next reactive cycle. By taking these into consideration, the adsorption energies of NO (Eads) on different types of graphene were calculated and are shown in ads for undoped graphene with defects was −5.89 eV meanwhile the Eads for graphene with pyridinic-N was only −0.12 eV. The more positive Eads of NO on graphene with pyridinic-N than those of NO on undoped graphene with defects, indicated that NO would drop from the graphene with pyridinic-N more easily. These results demonstrate that the pyridinic-N doping facilitated the desorption of NO as well as the regeneration of active sites. The adsorption of NO on the graphene with pyrrolic-N was also calculated but the result after optimization was unreasonable, implying that the C atom near pyrrolic-N atom was not the active site. Considering pyridinic-N existed in both of NGR1 and NGR2 but with 1.8 times higher content in NGR2, the excellent NORR performance of NGR2 compared to NGR1 implied a positive correlation between the content of pyridinic-N and the NORR activity. Thus, both the experimental results and DFT calculations indicated that pyridinic-N played the most important role in the NORR and that the facilitated desorption of NO might be a possible origin of the enhanced NORR performance of N-doped graphene.To further reveal the effect of N doping on the NORR activity, DFT calculations were performed. The enhanced adsorption of NOa et al. . Due to In summary, we prepared N-doped graphene and evaluated its electrochemical performance towards the NORR in an acidic solution. To the best of our knowledge, it was the first time that N doping carbon materials were adopted as NORR electrocatalysts. The N doping could significantly improve the electrocatalytic activity of graphene for the NORR, which was better than the activity of commercial Pt/C. Furthermore, our results indicated that Pyridinic-N played an important role in the NORR by facilitating the desorption of NO from reaction sites as shown by density functional theory calculations. The findings of this work shed lights on the new application of N doping carbon-based materials and would be valuable for the design of metal-free electrocatalyst for the NORR.


When adjusting for age, gender and smoking status, the odds ratio of having IPF when expressing mPD-L1 in alveolar and/or bronchiolar epithelial cells was 4.3 (CI: 1.3–14.3). Conclusion: Expression of mPD-L1 in epithelial cells in the lung parenchymal zones was detected in a consistent subgroup of patients with IPF compared to other interstitial pneumonias. Larger studies are needed to explore the role of mPD-L1 in patients with IPF.Background: Idiopathic pulmonary fibrosis (IPF) is the most common and severe form within the group of idiopathic interstitial pneumonias. It is characterized by repetitive alveolar injury in genetically susceptible individuals and abnormal wound healing, leading to dysregulated bronchiolar proliferation and excessive deposition of extracellular matrix, causing complete architectural distortion and fibrosis. Epithelial-to-mesenchymal transition is considered an important pathogenic event, a phenomenon also observed in various malignant neoplasms, in which tumor cells express programmed death-ligand one (PD-L1). The aim of this study was to assess the presence of PD-L1 in patients with IPF and other interstitial lung diseases (ILDs). Method: Patients with a clinically and radiologically suspected idiopathic interstitial pneumonia or other ILDs undergoing transbronchial cryobiopsy to confirm the diagnosis at the Department of Respiratory Diseases and Allergy, Aarhus University Hospital, were included in this prospective observational study. Cellular membrane PD-L1 expression in epithelial cells was determined using the DAKO PD-L1 IHC 22C3 PharmDx Kit. Results: Membrane-bound PD-L1 (mPD-L1) was found in twelve (28%) of the forty-three patients with IPF and in five (9%) of the fifty-five patients with other ILDs ( Idiopathic pulmonary fibrosis (IPF) is the most common and severe form of idiopathic interstitial pneumonia, with a mean survival time of 3–5 years without antifibrotic treatment. Patients suffering from IPF have an increased risk of peripheral lung cancer , with a IPF is characterized by a progressive scarring of the lung parenchyma due to dysregulated bronchiolar proliferation and excessive accumulation of extracellular matrix (ECM), resulting in architectural distortion and respiratory failure. The pathogenesis of these changes is still not fully understood, but it may be divided into three steps. The first step is microinjuries occurring in type 1 pneumocytes from environmental exposures  ,7,8 and In recent years, the discovery of immune checkpoint inhibitors for the treatment of patients with various types of cancer, including lung cancer, has revolutionized the prognosis of patients with cancer . One of In a recent pilot study, Jovanovic et al. showed that soluble PD-L1 serum levels in patients with IPF were significantly higher compared to healthy controls . MoreoveSimilarly, Celada et al. found that PD-1 expression on the surface of circulating CD4+ T cells was significantly higher in patients with IPF compared to healthy controls. Immunohistochemical staining of PD-L1 and PD-1 in lung biopsies from patients with IPF and healthy controls showed increased expression in lymphocytes in 12 patients . In anotThe aim of the present study was to analyze the expression of PD-L1 in epithelial alveolar and/or bronchiolar cells present in fibrotic areas in lung tissue obtained through cryobiopsies from patients with IPF and compare these to patients with other interstitial lung diseases (ILD). Analysis of blood concentration of soluble PD-L1 was not considered for this study, but blood samples were collected and stored for further investigation. Patients having cryobiopsies performed as part of an investigation for ILD at the Center for Rare Lung Diseases, Department of Respiratory Diseases and Allergy, Aarhus University Hospital, Denmark, were enrolled in the study. Only patients >18 years providing written informed consent could participate. Biopsies were not taken in patients with a documented significant lung function impairment  < 50%, lung transfer factor for carbon monoxide (TLco) < 35%), if body mass index was >35, if pulmonary hypertension with tricuspid gradient above 40 mmHg was evident or if the patient was suffering from other cardiac diseases or had other comorbidities increasing the risk of complications. Patients were also excluded if the cryobiopsy procedure was not possible for technical reasons reaching the high-resolution computed tomography (HRCT) scan selected area. A HRCT pulmonary function test (PFT) , analyzed in accordance with the Global Lung Function Initiative reference values , and theThe cryobiopsy procedure was performed as previously described . PatientThe frozen lung samples were thawed in isotonic saline, fixed in formalin and finally embedded in paraffin in accordance with standard procedures. pos) parenchymal epithelial cells (type II pneumocytes or bronchiolar cells) was graded using a semiquantitative approach: <25%, 25–49%, 50–74% and 75–100% of the epithelial alveolar and/ or bronchiolar cells. The intensity was graded in three categories . The pathologist was blinded to the final diagnosis.All specimens were serially sectioned. The first section was stained with hematoxylin–eosin; the others were used for immunohistochemistry. The pathologic report followed previously published criteria . A report-test or the Mann–Whitney test, as appropriate, according to their distribution. Categorical data are presented as proportions of the total population and analyzed with the chi2-test or Fisher’s exact test when the total number of one group was ≤5. A p-value of <0.05 was considered statistically significant. Stata IC  was used for statistical analysis.Normally distributed data are presented as mean values ± standard deviation (SD), while data with a non-normal distribution are presented as median values with interquartile ranges (IQR) or total range. Data were checked for normality by histogram and the quantile–quantile plot. Continuous data were analyzed with the unpaired p = 0.047) and traction bronchiectasis (p < 0.001) were identified more often in patients with IPF. Traction bronchiectasis was also found more frequently in mPD-L1pos IPF patients compared to the mPD-L1pos non-IPF patients (p = 0.003). When grouping the patients into mPD-L1pos and mPD-L1-negative (mPD-L1neg) (p = 0.007) and GAP index 2 more frequent in the mPD-L1pos IPF group compared to the mPD-L1neg IPF group . IPF was diagnosed in 43 (44%) patients, and 55 (56%) patients were diagnosed with other ILDs than IPF (non-IPF), e.g., twenty with hypersensitivity pneumonitis (HP), seven with non-specific interstitial pneumonitis (NSIP), two with desquamative interstitial pneumonitis (DIP), one with eosinophilic pneumonia and twenty-five with other ILDs. Baseline characteristics are presented in patients  (p = 0.0D-L1neg) , baselinroup see . When adpos in the epithelial alveolar and/or bronchiolar cells. In the non-IPF group, five cases were found (9%) (p = 0.015). Two of the non-IPF patients were diagnosed with HP, two with NSIP and one with DIP. The intensity of the mPD-L1 staining was weak (grade 1) in four of the patients and moderate (grade 2) in thirteen of the patients. In one patient, mPD-L1-positive cells were found in <25%, in six patients in 25–49%, in eight patients in 50–74% and in two patients in >75% of the cells  and a combination of patchy fibrosis and fibroblast foci p = 0.02) were more frequently detected in the mPD-L1pos patients. When comparing the IPF mPD-L1pos patients with the IPF mPD-L1neg patients, or the IPF mPD-L1pos with the non-IPF mPD-L1pos patients, we found no differences in histological elementary lesions. We found no association between honeycombing and expression of mPD-L1. No association was found either between lymphocytosis in BAL (lymphocytes > 15%) and PD-L1pos patients (p = 0.97) or between percentages of macrophages in BAL and PD-L1pos patients (p = 0.36), nor did we find an association between C-reactive protein levels and PD-L1pos patients (p = 0.66).When comparing the mPD-L1neg, one IPF mPD-L1pos and one non-IPF mPD-L1neg). Four patients withdrew from further participation in the study (one with IPF and three non-IPF). There was a small but significant difference in the change in TLco at the one-year follow-up between the patients with IPF (−1.5 ± 8) and the non-IPF patients (5.2 ± 12) (p = 0.003). There were no other significant differences in PFT or distance in 6MWT when comparing patients with IPF to non-IPF patients, mPD-L1pos patients to mPD-L1neg patients, mPD-L1pos IPF patients to mPD-L1neg IPF patients or PD-L1pos IPF to PD-L1pos non IPF patients (pos non-IPF patients were treated with immunosuppressants (steroids and azathioprine). During the one-year follow-up, four patients died  were treated with antifibrotics. At present, we do not know if the presence of mPD-L1 is a key step in a subset of patients with IPF or a marker associated with disease development or progression. Moreover, we do not know if the expression has a protective role in preventing further progression and fibrosis or if it leads to more fibrotic changes.pos patients might support this hypothesis, although traction bronchiectasis was found more frequently in the IPF mPD-L1pos patients. However, the number of patients in each group was low, and thus we cannot draw any conclusions. On the other side, the uncovering of pathological processes leading to disease development and progression might identify ILD subtypes based on different pathways. Splitting the diseases opens up the possibilities of targeted treatment and precision medicine, as has been seen in cancers. In recent years, the discussion of lumping and splitting patients with fibrotic ILDs and IPF has been frequently debated. Several studies have suggested that these diseases share common traits with respect to clinical course and treatment response to antifibrotics, probably due to different triggers resulting in the initiation of common fibrotic pathways ,33,34,35Until now, IPF has been regarded as a risk factor for lung toxicity with respiratory deterioration and acute exacerbations when considering anti-PD-1 or anti-PD-L1 immunotherapy in non-small cell lung cancer. The incidence of any grade of pneumonitis was found to be 1.3–3.6% in a meta-analysis by Khunger et al. ; in a reIdentification of a subgroup of patients in which expression of PD-L1 on membranes of epithelial cells in the injured lung areas could incite clinical trials with drugs already used to treat a variety of malignant tumors.There are limited data regarding PD-L1 and PD-1 expression in non-cancer patients. However, a recent study found an increased expression of PD-L1 in renal cells in a mouse after certain types of injuries .pos IPF and non- IPF patients.Our study has several limitations. First, the length of the follow-up time might have been too short to observe major disease progression. Most of the patients with IPF were diagnosed with well-preserved FVC and were treated with antifibrotics, which affects disease progression. Second, we were not able to perform a power calculation, and this may have affected the subgroup analysis in mPD-L1Our study also has several strengths. Cryobiopsies were performed in all patients by three experienced bronchoscopists, ensuring a high biopsy quality, and all patients were diagnosed after MDT conferences. Additionally, the pathologist (L.B.M.) was blinded to the final diagnosis, routinely assesses PD-L1 expression in lung cancer and is familiar with this analysis using a semiquantitative approach. All subjects, apart from one patient, agreed to participate in the study, which minimized selection bias. We demonstrated that the expression of mPD-L1 in epithelial cells in the alveoli and bronchiolar cells present in fibrotic areas was evident in a subgroup of patients with IPF. Larger studies with longer follow-up time are needed to explore the role of mPD-L1 in patients with IPF.


Graphene-thermopolyurethane (G-TPU) composite films were fabricated and the effects of the TPU initial concentration, characteristics of TPU, and graphene loading on the electrical, mechanical, thermal, infrared thermal response and near-infrared-light-assisted self-healing properties of the composite films were investigated in detail. The experimental results demonstrate that the comprehensive performances of the composite film are related to the initial concentration of the TPU solution and the characteristics of the TPU and the graphene loading. The composite film prepared from TPU solution with low initial concentration can have conductivity under the condition of low graphene content. However, the composite film prepared with appropriate initial concentration of TPU solution and high graphene loading is conducive to obtain high conductivity. After 60 s of near-infrared illumination, the temperature of the composite film first increases and then decreases with the increase in graphene loading until it reaches saturation. The near-infrared light thermal response of the composite film with high graphene loading is related to the initial concentration of TPU solution, while the near-IR thermal response of the composite film with low graphene loading is independent of the initial concentration of TPU. The surface micro-cracks of the composite film almost disappeared after 10 min of near-infrared illumination. The resistance of the conductive composite film increases after healed. The composite film prepared with low melting point TPU is more favorable to obtain high near-IR thermal self-healing efficiency. With the development of information technology, many intelligent electronic devices have been rapidly developed and widely used ,2,3,4. HIn recent years, the advancement of wearable electronic devices, which use self-activating and self-adjusting systems, has raised new requirements for self-healing polymers ,27,28. ARecently, nanomaterials, such as gold nanoparticles, carbon nanotubes, graphene (G) and oxide graphene (GO), are added into TPU to endow composites with excellent electrical conductivity, thermal conductivity, antibacterial and self-healing properties; the improvement of mechanical properties of composites has been widely studied ,35,36,37Previous works mainly focused on the effect of carbon nanomaterials on TPU self-healing behavior, but did not pay attention to the properties of the TPU matrix ,38,39. HN,N-Dimethylformamide (DMF) was purchased from Tianjin Yongda Chemical Reagent Co., Ltd., Tianjin, China; thermoplastic polyurethane (TPU) master batches model number: HF-3H85A-3 , HM85A , and E685C4 , respectively, were purchased from BASF (China) Company Ltd. Guangzhou Branch, Guangzhou, Guangdong, China; ALR CL87A  masterbatches were purchased from Lubrizol Estane Chemical Co. Ltd., Estane Lubrizol, Cleveland, OH, USA.Graphene powders  were purchased from the Chinese Academy of Sciences Chengdu Organic Chemistry Co., Ltd., Chengdu, Sichuan, China. −1 for 60 min by using a high-speed shear disperser. The G-TPU slurry was poured into the Teflon mold, removing bubbles and volatilizing most of the solvent on a 50 °C heating plate until the solvent obviously disappeared, and then it was dried in drying oven at 70 °C until the weight was constant. The G-TPU film was peeled off. G-TPU films with different properties were obtained by changing the initial concentration of TPU and the loading of graphene in the G-TPU film and TPU with different characterizations.Graphene-thermoplastic polyurethane (G-TPU) composite film was prepared by using a solution mixing and tape casting process . A certa−1, the heating rate was 20 °C × min−1, and the sample test was heated from −50 °C to 200 °C, then cooled to −50 °C to remove the thermal stress, and then heated to 200 °C. The DSC curves shown in the figure were the data after removing the thermal stress. Microstructures of films were characterized by using a scanning electron microscope  and optical microscope  with a digital camera. Resistivity was tested by using a four-point probe system . The resistivity of the sample was measured at six different sites and calculated from the average value of those measurements. An infrared thermal imager . The thermal conductivity of sample was measured by a DRL-III heat flow meter instrument  according to the standard ASTM D5470.Thermal analysis of the film was performed using a differential scanning . The weight of sample was 13–15 mg, Gas1: Nitrogen 50.0 mL × min−1 by a universal tensile testing machine .Mechanical measurement: tensile strength of sample (dumbbell sample with size of 50 mm × 16 mm × 4 mm) was measured according to GB/T 1040.5-2008 standard, and its extension rate was 50 mm × minHealing measurement: self-healing process was conducted by infrared lamp  as the light source; use a knife to make a 5 mm scratch on the surface of sample, and then put the damaged sample under infrared irradiation (IR) for a certain period of time.m ~163 °C) solutions with the initial concentrations of 10 wt%, 20 wt% and 30 wt% were prepared and their viscosities were 65 mpa·s, 300 mpa·s, and 1800 mpa·s, respectively. The G-TPU composite films were prepared according to the graphene loading in the composite film of 0, 0.1 wt%, 0.3 wt%, 0.6 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt% and 7 wt%, respectively. Due to its unique properties, graphene sheets are the promising functional fillers for polymer. As mentioned earlier, we have conducted some research works on graphene/graphene oxide to improve the properties of PU, including the effect of the PU initial concentration on the properties of composite film for the first time ,42,43,44g) and the melting point of the pure TPU film are −35.6 °C and 163 °C, respectively. With the increase in graphene loading, the Tg of the composite film first decreases and then increases. This may be related to the good dispersion of graphene in the composite film with low graphene loading and the good compatibility with the TPU matrix, which is conducive to the free movement of soft segments of TPU, resulting in the decrease in Tg; on the contrary, the composite film with high graphene loading has poor graphene dispersion, and too much graphene sheets hinder the free movement of the soft segments of TPU, resulting in the increase in Tg [To investigate the effect of graphene on the thermal property of the composite film, the thermal profiles of the pure TPU matrix (curve 1) and the composite films prepared with 20 wt% of TPU and the graphene loadings of 0.6 wt% (curve 2), 2 wt% (curve 3), 3 wt% (curve 4), 4 wt% (curve 5) and 5 wt% (curve 6) are discussed by DSC, respectively, as shown in se in Tg ,46. The −1 × K−1, 0.2123 W × M−1 × K−1 and 0.2117 W × M−1 × K−1, respectively. With the increase in the graphene loading, the thermal conductivity of the composite film first increases and then decreases, which is higher than that of pure TPU. The thermal conductivities of the composite film are prepared with 10 wt%, 20 wt% and 30 wt% of the initial TPU concentrations and 4 wt% graphene reach the maximum, which are 0.3788 W × M−1 × K−1, 0.3657 W × M−1 × K−1 and 0.347 W × M−1 × K−1, and increase by 80%, 72% and 64% respectively, meaning that graphene can improve the thermal property of TPU, which is related to the initial TPU concentration. Graphene sheets with good dispersion are conducive to the formation of thermal conductivity channels, so as to improve the thermal conductivity. This is consistent with the influence trend of the conductivity of the composite film.To further investigate the effect of graphene on the thermal property of the composite film, we measured the thermal conductivity of the composite films with different graphene loadings and initial TPU concentrations, as shown in Previous research proved that graphene sheets have good infrared photothermal response performance, thus researchers could prepare near-IR-induced thermal self-healing graphene-based polymer composites ,41,43. HIn order to explore the effect of initial TPU concentration on the mechanical properties of composite film, the composite films prepared with the TPU initial concentration of 10 wt%, 20 wt% and 30 wt% and graphene loadings of 0 wt%, 0.6 wt%, 1 wt%, 3 wt%, and 5 wt% were used to measure the tensile strength, as shown in In order to explore the self-healing performance of G-TPU assisted by near-infrared light, the composite films were prepared with 20 wt% of the initial TPU concentration and graphene loadings of 0 wt%, 0.6 wt%, 2 wt%, 4 wt% and 5 wt%, respectively. A 0.5 mm scratch was formed on the surface of the sample with a knife, and then the sample was exposed under the infrared lamp with an infrared light intensity of 3450 lux for different times; the morphology of scratch was observed and characterized by optical microscope and SEM. Usually, the ratio of tensile strength of the sample, with and without self-healing, indicates the self-healing efficiency ,38. FiguThe self-healing efficiency of thermoplastic materials is closely related to temperature. The composite films prepared with 20 wt% of the initial TPU concentration and graphene loading of 2 wt% were healed under IR irradiation for 10 min at 110 °C, 120 °C, 130 °C, 140 °C and 150 °C, respectively. In order to explore the multiple self-healing abilities of the composite film, we chose the composite film prepared from 20 wt% of the initial TPU concentration and 2 wt% of graphene sheets for five self-healing cycles by near-IR irradiation. In our previous work, we fabricated G-TPU composite films using TPU-A, TPU-B, TPU-C and TPU-D as the matrix, respectively, and discussed the effects of TPU with different characteristics on the microstructure, the electrical conductivity, thermal conductivity and the IR light thermal response performance of the composite film . Here, wIn order to examine the application of the self-healing the composite film, we chose the composite film with 5 wt% of graphene loading, which has good electrical conductivity, in order to form a circuit with a 0.5 W light emitting diode (LED) bead. Graphene-thermopolyurethane (G-TPU) composite films were fabricated and the effects of the TPU initial concentration, the characteristics of TPU, and graphene loading on the electrical, mechanical, thermal and infrared thermal response, as well as the near-infrared-light-assisted self-healing properties of the composite films, were investigated in detail. The experimental results demonstrate that graphene improves the mechanical and thermal performances of the composite films and endows TPU with excellent electrical conductivity, infrared thermal responses and multiple high-efficiency near-infrared self-healing properties. However, the comprehensive performances of the composite film are also related to the initial concentration of the TPU solution and the characteristics of the TPU. The proper initial concentration of TPU solution and TPU masterbatch with a low melting point are beneficial to obtain composite films with excellent comprehensive properties. The composite film prepared with a low initial concentration of TPU solution can obtain electrical conductivity at low graphene loading, but the composite film with good electrical conductivity at high graphene loading requires an appropriate initial concentration of TPU. The near-IR thermal response of the composite film increases with the graphene loading until it reaches saturation. The near-IR thermal response of the composite film with high graphene loading is related to the initial concentration of TPU solution. The self-healing performance of the composite film with 2 wt% of graphene loading is the best and the self-healing efficiencies of the five self-healing cycles are 99%, 92%, 81%, 72% and 62%, respectively. The composite film prepared from the low melting point TPU is more favorable for obtaining high near-IR thermal self-healing efficiency.


Pinus tabuliformis Carr. bare-rooted seedlings grown outdoors in pots were irrigated with a graphene solution over a concentration range of 0–50 mg/L for six months. Graphene was found to stimulate root growth, with a maximal effect at 25 mg/L. We then investigated root microstructure and carried out transcript profiling of root materials treated with 0 and 25 mg/L graphene. Graphene treatment resulted in plasma-wall separation and destruction of membrane integrity in root cells. More than 50 thousand of differentially expressed genes (DEGs) were obtained by RNA sequencing, among which 6477 could be annotated using other plant databases. The GO enrichment analysis and KEGG pathway analysis of the annotated DEGs indicated that abiotic stress responses, which resemble salt stress, were induced by graphene treatment in roots, while responses to biotic stimuli were inhibited. Numerous metabolic processes and hormone signal transduction pathways were altered by the treatment. The growth promotion effects of graphene may be mediated by encouraging proline synthesis, and suppression of the expression of the auxin response gene SMALL AUXIN UP-REGULATED RNA 41 (SAUR41), PYL genes which encode ABA receptors, and GSK3 homologs.Graphene has shown great potential for improving growth of many plants, but its effect on woody plants remains essentially unstudied. In this work,  Graphene is an important carbon nanomaterial with unique physical and chemical properties, such as a colossal surface area, robust thermal and electrical conductivity, and good mechanical strength, which make it a chosen material for nanoelectronics , biomediGossypium hirsutum [Catharanthus roseus [Aloe vera [It is generally believed that the effect of graphene on plant growth is associated with its physiochemical properties, the application method used, the level and duration of exposure, and the plant species being examined . Numerouhirsutum , Catharas roseus , Arabidos roseus , watermes roseus  and Aloeloe vera  at conceloe vera . Graphenloe vera . Moreoveloe vera , 15–17.Notwithstanding these positive effects, it is reported that graphene may be detrimental to plants under certain conditions. The sharp edges of graphene may physically cut cell membranes and compromise their integrity . In addiBrassica napus L. [IAAs and ARFs were affected by graphene oxide treatment in tobacco [Exogenous stimuli can change plant gene expression profiles, and so provide important indicators for their effects. Graphene oxide treatment changes the transcription level of auxin and abscisic acid synthesis genes in napus L. . The tra tobacco . RNA-seq tobacco .Pinus tabulaeformis, one of the most widely distributed and important afforestation tree species in northern China, has played an important role in conserving soil and water and improving the environment [Pinus tabulaeformis was used to study the events occurring in woody plants in response to graphene treatment using physiological experiments, cytological observations and RNA sequencing. Our study enriches the understanding of the biological effects of graphene and provides a theoretical basis for the application of graphene in agroforestry.To our knowledge, previous studies on the biological effects of graphene have focused on herbaceous plants, and woody plants, which have distinct physiological structures and growth processes, have not been well investigated. Because of the advantages of strong adaptability, drought resistance, cold resistance, and ability to grow on barren land, ironment . Here, PAll the chemicals and reagents used in this study were analytically pure. Graphene suspension was produced by our laboratory as previously described . In ordeP. tabulaeformis bare-rooted seedlings of similar size were selected for experiments. There were 20 ± 3 plants in each group, among which 10 plants were selected for morphological and cytological analysis, and the remaining were used for RNA sample. Every plant was irrigated with 150 mL graphene solution once a month for 6 months.A graphene suspension with a concentration of 5 g/L was diluted in ultrapure water to different concentrations . Two years old pot-grown The fresh weight (FW) of the root tissue was determined and then root morphology analysis was conducted using a root scanner . The root tissue was then dried at 65°C for 48h and dry weight (DW) was determined . The watRoots were washed in water and placed into 10 mL centrifuge tubes containing 2.5% (v/v) glutaraldehyde dissolved with 50 mM phosphate buffer (pH = 7.2) for primary fixation. The following sample processing and preparation experiments were entrusted to the Electron Microscope and Mass Spectrometry Analysis Platform of the Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences. Images were obtained using a transmission electron microscope . The average root cell area was determined using Image J software to measure the two outermost cell areas in the transmission electron microscope images.https://bigd.big.ac.cn/) under accession number CRA004280. The longest transcripts were chosen as unigenes for subsequent analysis. A final unigene set was obtained by clustering with CD-HIT software [Three biological replicates were performed for both the control (CK) and graphene treatment groups. Cleaned root tissue was wrapped in aluminum foil and immediately immersed in liquid nitrogen. The following experiments, including RNA extraction, cDNA purification, library construction, sequencing and bioinformatics analysis were commissioned to OE Biotech Co., Ltd. . The sequencing was conducted using an Illumina HiSeq X Ten sequencer. Raw data were processed with Trimmomatic software to obtain clean reads , which wsoftware  to removsoftware  was usedsoftware . Genes wsoftware . DEGs we® RT reagent Kit . The expression of TUBB (TRINITY_DN15602_c0_g1_i1_1), which is predicted to encode the tubulin beta-5 chain, was used as an internal control. Primers were designed using the NCBI  Primer-BLAST program (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and are listed in S1 Table in Total RNA was extracted from CK and 25 mg/L graphene treated groups with the RNAprep pure plant kit , digested with DNase Ⅰ and reversed transcribed into cDNA using PrimeScriptEach experiment had three biological replicates, and the results are presented as mean ± standard deviation (SD). Significant differences between means were analyzed mainly by one-way analysis of variance using the SPSS 21 software. Significance differences were determined by a least significant difference (LSD) test at a 0.05 probability level.Scanning electron microscopy was used to determine the morphological characteristics of the graphene used in this study. At low power, the graphene presented a uniformly distributed lamellar structure, with a scale less than 1.70 × 2.77 μm . At highP. tabulaeformis. Graphene solutions ranging from 0–50 mg/L were used to irrigate P. tabulaeformis bare-rooted seedlings once a month. No visible differences were seen in the aerial parts between treatment and CK groups after 6 months  and the number of uniquely mapped clean reads was 33.72–40.06 million (69.0% - 71.2%) , Picea sitchensis (28.39%), Oryza sativa (8.74%), A. trichopoda (2.01%), Nicotiana tabacum (1.59%) and N. glutinosa (1.51%).The expression levels of unigenes were calculated by fragments per kilobase of transcript per million fragments mapped (FPKM) method and normalized using DESeq software. The significance threshold for differential expression was set as |log2(FoldChange)| > 1 and ant DEGs , of whicP. tabulaeformis root system, GO enrichment analysis were conducted with DEGs annotated from plant databases. A total of 4871 DEGs, including 1919 up-regulated and 2952 down-regulated, were aligned and classified using the GO database. To understand the effects of graphene on the To investigate the plant-related pathways affected by graphene, 1706 DEGs (694 up-regulated and 1012 down-regulated) annotated to plant database were also subjected to KEGG pathway analysis. Consistent with GO enrichment analysis, the DEGs up-regulated were enriched in categories related to plant hormone related signal transduction, while the DEGs down-regulated were enriched for the category ‘plant–pathogen interaction’ . The enrOur observations using transmission electron microscopy to examine roots of graphene treated roots suggested the plants may be experiencing mechanical stress. This was further suggested by GO term enrichment analysis, which showed that up-regulated DEGs were enriched for terms related to responses to abiotic stress, especially salt stress. We further analyzed the DEGs related to responses to different abiotic stresses, including salt stress, water deprivation, cold, osmotic stress, cadmium ion and heat. Of the total of 6477 DEGs annotated to plant databases, there were 176 genes related to the salt stress response, including 99 up-regulated and 77 down-regulated, which made up the largest proportion of the different abiotic stresses . In addiSAP18 [GOLS [NFX1 [MYB3, MYB2, MYB4, NAC22, bHLH148, SAP18 (Sin3A Associated Protein 18), GOLS  and NFX1 (NF-X-LIKE 1) are up-regulated after six months of graphene treatment. MYB3 was function in regulating anthocyanin biosynthesis and flower development in apple [MYB2 was involved in the patterning of proanthocyandin and anthocyanin pigmentation in Medicago truncatula [MYB4 plays dual roles in flavonoid biosynthesis [NAC22, which down-regulated by virus infection, might be related to the health stage maintenance [OsbHLH148, which induced by dehydration, high salinity, low temperature and wounding, leads to elevated tolerance to drought stress in rice [SAP18, encoding a histone deacetylase complex subunit, was function in transcription regulation [TsGOLS2, a galactinol synthase, conferred enhanced tolerance to high salinity and osmotic stresses in arabidopsis [AtNFXL1 gene, encoding a NF-X1 type zinc finger protein was required for growth under salt stress [in vivo.Many transcription factors are thought to be closely related to plant stress response, such as MYB family, NAC family, bZIP family, bHLH family, Zinc finger family, AP2/ERF family, and so on . In addiSAP18 , GOLS [318 [GOLS  and NFX1LS [NFX1  were repin apple . MYB2 wauncatula . Arabidoynthesis . Rice NAntenance . Overexp in rice . SAP18, gulation . Overexpbidopsis . And thet stress . These cMany studies revealed that plant hormone signaling pathways, including abscisic acid (ABA), ethylene (ET), auxin, gibberellins (GAs), cytokinins (CKs), brassinosteroids (BRs), jasmonic acid (JA), and salicylic acid (SA), play key roles in responding to various external stresses and regulating plant growth and development . KEGG paLOX, and PAO, respectively. And ROS scavengers include peroxidase, catalase and superoxide dismutase encoded by PERs, CATs, and SODs, respectively [GR, was critical to resisting oxidative stress [PERs, 8 CATs, 20 SODs, 3 GRs, 3 LOXs, and 3 PAOs genes up-regulated after graphene treated , were differentially expressed [PR1 (Pathogenesis- Related protein 1) homologous genes were down-regulated. The down-regulated expression of a large number of R genes suggests that graphene treatment might lead to a decrease in plant immune activity. The hormones SA and JA are recognized as being important for plant immunity. After graphene treatment, the number of upregulated and downregulated DEGs related to SA and JA activated signaling pathway were almost equal , based on PRGdb  gene, encoding pyrroline-5- carboxylate (P5C) reductase, was up-regulated after graphene treatment (GLYCOGEN SYNTHASE KINASE 3 (GSK3) genes ARABIDOPSIS THALIANA SHAGGY-RELATED KINASE 11 (AtSK11) and AtSK12 are involved in the root growth response that occurs upon mild osmotic stress [SMALL AUXIN UP-REGULATED RNAs (SAURs) auxin-responsive genes are involved in the regulation of adaptive growth under abiotic stress [SAURs differently expressed after graphene treatment. SAUR41, which is considered as a positive regulator for cell and hypocotyl elongation, was upregulated by graphene [PYLs promote plant growth and productivity [PYLs . The changed expression of these stress-responsive genes may therefore underlie the observed promotion of root growth in response to graphene treatments.Proline, the accumulation of which correlates with tolerance to drought and salt stress in plants, has been shown to affect root growth by controlling cell division , 53. Thereatment , which mc stress . As showc stress . There agraphene . Under agraphene . Mutatiouctivity . GraphenPR1 genes, P5CR, SAUR41, three GSK3 homologous genes and three PYLs were examined by real time PCR, to validate the RNA-seq results. The observed variation in expression of these genes in response to graphene treatment was consistent with that of transcriptome sequencing Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.