For the systematic perturbation of protein–protein interactions, we designed and synthesized tetra-substituted hexahydro-4H-pyrazinotriazine-4,7(6H)-diones as β-turn mimetics. 5c{3,9} stabilizes the direct interaction between LRS and RagD and activates mTORC1 in living cells.  H-pyrazinotriazine-4,7(6H)-diones as β-turn mimetics. We then devised a new synthetic route to obtain β-turn mimetic scaffolds via tandem N-acyliminium cyclization and constructed a 162-member library of tetra-substituted pyrazinotriazinediones with an average purity of 90% using a solid-phase parallel synthesis platform. Each library member was subjected to ELISA-based modulator screening for the LRS–RagD interaction, which plays a pivotal role in the nutrient-dependent mTORC1 signalling pathway. Western blot analysis of phosphorylated S6K1 as well as FRET-based imaging confirmed that 5c{3,9} stabilizes the direct interaction between LRS and RagD and activates mTORC1 in live cells under leucine-deprived conditions. Thus, 5c{3,9} can be used as a new research tool for studying the non-canonical role of LRS.For the systematic perturbation of protein–protein interactions, we designed and synthesized tetra-substituted hexahydro-4 Protein–protein interactions (PPIs) play critical roles in a myriad of biological processes and many complex diseases, including cancervia the specific binding of small molecules at the hot spots of the protein partners.A recent structural analysis of PPI interfaces revealed that not all residues at the PPI interface were critical, but rather that small “hot spots” conferred most of the binding energy.Because of the conformational adaptivity of hot spots under physiological conditions, small-molecule-based mimetics of secondary structures may be useful in studies of the biological processes of PPIs with unknown structures or as potential candidates for first-in-class therapeutics. Although a few β-turn mimetics have been reported as PPI modulators, these motifs are frequently observed in the loop regions of protein domains and are thought to be important in PPIs.Among the PPIs involved in various biological processes, we are interested in specific PPIs related to the mechanistic target of rapamycin complex 1 (mTORC1), which is a serine/threonine kinase that controls mRNA transcription and translation by integrating various environmental changes and signals such as growth factors, nutrients and energy status. Thus, mTORC1 modulates cell growth and proliferation and the dysregulation of mTORC1 is closely related to many diseases such as cancer and diabetes, making mTORC1 an attractive therapeutic target.H-pyrazinotriazine-4,7(6H)-dione 1. A small-molecule library of β-turn mimetics was constructed using a parallel synthetic strategy involving both solid- and solution-phase reactions. We also maximized the molecular diversity of our peptidomimetic library to occupy the potential chemical space for the specific modulation of protein–protein interactions. In order to determine whether the β-turn mimetics would perturb protein–protein interactions, we conducted an ELISA-based biological evaluation of our β-turn mimetic library against the specific interaction between LRS and RagD and identified a novel PPI stabilizer targeting the LRS–RagD interaction.Herein, we report the design and synthesis of a β-turn mimetic hexahydro-4i, i + 1, i + 2, and i + 3. One of the important features of the β-turn structure is that the distance between the Cαi and Cαi+3 atoms is less than 7 Å. Depending on the dihedral angles ψ and φ of the i + 1 and i + 2 residues, respectively, nine β-turn types defined by Hutchinson and Thornton are widely used: types I, I′, II, II′, VIa1, VIa2, VIb, VIII, and IV.et al. reported a strategy for designing trans-pyrrolidine-3,4-dicarboxamide as a β-turn mimetic structure as well as investigating its biological activities.The β-turn is one of the three major secondary structural motifs found in proteins and peptides, particularly in the reverse direction of polypeptide strands. The peptidomimetics of β-turns have been investigated to identify small-molecule modulators that disrupt β-turn-mediated recognition events.H-pyrazinotriazine-4,7(6H)-dione (1), as a β-turn mimetic to produce the desired conformation that could maintain its β-turn structural features with limited conformational flexibility under physiological conditions. The rigid bicyclic scaffold 1 allowed several slightly different conformers to cover the geometric β-turn structures and the resulting conformational restriction may have induced a higher binding affinity toward biopolymers because of its prepaid entropic penalty.1. When we calculated the distances between each atom of the energy-minimized conformer of its representative analog 7, the distance and geometry of the substituents were well-matched with the designated α-carbon of a β-turn motif, confirming that our β-turn mimetic scaffold 1 fits well into the geometric analysis data of β-turns observed in 10 245 reported structures in the protein data bank  showing excellent conformational similarity. The root-mean-square deviation (rmsd) value of compound 7 compared with each α-carbon of phospholipase A2 was 0.643 Å for five atom positions . Based on these results, we were confident that our pyrazinotriazinedione scaffold 1 mimicked the natural β-turn structures with limited conformational flexibility. In addition, our β-turn mimetic 1 showed a high F3sp value (0.67) with two stereogenic centers, which may allow selective binding toward certain target proteins to perturb PPIs.For β-turn mimicry, we designed and synthesized a fused bicyclic scaffold, tetra-substituted hexahydro-4ata bank .29 As fiH-pyrazinotriazine-4,7(6H)-dione 1 is shown in I) containing the R1 and R2 groups and the amine partner (II) on a solid support containing the R3 and R4 groups. To ensure the efficiency of the high-throughput parallel synthesis, we synthesized the carboxylic acid partner (I) in the solution phase, and the subsequent amide coupling with the amine partner (II) allowed for the formation of the linear precursor on a solid support. The final acid-catalyzed cleavage step from the solid support was designed for the in situ formation of N-acyliminium intermediates from aldehydes and an amide nitrogen, followed by their tandem stereoselective cyclization to obtain the β-turn mimetic scaffold 1 with a new stereogenic center. This pyrazinotriazinedione 1 contained four modifiable sites to maximize the molecular diversity, which could mimic the functionalities of side-chain moieties in natural amino acids for the specific recognition events in various PPIs.The retrosynthetic analysis of tetra-substituted hexahydro-4A-1 & A-2), we used two different hydrazines (methyl and tolyl) for diversification at the R1 position. Since a large quantity of acid partners is required for library construction, the synthesis of the acid partners was carried out in the solution phase . The remaining internal nitrogen was further decorated with t-butyl bromoacetate via SN2 reaction in the presence of KHCO3 at an elevated temperature. Due to the limited nucleophilicity of the internal nitrogen in tolylhydrazine, its modification step with t-butyl bromoacetate suffered with the formation of a diazene moiety as a major by-product, which lowered the overall yields regardless of the types of isocyanates used. For methylhydrazine, Boc-protected methylhydrazine was modified with three different isocyanates  (iii), followed by Boc deprotection using 4 N HCl in 1,4-dioxane (iv). After the SN2-type modification of the internal nitrogen with t-butyl bromoacetate, six different carboxylic acid partners (A-1 & A-2) were obtained through acidic hydrolysis on a gram scale in 3–4 steps with moderate overall yields (30–51%).To generate carboxylic acid partners . Next, solid-supported secondary amines 2 were coupled to various Fmoc-protected natural or unnatural amino acids  under the HCTU-based amide coupling conditions (vi). After the deprotection of Fmoc amines 3 on a solid support using 20% piperidine (vii), the resulting amine partners 4 were coupled with carboxylic acid partners (A-1 or A-2) under HOBt and DIC-mediated amide coupling conditions (viii or ix), respectively. After completion of the amide coupling monitored using ninhydrin testing, the final acidolysis step (x) involving neat formic acid allowed the formation of bicyclic β-turn mimetic scaffold 1 in excellent purities via the N-acyliminium intermediate shown in As shown in 7–14) were obtained without further purification with good purities and were fully characterized using 1H, 13C, and gCOSY NMR spectroscopy along with LC/MS and chiral HPLC analysis  enough to accommodate a series of hydrophobic substituents and maintain the drug-likeness of the resulting β-turn mimetics (clog P range from 3.45 to 7.47).As shown in 1 in a stereoselective manner. In fact, a new stereogenic center was generated via two different trajectories of the nitrogen nucleophile (pathways a and b) during the tandem N-acyliminium cyclization step under neat formic acid  NMR experiment to confirm the absolute configuration of the β-turn mimetic pyrazino-triazinedione scaffold. As shown in a at the new stereogenic center and Hb at the R4 position, and concluded that the nitrogen nucleophile attacked the top face of the N-acyliminium intermediate (pathway a) to avoid the steric repulsion of bulky R4 residues during tandem cyclization.Using this synthetic route, a new chiral center was generated in bicyclic β-turn mimetic scaffold acid see . Howeverin vitro instability, GST-RagD was roughly purified using a glutathione column and used immediately without freezing. The specific interaction between LRS and RagD can capture RagD-GST at the surface of each well, which was detected using anti-GST antibody. The level of small-molecule-based modulation toward the LRS–RagD interaction was quantified based on the quantity of bound RagD-GST, which was measured by determining the enzyme activity conjugated to the secondary antibody.To identify small-molecule modulators of the LRS–RagD interaction, we conducted ELISA-based screening with purified LRS coated on 96-well plates and each well was treated with each β-turn mimetic library member and GST (Glutathione S-transferase)-tagged RagD simultaneously for 3 h. Due to its 1 with hydrophobic and aromatic substituents stabilized the LRS–RagD interaction more effectively  to examine the interaction between CFP (Cyan Fluorescent Protein)-fused LRS (LRS-CFP) and YFP (Yellow Fluorescent Protein)-fused RagD (RagD-YFP) in live cells: the extent of the LRS and RagD direct interaction was measured as the intensity of the FRET signal between CFP and YFP -diones to mimic the β-turn structure, a key secondary structural motif. Distance calculations of energy-minimized conformers and the alignment of model compound 7 with side chains of a biologically active β-turn motif confirmed that our tetra-substituted β-turn mimetic bicycle 1 was conformationally similar to the natural β-turn structure. We then developed a robust synthetic pathway to obtain the β-turn mimetic scaffolds via tandem N-acyliminium cyclization and constructed a 162-member library of tetra-substituted pyrazinotriazinediones as β-turn mimetics with an average purity of 90% using a solid-phase parallel synthesis platform. In this synthetic route, a new chiral center was generated in a diastereoselective manner, which was confirmed using 2D NOE. After library construction, we examined whether these β-turn mimetic compounds could modulate PPIs and identified a series of compounds that could effectively stabilize the LRS–RagD interaction using ELISA-based screening. The cellular interaction between LRS and RagD directly mediated the leucine-dependent activation of mTORC1 and resulting biological outcomes. The western blot analysis of phosphorylated S6K1 and FRET imaging in live cell revealed that 5c{3,9} stabilized the direct interaction between LRS and RagD and activated the leucine-dependent mTORC1 signalling pathway in living cells. With this mechanism of action, 5c{3,9} can serve as an excellent research tool for studying the non-canonical role of LRS under leucine-deficient conditions and as a lead structure for examining disease models that require the selective activation of anabolic processes for wound healing or the restoration of muscle loss.For the systematic perturbation of PPIs, we designed and synthesized tetra-substituted hexahydro-4Supplementary informationClick here for additional data file.


A highly flexible porous ionic membrane (PIM) is fabricated from a polyvinyl alcohol/KOH polymer gel electrolyte, showing well‐defined 3D porous structure. The conductance of the PIM changes more than 70 times as the relative humidity (RH) increases from 10.89% to 81.75% with fast and reversible response at room temperature. In addition, the PIM‐based sensor is insensitive to temperature (0–95 °C) and pressure (0–6.8 kPa) change, which indicates that it can be used as highly selective flexible humidity sensor. A noncontact switch system containing PIM‐based sensor is assembled, and results show that the switch responds favorably to RH change caused by an approaching finger. Moreover, an attachable smart label using PIM‐based sensor is explored to measure the water contents of human skin, which shows a great linear relationship between the sensitivity of the sensor and the facial water contents measured by a commercial reference device. And the mechanical properties of the obtained PIM samples were also tested in a stretching apparatus (Instron 5900) as shown in Figure An as‐prepared highly flexible PIM sample (diameter: 10 cm) with PVA:KOH mass ratio of 6:3 was shown in I and I0 are the electrical currents at a given and initial humidity level, respectively . The real‐time dynamic response curve in Figure3 solutions were sealed in glass bottles at 25 °C to obtain constant RH conditions of 6.3%, and 95%, respectively). Moreover, the stability of the humidity sensor has been tested under the same detected humidity condition (RH: 81.25%) in 4 d after the sample exposed in ambient environment, and the result in Figure S3 in the Supporting Information shows that the four humidity sensing tests of the PIM sensor are basically consistent (sensitivity ±2.5%) with each other, indicating its performances are stable under the ambient condition of CO2 and other gases existed. The 3D porous structure of PIM with a lot of active sites can ensure high sensitivity, good stability, and a wide range of dynamic response, on which water molecules can connect with the sensitive material as the capillary condensation increases at different levels.The humidity response characteristics of this PIM‐based flexible sensor  were evaluated by measuring its electrical current under different RH conditions at room temperature.Previous studies have demonstrated that temperature usually has great impact to the performances of a humidity sensor. For example, carbon nanotube (CNT)‐based sensors are very sensitive to temperature because the binding and activation energies between water molecules and CNT are highly dependent on temperature.The humidity performances of PIMs with various mass ratios and thicknesses were systematically investigated. The sensitivities of the four as‐prepared products were determined at PVA:KOH mass ratios of 6:0, 6:1, 6:2, 6:3, and 6:4 . The results infer that the ratios can boost the moisture adsorption process as the conductive ability of PIM changes with additions of the KOH  in the PVA molecular chain is active in this process, the water molecule will reacted with the hydrophilic sites producing “bonded” water based on the diffusion of free volume, water/polymer interaction, resulting in the inner porous surface of the PIM is covered by a monolayer of water molecules.R2 << R1); The electrical impedance spectroscopy data of the sensor from dry air to 61.42% RH for several cycles were displayed in Figure S8 in the Supporting Information, which were also varied along with the RH change, demonstrating the excellent ionic conduction of the sensor. The increase in conductance is related to the combined influence of chemical and physical adsorption processes, but the chemical process proceeds more slowly than the physical process, so the increased conductance is mainly caused by an increase in physical adsorption.The basic humidity‐sensing response mechanism of the PIM‐based sensor can be explained as follows  into voltage shifts, so that the amplified result was automatically displayed by the on–off and degrees of brightness of a light‐emitting diode (LED) light, as shown in the video . And the sensitivity of the noncontact devices can be optimized through dosing water‐retention material and reducing the number of external circuits.Because of the high sensing performance of the PIM‐based humidity sensor, the flexible sensor was can be used to multifunctional applications. For example, it can be applied to detect the moisture around fingertips. A commercial RH sensor was used to calibrate the RH values of the fingertips. The results in FigureR2 = 90.67%) between the sensitivity of the PIM‐based sensor and the actual water contents data measured by Sensirion EX‐H4. Using this relationship, the real‐time water content of a person's skin can be accurately predicted with this smart label. For example, our PIM‐based sensor was applied to check the water contents of the facial skin in the morning, and a commercial device (Skin Moisture Detection Device SQNO.1) was set as a reference  film substrate, which can ensure the humidity of skin can effectively interact with the PIM without directly contacting with the skin . And another nonporous PDMS film was used as outer layer packaging to prevent the interference from the RH in surrounding environment Figure5a. A h3A PIM with well‐defined 3D porous structures was prepared by naturally evaporating the PVA/KOH polymer gel electrolyte, and its humidity sensing properties showed that a PVA/KOH mass ratio of 6:3 and thickness of about 70 µm were optimal process. Characterizations reveal that the conductivity of PIM‐based sensor changed more than 70 times as the RH value increased from 10.89% to 81.75%, with rapid response (0.4 s) and recovery times (2.6 s). It is noteworthy that this sensor was insensitive to temperature and pressure change, indicating that it is a successful candidate for highly selective flexible humidity sensors. Furthermore, the applications of the PIM‐based sensors were successfully demonstrated. A noncontact smart switch can respond rapidly to an approaching finger, and an attachable smart label was developed to real‐time access the water content of human skin. These performances will offer the tremendous opportunity to apply the PIM‐based sensors to multifunctional intelligent wearable systems.4Preparation of the PIM Sample and the PIM‐Based Humidity Sensor: The PVA/KOH porous ionic membrane was fabricated using a solution‐casting process at ambient conditions. A PVA solution was produced by mixing PVA (6 g) in deionized water (115 mL) and stirring continuously at 85 °C for about 2 h. Then, a freshly prepared cold aqueous solution (5 mL) of KOH (3 g) was added drop‐wise to the prepared PVA stock solution. No other additives were used to promote the reaction, thereby avoiding the effects of associates or impurities. The prepared PVA/KOH polymer electrolyte mixture was then cast on a glass plate and dried at room temperature, and, once dry, the PIM membrane was peeled off the glass plate. The amount of KOH in the electrolyte solution ranged from 1 to 4 g, and the thickness of the PIM samples, measured by SEM, ranged from 16 to 125 µm. Finally, the PIM‐based humidity sensor was produced by fixing a flexible electrode on the surface of the cutting membrane with silver paste.Humidity Sensing Test for the PIM‐Based Sensor: Figure S10 in the Supporting Information shows the measurement set‐up. Wet (water‐saturated) and dry air were mixed under different mass ratios to obtain various stable RH conditions ranging from 10.89% to 81.75%. The air was water‐saturated by bubbling dry air through saturated salt solutions sealed in a flask. The RH value of the test environment was adjusted by mixing moist and dry air inside the measurement chamber (0.7 cm3) at different flow rates, controlled by flow controllers. The actual humidity values were confirmed with a high‐accuracy general RH sensor placed inside the measurement chamber (Sensirion EX‐H4). Changes in the current were measured with a digital source meter (Keithley 2400). The data acquisition step was 10 s, based on which it took over 15 s to fill/evacuate the testing chamber.Assessing the Detective Limits and Response and Recovery Times: Various constant RH conditions were achieved by humidity bottles containing saturated solutions of different metal salts. LiBr, LiCl, MgCl2, Mg(NO3)2, NaCl, KCl, and KNO3 were sealed in glass bottles at 25 °C to obtain RH conditions of 6.3%, 11%, 33%, 54%, 75%, 85%, and 95%, respectively, thereby facilitating sudden changes in the environmental RH by transferring the sensor between the dry air and the achieved constant RH. The true value was checked by a commercial sensor (Sensirion EX‐H4). During detective limits and times testing, the PIM sensor was transferred between the RH vessels as quickly as possible to lower the risk of measurement error.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.SupplementaryClick here for additional data file.


The demand for transparent conductors is expected to grow rapidly as electronic devices, such as touch screens, displays, solid state lighting and photovoltaics become ubiquitous in our lives. Doped metal oxides, especially indium tin oxide, are the commonly used materials for transparent conductors. As there are some drawbacks to this class of materials, exploration of alternative materials has been conducted. There is an interest in films of carbon nanomaterials such as, carbon nanotubes and graphene as they exhibit outstanding properties. This article reviews the synthesis and assembly of these films and their post-treatment. These processes determine the film performance and understanding of this platform will be useful for future work to improve the film performance. As electronic devices, such as touch screens, displays, solid state lighting and photovoltaic devices become more prevalent in our lives, the demand for transparent conductors increases. Doped metal oxides, especially indium tin oxide (ITO) ,2 are ofConducting polymers have exhibited good electrical, optical and mechanical properties . HoweverCarbon nanotube (CNT), a one-dimensional material, is extensively studied by various groups due to its attractive properties. CNT exhibits low electrical resistivity ,25 and hVarious techniques have been developed to synthesize CNTs. Some common techniques include arc discharge , laser aCarbon nanotubes (CNTs) can be dispersed in solution via three main ways: dispersion in organic solvents , dispersCarbon nanotubes (CNTs) are often dispersed in solution by ultrasonication. This step is essential as it breaks down the CNT aggregates to disperse them in the solvent. As the process is very harsh, defects are introduced, which can degrade the electrical conductivity of the CNT . BesidesCarbon nanotube (CNT) films formed directly via chemical vapor deposition (CVD) ,42 generAnother important factor that determines the carbon nanotube (CNT) film property is the characteristics of individual CNTs. Synthesized CNTs are not uniform in conductivity type, length and diameter. Longer and larger diameter metallic CNTs are more desirable for transparent conductor applications. The lower resistivity of metallic carbon nanotubes (CNTs) relative to semiconducting CNTs is attributed to the longer mean free path in metallic CNTs. Ballistic conduction in metallic CNTs can span up to micrometer range but semiconducting CNTs have a series of barriers to conduction along their lengths ,44. ResiBesides the lower sheet resistivity of metallic CNTs, the improvement in electrical conductivity of a film based on metallic CNTs only is attributed to the lower junction resistance between two metallic CNTs compared to junction resistance between one metallic CNT and one semiconducting CNT . The junHence, sorting of carbon nanotubes (CNTs) or selective synthesis of metallic CNTs is very crucial. Various groups have explored different means of sorting or selective synthesis, such as selective chemical functionalization ,51, seleLarger diameter CNTs are preferred for transparent conductors because they carry more current than smaller diameter CNTs ,57. In fav1.46 [Another important factor that influences the conductivity of the CNT film is the tube length of the CNTs ,63. The av1.46   and transmittance (T) of carbon nanotube network are related byDC and σOp are the electrical and optical conductivities respectively [DC/σOp is desired for a low sheet resistance and high transparency network. The electrical conductivity, σDC, is found to correlate with the network morphology. The mean diameter of the CNT bundles (bundled due to agglomeration of solution processed CNTs) has an inverse relationship with σDC. This is because the smaller the bundle, the higher the junction density and number of electrical pathways in the network. Hence, debundling of CNTs is desirable [Sheet resistance  grown CNTs has proven to be an effective method to transfer CVD grown CNTs. The method is deterministic and the CNTs can be completely transferred from a donor substrate to a receiving substrate . It is eAfter the assembly of the CNT film, post deposition treatment is often carried out to improve the electrical conductivity by improving the junction resistance between two CNTs ,71, remoThe junction resistance between two CNTs is expected to decrease when they are joined. Two CNTs can be joined by Joule heating . By cont3 [The electrical conductivity of CNT network can also be improved by removing surfactants often used to disperse CNTs in solution. Some surfactant residues are deposited together with CNTs during CNT film assembly. As the surfactant is insulating, the electrical conductivity of the CNT film is compromised. Hence, after CNT film assembly, removal of the surfactant improves the electrical conductivity. Surfactant can be removed by washing with water, followed by acetone  or by ac3 .2 shifts the Fermi level closer to the valence band and conductivity improves [3, SOCl2 or I2 also prove effective [Another commonly used method to improve the electrical conductivity of CNT network is by doping. Various types of dopants have been studied and found to improve the electrical conductivity by different extents. Doping of CNTs by vapor phase reactions with bromine (electron acceptor) or potassium (electron donor) has yielded decreased resistivity by a factor of 30 . Doping improves . P dopinffective ,77. Anotffective ,79. HoweGraphene, the two-dimensional allotrope of carbon, has generated much interest because of its high mobility , transpaSolution processed graphene films have been studied extensively because their production via roll-to-roll processing can potentially be scaled up for commercial applications. There are two main forms of precursors to graphene in solution: graphitic precursors ,84,85,86et al. [i.e., the width and length) tend to be <400 nm, which will increase the number of inter-flake interfaces when a large surface area film is formed from such dispersions. The large number of inter-flake interfaces present will decrease the electrical conductivity of the film formed. Thin films can be formed from graphite dispersions via various techniques like vacuum filtration [As graphite is hydrophobic, surfactants are often used to assist the graphite to disperse in organic solvents. Lotya et al.  demonstrltration ,85, spraltration ,85 or Laltration . Another group  reportedAnother form of precursor to graphene in solution is graphite oxide precursors. By oxidizing graphite via the Hummers method  or a modet al. compared the sheet resistance and transparency of films formed via various reduction treatments [Becerril eatments . Three teatments . et al. also reported that incomplete reduction has detrimental effects on the electrical properties of reduced graphene films [3 bonds with the carbon atoms in the basal plane. The sp3 bonds disrupt the transport of charge carriers delocalized in a sp2 network. Hence, the electrical conductivity is lower in a partially reduced film relative to a completely reduced film. Mattevi ne films . The reset al. [Besides complete reduction of graphene oxide films, large graphene sheets are desired as they decrease the inter-flake interfaces. Large-scale graphene sheets with an area up to 20 × 40 µm have been demonstrated by Tung et al.  as shownThe main advantage of solution processed films is their compatibility with large scale and low cost roll-to-roll processing. The key disadvantages are the incomplete reduction of graphene oxide and the small dimensions of the graphene sheets formed. Development of methods to overcome these issues will result in films with better electrical conductivity, which is an important figure of merit for transparent conductors.Graphene films can also be formed by chemical vapor deposition (CVD) growth. The process usually involves breaking down a gaseous carbon feedstock  in hydrogen gas at high temperature on a metal catalyst to form a graphene film. Graphene film growth via CVD is very appealing because large scale growth is possible and the electrical conductivity of CVD grown graphene films is generally better than that of solution processed graphene films. et al. [et al. has also demonstrated very outstanding sheet resistance of 30 Ω/sq and 90% transmittance. High quality graphene films with dimensions up to 30 in have been demonstrated by Bae et al. . This isDespite the impressive progress in the field, some challenges remain to be overcome. CVD grown graphene films that demonstrate very low sheet resistance are often doped chemically. However, chemical doping is unstable and the sheet resistance of graphene films increases with time. This will be discussed in greater detail in a subsequent Grain boundaries in graphene films have been found to decrease the local work function, leading to potential barriers that scatter charge carriers by both backscattering and intervalley carrier scattering . The caret al. reported an approach that achieves grain size up to tens of micrometers by pre-patterning seed crystals to control the graphene nucleation [Yu cleation . In the et al. found that high temperature, low partial pressure and methane (carbon feedstock for graphene growth) flow rate during CVD growth result in fewer nucleation sites. Hence, they proposed a two-step CVD process [Other groups have also adopted a similar strategy of decreasing the nucleation sites to increase the grain size and decrease the grain boundaries by various clever methods. Li  process . The firMillimeter-sized grains have been demonstrated by various groups ,107,108 Besides grain boundaries, wrinkles in graphene films can impede electrical transport across the folds . WrinkleA graphene film grown via chemical vapor deposition (CVD) is often grown on a metal foil or metal film on substrate. Graphene film, which is used for transparent conductor applications has to be transfer printed from the growth substrate to a transparent substrate. Polymeric substrates are often used as they are flexible and transparent. It is not feasible to grow graphene film directly on a polymeric substrate via CVD because the polymeric substrate degrades under the high temperature employed during CVD. In this section, we discuss the various techniques developed to transfer print graphene films and relate the transfer printing process to the quality of the film transferred. The transfer printing process ,119,120 et al. reported dry and wet transfer techniques using PMMA, as illustrated in Various types of supporting substrates have been investigated. They include poly(methyl methacrylate) PMMA ,115, polIn the dry transfer process, PMMA is spin-coated onto graphene film grown on copper foil. A PDMS frame was used to support the PMMA/graphene film sandwich while etching the copper foil using ammonium persulfate. This copper etchant is preferred over iron(III) nitrate, because it does not leave behind contamination residues like iron oxide. After the copper foil is completely etched, the graphene film is transferred to a receiving substrate. Heat treatment above the glass transition temperature of PMMA is performed after the graphene transfer to improve the contact between the graphene film and the receiving substrate. This is achieved by softening the PMMA so that the gap between the graphene film and the receiving substrate is reduced. The improved adhesion of the graphene film to the receiving substrate prevents cracks and tears from forming when the PMMA is removed. The wet transfer process is similar, as illustrated in et al. [Liang et al.  also repi.e., ~275 Ω/sq), but subsequent transfers quickly decrease the sheet resistance. Hence, it was postulated that the adhesion between the first layer of film transferred directly onto the receiving substrate and the receiving substrate is poor, which results in mechanical damage of the film when the thermal release tape is removed. Hence, the sheet resistance is poor. Subsequent transferred layers do not interact directly with the substrate, so the sheet resistance is lower.The importance of adhesion between the transferred graphene film and receiving substrate is emphasized in . When trIt is evident that the transfer printing process of graphene films to an arbitrary receiving substrate strongly influences the quality of the transferred film. This is because the transfer process can introduce residual contaminants and mechanical damage to the transferred film . Hence, 3 in nitromethane, HNO3 in nitromethane and HCl. These strong oxidizing agents withdraw electrons from the graphene film and increase the doping density, leading to a decrease in sheet resistance [3 in nitromethane, the doping effect is transient. The sheet resistance increases by ~100% after 80 days in ambient condition at room temperature [x, also suffers sheet resistance degradation with time [i.e., only 8% decrease in sheet resistance). Typical sheet resistance of a transferred (via PMMA) graphene film is ~125 Ω/sq. This sheet resistance is low enough for certain applications, such as touch screens. However, when integrated into a solar cell or large area display, it will not be suitable as the series resistance is too high. Wet doping agents can be used to enhance the electrical conductivity of the graphene films ,122,123,perature . Anotherith time . A self-et al. reported a novel method of using nonvolatile ferroelectric polymer poly(vinylidenefluoride-co-trifluoroethylene) (P(VDF-TrFE)) gating to decrease the sheet resistance of the graphene film [Ni ene film . The advAlthough single layer of chemical vapor deposition (CVD) grown graphene film has relatively low sheet resistance <500 Ω/sq, the search for a scalable, non-volatile and highly effective method to dope graphene continues so that graphene films can be used in more commercial applications . The scaVarious groups have also explored hybrid films ,133,134.As demand for transparent conductors in flexible devices is expected to be high in future, alternative materials to substitute metal oxides are sought. Films of carbon nanomaterials have been identified as one potential class of substitute materials. More work remains as the sheet resistance of carbon nanomaterial films is still unable to reach <10 Ω/sq at 90% transparency refer to  below, wNonetheless, the films, which have been demonstrated may be suitable for less demanding applications, such as touch panels. Further exploration in their stability in devices over time is necessary. The scalability, reproducibility and cost effectiveness of integrating them into practical devices also have to be carefully evaluated. Finally, the success of practical application of carbon nanomaterial films in transparent conductors is also dependent on the development of competing alternative materials, such as thin metal films, metal nanowire films, conducting polymers and various other forms of hybrid films.


The U3 small nucleolar RNA (snoRNA) is an essential player in the initial steps of ribosomal RNA biogenesis which is ubiquitously present in Eukarya. It is exceptional among the small nucleolar RNAs in its size, the presence of multiple conserved sequence boxes, a highly conserved secondary structure core, its biogenesis as an independent gene transcribed by polymerase III, and its involvement in pre-rRNA cleavage rather than chemical modification. Fungal U3 snoRNAs share many features with their sisters from other eukaryotic kingdoms but differ from them in particular in their 5’ regions, which in fungi has a distinctive consensus structure and often harbours introns. Here we report on a comprehensive homology search and detailed analysis of the evolution of sequence and secondary structure features covering the entire kingdom Fungi. The U3 small nucleolar RNA (snoRNA) is a box C/D snoRNA with an exceptional structure. Like many other snoRNAs it is involved in pre-rRNA processing. However, in contrast to typical box C/D snoRNAs ,2 it doepansions . In addipansions .Saccharomyces cerevisiae), while the hinge between the first and the second hairpin binds to the 5S. cerevisiae U3 genes, which are excised by the canonical pre-mRNA splicing machinery [Neurospora crassa [Fungal U3 snoRNAs deviate from the eukaryote consensus in particular close to their 5a crassa . In conta crassa . So far,a crassa ,12,13 arhttp://www.bioinf.uni-leipzig.de/publications/supplements/16-018). The 54 fungal U3 snoRNAs from 52 distinct organisms reported in [muscle [infernal v1.1 [muscle alignments comprising both previously published U3 sequences and the homologs detected in the initial infernal search. Novel sequences found by these CMs were furthermore added to the alignments.We surveyed a total of 147 fungal genomes from Microsporidia, Mucoromycotina, Blastocladiomycota, Basidiomycota, and Ascomycota; a complete list is supplied in the Supplemental Material  database.Structural models were computed and iteratively improved using mLocARNA , RNAz [1NA [RNAz , as well package  as well RNAduplex a component of the ViennaRNA package [RNAduplex does not handle complex internal structures of the interaction partners. As a control we used random sequences with the same length and nucleotide background distribution as the 18S rRNA fragments.Base pairing between U3 and potential target sites were computed with  package  designed package . Homologmeme [Introns were identified using meme  to retri species  reports Schizosaccharomyces japonicus and Allomyces macrogynus with 11 and 10, resp., nearly identical paralogs. Duplications are evolutionarily recent and appear to be species- or genus-specific. There is no evidence for diverged families of paralogs. Instead, paralogous copies are presumably subject to concerted evolution [U3 snoRNA genes were identified in all 147 genomes included in this study, supporting the indispensable nature of U3 in rRNA biogenesis. We found a total of 310 genes, i.e., the U3 snoRNAs, similar to snRNAs, tend to appear in multiple genomic copies. Almost all investigated genomes contain three or fewer copies. Notable exceptions are volution  similar The secondary structures of U3 snoRNAs vary substantially between kingdoms [Rhodotorula minuta has a 50 nt insert in the loop of stem M6, the yeast Kluyveromyces lactis shows an even larger 80 nt insert in M8, while another yeast, Candida glabrata has an extra stem of about 50 nt between M9 and M10.The characteristic sequence motifs offer no surprises in the fungal kingdom. Motif ATACTY is nearly perfectly conserved, while A = GYATCW is more variable. Both C and CRTGATGA typical for box C/D snoRNAs. As in other box C/D snoRNAs, box CWYGATGA. The pattern for box B is quite variable at both ends but conserves the core motif AGYGA. Box D = CTGA is very stringently conserved and matches the pattern perfectly in almost all of the 310 sequences. Not unexpectedly, box DThe guiding potential of the predicted U3 snoRNA genes can be quantified by the number of base pairs and the interaction energy between the 5Kluyveromyces genus [Baudoinia compniacensis, Cucurbitaria berberidis, and both U3 genes in Rhodotorula graminis). In eight genomes we observed multiple copies of the U3 gene of which at least one is intronless and another one contains an intron, e.g., Coprinopsis cinerea, a basidiomycete, carries two U3 transcripts where one is intron interrupted. The remaining seven species are spread over Dothideomycetes , Leotiomycetes , and Sordariomycetes .Of the 310 genes from 147 organisms 138 from 78 genomes are interrupted by at least one intron. Intron-bearing genes are not found in early branching lineages such as Microsporidia, Mucoromycotina, Chytridiomycota, or Blastocladiomycota. They are broadly distributed across the Basidiomycota but not present in Taphrinomycotina. In Pezizomycotina they are found in the vast majority of analyzed organisms. Among the Saccharomycotina, only Saccharomycetaceae have introns. U3 genes both with and without introns are present in the es genus . A more GT-AG3GC-AG3Pyrenophora tritici-repentis and 5GC-GG3Nadsonia fulvescens. Notably, GC-AG junctions have been observed a common exception also in large surveys of fungal introns [GTRWGT, where the underlined nucleotides are absolutely conserved. Almost all of the remaining donor have a mismatch in exactly one of the other three positions. All except one acceptor sites share YAG with a strong preference of CAG over TAG (129 versus 8 introns). The patterns conform very well to the consensus motifs derived from large sets of introns [AYRCTR [TRACRC in 129/138 cases. The distance between branch and acceptor sites is strongly constrained, varying between 6 to 36 nt in fungal intron [S. cerevisiae and N. crassa [The U3 introns have an average length of 92 nt, approximately matching the typical intron length in most fungi . Almost  introns ,25. Near introns ,26. The  [RCTRAY . The prel intron ,27. The Baudoinia compniacensis, which is inserted in stem M3 upstream of box CThe introns are inserted almost exclusively in the most-5 motifs  . 133 of Penicillium or Saccharomyces genera, for example exceed 75%. Between Aspergillus and Penicillium we still observe between 40% and 60%. On the other hand there is also evidence for intron sliding [The tight clustering of intron locations in the U3 snoRNA sequence suggests that at least large groups of the introns are homologous, dating back to the same insertion event. Due to the rapid rate of sequence evolution in introns direct evidence for a common origin can be obtained from sequence similarity however only for closely related species. Introns with the  sliding . The intS. cerevisiae strains, which found that typically effects of individual mutations are small but may have very large epistatic effects acting in particular to maintain base pairing patterns [The U3 snoRNA is much better conserved in fungi and in eukaryotes in general than all other box C/D snoRNAs. Despite the presence of several important, well-conserved sequence motifs and well conserved core secondary structure elements, it shows extensive variability and clade-specific peculiarities. Among its well-preserved key features is also the propensity of its 5patterns .Fungal U3 genes are the only snoRNAs that are frequently interrupted by introns. These share canonical pre-mRNA splice sites and are clearly processed by the spliceosomal machinery. Introns have been inserted almost exclusively in the 5U3 introns appear only in the crown group fungal lineages. Introns may have been inserted only a single time in fungal history . If thisAlthough the 5


In addition, survival analysis showed that DLBCL patients who co-expressed PD-L1 and p-AKT had significantly poorer outcome than patients with single positive or both negative expression (P<0.05). In vitro, total PD-L1 and membrane PD-L1 (mPD-L1) proteins were overexpressed in five DLBCL cell lines by western blot and flow cytometry. We observed that AKT/mTOR pathway was activated in DLBCL cells after stimulated with human recombination PD-1/Fc. Taken together, these results suggested that the combination of PD-1/PD-L1 antibodies and AKT/mTOR inhibitor might be a promising and novel therapeutic approach for DLBCL in the future.Programmed death-1 (PD-1) /programmed death-ligand 1 (PD-L1) engagement usually leads to diminished antitumor T-cell responses, which mediates the immune escape of tumor cells. However, little is known whether PD-1/PD-L1 could directly activates intracellular oncogenic signaling pathways in tumor cells. The purpose of this study is to investigate whether intracellular AKT/mTOR signaling could be directly activated by PD-1/PD-L1 during the malignant progression in diffuse large B-cell lymphoma (DLBCL). Detection of the expression of PD-L1 and p-AKT by immunohistochemistry (IHC) showed that both proteins were overexpressed in 54% and 48% DLBCL cases, respectively. Spearman test showed that PD-L1 expression was correlated with p-AKT expression (R=0.244, χ Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL) accounting for approximately 30-40% of newly diagnosed NHL cases . It is aProgrammed death ligand 1 , a member of the B7 family, is an important ligand of programmed death 1 , which is an immune inhibitory receptor expressed on the surface of T cells, B cells and monocyte upon activation . RecentlInteraction between PD-1 and PD-L1 leads to the suppression of T-cell function and further facilitates immune escape of tumor cells. However, apart from evading host immunity, little is known about the intracellular signal transduction in tumor cell after PD-L1 binding to PD-1. PD-L1 is a type I transmembrane protein with 290 amino acids, consisting of immunoglobulin V-like and C like domains, a hydrophobic transmembrane domain, and a cytoplasmic tail domain . SeveralIn this retrospective study, due to lack of tumor tissue or clinical data of some cases, only 100 DLBCL patients between Jan 2008 and Dec 2011 in our hospital were evaluable. Clinical characteristics of the patients were summarized in Table Positive PD-L1 expression was found in 54 DLBCL cases (54%) by IHC. Representative IHC staining patterns of negative and positive PD-L1 expression were given in Figure 2=5.260; P=0.026, Table 2=5.702; P=0.027, Table 2=5.962; P=0.017, Table We also found that positive PD-L1 expression was significantly associated with pathological subtype  and 5 years OS (P=0.007) compared to patients with both negative expression of PD-L1 and p-AKT (Table Median follow-up duration was 52.4 (ranging from 1.5 to 89.1) months. All of the 100 patients were available for the 3-year overall survival analysis, however only 72 patients could be done for the 5-year overall survival analysis. The results revealed that 3-year and 5-year overall survival rates were 73% and 59.2%, respectively. Clinically, some DLBCL patients were only treated with CHOP/CHOPE. But other DLBCL patients were treated with standard R-CHOP. Considering the influence of different therapy schemes on DLBCL patient prognosis, we separately analyzed 61 patients who adopted CHOP/CHOPE regiment and 39 patients who adopted R-CHOP regiment. Kaplan-Meier survival curves of 61 patients who adopted CHOP/CHOPE regiment showed that DLBCL patients with either positive PD-L1 or p-AKT expression corresponded with a significantly shorter 3 years OS  and 5 years OS (P=0.014) compared to patients with both negative expression of PD-L1 and p-AKT , pathological pattern , clinical stages , IPI scores , chemotherapy regimens , PD-L1 expression , p-AKT expression , co-expression of PD-L1 and p-AKT  were associated with poor prognosis in DLBCL patients. To determine the prognostic value of PD-L1 and p-AKT expression, multivariate cox regression models for comparison with prognostic factors were applied. We found that clinical stages , chemotherapy regimens , PD-L1 expression  and p-AKT expression  were the independent prognostic factors negatively impacting survival.Subgroup analyses were performed according to gender, age, pathological pattern, clinical stages, IPI scores and chemotherapy regimens, PD-L1 expression, p-AKT expression and co-expression of PD-L1 and p-AKT. The results of univariate and multivariate cox analysis were summarized in Table In short, PD-L1 expression was correlated with p-AKT, and the co-expression of PD-L1 and p-AKT would result in a poorer prognosis compared with single positive or both negative expression of PD-L1 and p-AKT in DLBCL. Even PD-L1 and p-AKT were the two independent prognostic factors negatively impacting survival.We selected five human DLBCL cell lines to determine the total PD-L1 expression by western blot and mPD-L1 expression by flow cytometry. The results indicated that all of the five DLBCL cell lines expressed PD-L1 Figure  and mPD-To further determine whether PD-1/PD-L1 binding could directly activate the intracellular AKT/mTOR signaling in tumor cells, DLBCL cell lines were treated with human recombinant PD1/Fc for 24h and 48h. Total and phosphorylated AKT expressions were evaluated by western blot. Results revealed that p-AKT level was significantly up-regulated in three DLBLC cell lines including DHL-16, U2932 and Ly-3, suggesting that the intracellular AKT/mTOR pathway in DLBCL cells was activated Figure . Taken tP =0.0323) [P<0.05). Therefore, PD-1 or PD-L1 blockage might be a novel therapeutic approach for DLBCL and other tumors. Several clinical trials carried out in the past two years had revealed that both anti-PD-1 antibodies  and anti PD-L1 antibodies (such as MPDL3280A) had shown significant antitumor effects in patients with metastatic melanoma [With the advent of rituximab, the OS of DLBCL patients has been significantly improved. However, still 30~40% DLBCL patients inevitably develop into relapsed/refractory disease. So further studies are needed to investigate the underlying mechanism and develop novel therapeutic approaches for DLBCL. Recent studies have highlighted the role of PD-1/PD-L1 pathway in negatively regulating host antitumor response. As the major ligand of PD-1, PD-L1 is widely expressed and correlated with poor prognosis in many human cancers, including melanoma , lung ca=0.0323) , which wmelanoma , bladdermelanoma , and lunmelanoma .P=0.033, P=0.028), and R-CHOP for 5-years OS (P=0.023), but not R-CHOP for 3-years OS (P=0.051).DLBCL tumors have several important oncogenic signaling pathways which make DLBCL to be a heterogeneous disease and impact overall survival of DLBCL patients. The AKT/mTOR signaling pathway has been demonstrated to be constitutively activated in DLBCL , and AKT2=5.962; P=0.017). In addition, we found that the DLBCL patients who co-expressed PD-L1 and p-AKT had the worst clinical outcome compared with the patients who were single positive or both negative expression of PD-L1 and p-AKT for 3-years and 5-years OS (P<0.05). So we hypothesized that PD-1/PD-L1 binding might directly activate the intracellular AKT/mTOR oncogenic signaling in tumor cells to promote DLBCL progression. To confirm this hypothesis, we selected five DLBCL cell lines, which overexpressed PD-L1 protein and mPD-L1, to be stimulated with human recombinant PD-1/Fc protein for 24h and 48h. Interestingly, we found that level of p-AKT protein was significantly up-regulated with a time-dependent manner, suggesting that PD-1/PD-L1 binding could directly activate the intracellular AKT/mTOR oncogenic signaling in DLBCL tumor cells. Otherwise, Parsa et al found that expression of the gene encoding PD-L1 increased after loss of PTEN gene and activation of PI3K pathway in human glioma [In our study, spearman test showed that PD-L1 expression was correlated with p-AKT expression in DLBCL tissues in the Tianjin Medical University Cancer Institute and Hospital  from Jan 2008 and Dec 2011 were studied. Archived FFPE tumor tissues were obtained from our Department of Pathology and these cases were reclassified according to the 2008 WHO classification and Hans algorithm by experienced hematopathologists. In addition, 10 specimens of normal lymph gland tissue obtained from patients with reactive hyperplasia of lymph node were used as normal controls. All clinical information was obtained by reviewing the patients’ medical charts. The study and all protocols below were approved by the Ethics Committee of TMUCTH.IHC staining for PD-L1 and p-AKT proteins were performed using the streptavidin–peroxidase method (SP method). Briefly, the paraffin-fixed slides were dewaxed in xylene and rehydrated through graded alcohols. Antigen retrieval was respectively carried out using EDTA buffer (pH 8.0) for anti-PD-L1 and citric acid buffer (pH 6.0) for anti-phospho-AKT (Ser473) in 120°C for 2 minutes and 30 seconds. Endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide for 10 minutes at room temperature in dark place. Nonspecific binding of the primary antibody was blocked by incubating the slides with 10% normal horse serum for 30 minutes at 37°C. Then they were incubated with the primary antibodies including rabbit anti-PD-L1 polyclonal antibody  and rabbit anti-phospho-AKT (Ser473) polyclonal antibody  at 4°C overnight. And then they were incubated with secondary anti-rabbit IgG/HRP at 37°C for 30 minutes. Subsequently, for visualisation of the antigen, the sections were performed with the chromagen 3, 3′-diaminobenzidine. The slides were counterstained with hematoxylin and mounted under coverslips.Percentages of PD-L1 positive tumor cells and staining intensity were evaluated for each slide. Staining for PD-L1 was considered high expression, if ≥5% of the tumor cell population showed 2+ or 3+ membrane staining. In addition, if ≥20% of the total tissue cellularity showed 2+ or 3+ membrane or cytoplasmic staining in malignant and/or nonmalignant cells, it was considered to have a microenvironment positive for PD-L1 . p-AKT eThe DLBCL cell lines: TMD8, U2932, SUDHL-16 were cultured in RPMI-1640 medium  containing 10% fetal bovine serum . OCI-LY3 (LY3) and OCI-LY7 (LY7) were cultured in IMDM medium  supplemented with 15% fetal bovine serum . All medium were supplemented with 1% penicillin-streptomycin and all cells were incubated at 37°C in 5% CO2.For flow cytometry, DLBCL cell lines were harvested, washed, and then labeled with fluorochrome-conjugated PE anti-Human CD274   for 30 minutes at 4°C in dark place. Then the mPD-L1in all samples was measured by flow cytometry . Results were analyzed with FlowJo software (version 7.6).All five cell lines were cultured in complete medium containing 100ug/ml human recombinant PD1/Fc  for 24h and 48h. And then total proteins were isolated and run using western Blot to detect the total AKT and p-AKT expression.Cells were harvested and suspended in RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF). 20-50μg total protein (depending on different proteins) were separated by sodiumdodecyl sulfate–polyacrylamide (SDS–PAGE) gel electrophoresis and then transferred to polyvinylidene difluoride membranes , which were then blocked in 5% non-fat milk in Tris-buffer saline-Tween (TBST) for 1 h. The immunoblotting was performed by incubation with the primary antibodies including PD-L1 , p-AKT and total-AKT , and β-actin , which was used as an endogenous protein for normalization at 4°C overnight. Blots were then washed and incubated with a 1:3000 dilution of Goat anti-Rabbit IgG H&L (HRP)-conjugated secondary . Signals were detected by enhanced chemiluminescence Plus reagents  and formed images by Chemiluminescence Imaging System. Signal quantification was obtained using Quantity One software  and normalized to β-actin.Clinical, pathological, and chromosomal charac- teristics of the patients were compared using chi-square tests or Fisher's exact tests. The Kaplan-Meier method was used to estimate the over survival (OS) distributions, and the log-rank test was performed to compare the survival difference. Univariate analysis were performed with log-rank test. Multivariate-adjusted Cox regression models were used to evaluate independent prognostic factors. Difference was considered significant when the P value was <0.05 and all reported P values were two sided. These statistical analyses were performed with SPSS 17.0 statistical software .


In this work, the relative dielectric permittivity of graphene oxide (GO), both its real and imaginary parts, have been measured under various humidity conditions at GHz. It is demonstrated that the relative dielectric permittivity increases with increasing humidity due to water uptake. This finding is very different to that at a couple of MHz or lower frequency, where the relative dielectric permittivity increases with decreasing humidity. This GO electrical property was used to create a battery-free wireless radio-frequency identification (RFID) humidity sensor by coating printed graphene antenna with the GO layer. The resonance frequency as well as the backscattering phase of such GO/graphene antenna become sensitive to the surrounding humidity and can be detected by the RFID reader. This enables battery-free wireless monitoring of the local humidity with digital identification attached to any location or item and paves the way for low-cost efficient sensors for Internet of Things (IoTs) applications. It was established that oxygen functional groups in GO drive intercalation of water molecules between individual GO sheets, which results in an increase in the inter-layer spacing in GO thin films5. The presence of inter-layer water in a GO film can be crucial for a number of its properties, such as electrical conductivity7, molecular permeation9, mechanical10 and dielectric properties11.Graphene oxide (GO) is a chemical derivative of graphene functionalised with hydroxyl and epoxy groups. GO is a hydrophilic material and is capable to absorb a significant amount of water. Its water uptake depends strongly on the humidity of the environment and was previously studied by X-ray and neutron diffraction and 12. Of particular interest would be the relative dielectric properties of multi-layered GO as a function of water uptake, considering the low intrinsic relative permittivity εr of GO and the high 12. While the equivalent circuit technique works well at low frequency, it is not suitable for high frequency, such as at GHz, due to parasitic effects. In this work the GO relative dielectric permittivity was obtained based on the measured transmission and reflection parameters (S-parameters) at GHz. There is no need of equivalent circuit model for the GO.Multi-layered GO electrical and dielectric properties under various humidity conditions have been studied at low frequency6. We experimentally determine both the real and imaginary parts of the GO relative dielectric permittivity at GHz. The findings in this work are very different to those reported in12 – both the real and imaginary parts of the GO relative dielectric permittivity decrease with decreasing humidity, from ∼17 at 100% RH to 12 at 10% RH  and from 6 at 100% RH to 2 at 10% RH (the imaginary part of the relative permittivity). In particular, the imaginary part  method, free space method, resonator cavity, etc.14. However, all these methods do not suit permittivity measurement for small and thin piece of GO under different humidity environments.The electrical property of GO can be completely characterized by its relative dielectric permittivity, Here, to measure the relative permittivity of the GO layer under various humidity conditions, a resonator circuit was designed Fig. , with GO16. The saturated salt solutions used were LiCl (RH-11%), K2CO3 (RH-43%), Mg (NO3)2 (RH-55%), NaCl (RH-75%) and K2SO4 (RH-98%) aqueous solutions prepared by dissolving excess amount of salts in deionised water. Before each measurement with each particular salt, the humidity was set to be stabilised for at least 48 hours. All measurements are done at 24 °C. When the electrical property of GO (such as its permittivity) changes with humidity, it alters the loading of the resonator and results in a shift of the resonance frequency as well as change of the backscattering phase.Both the GO and the calibration circuits were placed in a hermetic container  for the samples with and without printed GO layer are displayed in Fig. The measured transmission coefficients S1 for theFrom the measurement results, it becomes clear that the sample with GO layer has responded to the humidity change, whereas the sample without GO hasn’t. The different responses of these two resonators can only be caused by the change of GO electrical properties due to water uptake. For the resonator with GO layer, it can be observed that the resonance shifts to lower frequency and its fractional bandwidth increases as the humidity rises. This reveals that both the real  antenna. Instead of using GO capacitor to sense the humidity12, the phase shift of the backscattering signal due to the humidity change was detected by the RFID reader. The GO sensor is battery-free, wireless and fully printable. Battery-free wireless sensing is in the heart of IoTs technology19, allowing collection of information about the immediate state of the object without the need of batteries. Here we demonstrate a battery-free RFID humidity sensor by combining printable graphene RFID antenna with GO coating.It’s well known that GO is sensitive to humidityRFID antennas are sensitive to changes in the environment due to proximity effects. When a layer of GO is printed on top of an RFID antenna – the resonance frequency of the latter becomes sensitive to the permittivity of the GO layer (and thus to humidity) as it alters the antenna impedance. Other parameters, such as the phase of the backscattered signal, change as well, which can be easily detected by the RFID reader. The operating principle and use of the phase of the backscattered signal to measure the relative humidity (RH) with the GO-coated printed graphene RFID antenna, shown in Fig. 21.When a RFID reader transmits an electromagnetic wave signal  to an RFID antenna, the antenna draws energy from this forward signal and activates the RFID chip on the antenna. The backscattered signal is both amplitude and phase modulated by the RFID chip through varying the chip’s input impedance. Modulation occurs as the RFID chip rapidly switches between two discrete impedance statesThe operating principle and equivalent circuit for the antenna’s amplitude and phase modulation are schematically shown in Fig. Va on the antenna in Fig. 22PAnt is the power available at the antenna port, Za is the antenna impedance. The switching between the two input impedance states ZC1 and ZC2 generates two different currents at the antenna port, which can be calculated as:22Different to a normal antenna, impedance of a RFID antenna is typically designed to conjugately match to the higher impedance state of the chip in order to maximize the collected power. The equivalent open source voltage 23). The resistance of the GO coating is still several orders of magnitude higher than the resistance of the printed graphene RFID antenna in this experiment (fractions of Ohm24), so the effects of the change of the GO conductivity can be ignored and only the change in dielectric property are taken into account. The GO dielectric property change alters the antenna impedance Za. As Za changes so do I1and I2, causing the backscattered signal phase varies accordingly. The backscattered signal phase change can be detected by the RFID reader. In this work, the backscattered signal phase was measured using Voyantic Tagformance under various humidity conditions and depicted in Fig. 25.When the humidity changes, the GO layer on the RFID antenna changes its dielectric property. At high humidity, the ionic conductivity due to the intercalated water increases and even pristine GO becomes conductive but only poorly (mega ohms resistance at 100% RH and Giga ohms at 0% RHRH) can be observed, unambiguously demonstrating the effectiveness of wireless printed graphene enabled battery-free RFID GO humidity detection.From Fig. 28. In those reported works, the minimum power-on-tag was measured and the resonance frequency was then extracted from the minimum power-on-tag. This technique requires the reader to scan the whole allocated UHF RFID frequency spectrum and post-measurement data processing to find out the minimum power-on-tag and resonance frequency. In this work, the backscattered signal phase was measured. The advantage of measuring the backscattered signal phase is that there is no need for the reader to scan the whole allocated frequency spectrum in order to find out the resonance frequency. As it can be seen in Fig. It is worth to notice that the technique used here to detect the humidity change is very different to that employed in other reported printed battery-free UHF RFID sensors7) and decreases with decreasing humidity at GHz. Furthermore the dielectric property has been used to design and build a RFID sensing tag which can act as a battery-free wireless humidity sensor, by coating GO layer on top of the printed graphene RFID antenna. Such combination can form bases for future energy harvesting enabled RFID sensors for IoTs applications. Furthermore, backscattered signal phase rather than minimum power-on-tag or resonance frequency has been used to detect the humidity change, which can significantly simplify and speed up the monitoring process.We have experimentally extracted the GO relative dielectric permittivity under various humidity conditions at GHz. The measurement results clearly reveal that the GO dielectric property  changes with the humidity but in a different manner as it does in a couple of MHz or lower frequency. Most distinguishingly, the relative dielectric permittivity does not have large changes , etc. The S-parameters were used to extract the relative dielectric permittivity in this work.30. In brief, 4 grams of graphite was mixed with 2 grams of NaNO3 and 92 mL of H2SO4. KMNO4 was subsequently added in incremental steps in order to achieve a homogeneous solution. The temperature of the reaction was monitored and kept near 100 °C. The mixture was then diluted by 500 mL of deionised water and 3% H2O2. The resulting solution was washed by repeated centrifugation until the pH value of the solution was around 7. The GO was then diluted to the required concentration. Lateral size of GO flakes is about 500 μm × 500 μm.Modified Hummers method was employed to prepare GO. The typical oxygen content for GO produced by this technique is around~ 30–40%31.The water uptake with two different flake sizes (0.5 μm and 10 μm) has been measured by monitoring the weight change of GO exposed to different humidity conditions. The results show that the small and large flakes only have a few percentage decrease in mass uptakes indicating similar hydration behaviour (see Supporting Materials, Fig. 32. The lateral SEM view of the GO coated on printed graphene on paper substrate is shown in Fig. For the purpose of coating GO on a printed graphene RFID antenna, a 10 grams per litre viscous GO solution was used. This allowed direct screen printing of the GO on the antenna, which was left to dry overnight in a fume hood under continuous air flow. The printed graphene RFID antenna is made with screen printing and rolling compressionAll data generated or analysed during this study are included in this published article and its supplementary information file.Supplementary Information


Programmed death ligand 1 (PD-L1) expression is a predictive biomarker of the success of PD-1/PD-L1 inhibitor therapy for patients with advanced non-small cell lung cancer (NSCLC) but its role as a prognostic marker for early stage resectable NSCLC remains unclear. Here, we studied PD-L1 expression and tumor infiltrating lymphocytes (TILs) in surgically resectable NSCLC and correlate the finding with clinicopathological features and patient outcomes. Total of 170 archival samples of resectable NSCLC were probed for PD-L1 expression using the clone 22C3 pharmDx kit. The PD-L1 expression was determined by the Tumor Proportion Score (TPS) and classified into TPS <1%, TPS 1 to 49% and TPS ≥50%. The scoring of TILs was from hematoxylin & eosin stained tissue sections using a system for standardized evaluation of TILs in breast cancer. PD-L1 expression was compared with clinical pathological characteristics and survival outcome. Expression of PD-L1 scores of TPS ≥50%, TPS 1 to 49% and TPS <1% were observed in 10.6%, 24.7% and 64.7% of the 170 archival samples, respectively. Positive PD-L1 expression was significantly higher in patients with squamous carcinoma, in those with higher TNM stage and with the presence of TILs. Neither the PD-L1 expression, TIL status, nor their combination was an independent prognosis biomarker of survival when the data was subjected to either univariate or multivariate analysis. The incidence of PDL1 expression appears to be lower in patient with early stage resectable lung cancer. PD-L1 expression and TILs are not prognostic indicators of survival outcome in this population. Blockade of immune checkpoints has recently emerged as a novel treatment for various cancers. In particular, monoclonal antibodies targeting programmed cell death 1 (PD-1) or its ligand (PD-L1) have been extensively studied in lung cancer, and their roles as first-line –6 treatmThe recent clinical series had reported various degrees of PD-L1 expression in lung cancers ranging from 7.4% to 72.7%, which was well reviewed by Mino-Kenudson . The samin vivo [Primary mechanism of PD-L1 on tumors is innate immune resistance and adaptive immune resistance. Interferon gamma (IFN-γ) is released by CD8+ T-cells and is a major inducer of PD-L1 expression in vivo , 17. In in vivo , 18–22. To address the issue, we investigate association with clinicopathologic characteristics and the prognostic value of PD-L1 expression measured by a commercial 22C3-PD-L1 immunohistochemistry diagnostic assay with a Dako platform in patients with surgically resectable NSCLC. We have also explored the immune microenvironment by studying the association between PD-L1 expression and tumor lymphocyte infiltration.A total of 170 patients were eligible for study and their characteristics are summarized in Table PD-L1 was commonly expressed at the cell membrane of cancer cells, and only in selective cases in the cytoplasm. Heterogeneous distribution of PD-L1 staining was observed within a single section of tumor tissue, with some areas being dominated by cells with strong PD-L1 expression, whereas other areas were characterized by cells lacking PD-L1 expression. Representative examples of PD-L1 Tumor Proportion Score (TPS) <1%, TPS 1 to 49%, and TPS ≥50% are shown in Figure Expression of PD-L1 was correlated with the clinicopathological characteristics by univariate analysis in a two-level classification of tumors that were negative (TPS <1%) vs. positive (TPS ≥1%) for PD-L1 expression (Table p=0.009). In this study, higher tumor stage was significantly associated with PD-L1 expression in univariate analysis, stage I 20.0%, stage II 41.9%, and stage III 41.6%, p=0.031. In the multivariate binary logistic analysis, squamous carcinoma tumors and higher TNM stage was confirmed as significantly independent factors for higher incidence of PD-L1 expression , p=0.004, by multivariate analysis (Table TILs were observed in 141 82.9%) of the tumors  (range: 1.4 to 104 m) and 89 patients had cancer-related deaths.p=0.202  vs. 57.9 months , HR 1.32 , 2 Figure . Using t2 Figure . Similar6 Figure . Further8 Figure . The una8 Figure ; Table 2We studied PD-L1 expression and TILs in 170 patients with resectable NSCLC and found neither the PD-L1 expression, TILs status, nor their combination to be an independent prognosis biomarker. The incidence of positive PDL1 expression is lower than expected. This could be related to the early stage of disease.We noticed that many clinical series of lung cancers had reported various amounts of PD-L1 expression and correlate expression with clinicopathologic characteristics and/or survival leading to conflicting results . One of Among the above PD-L1 antibodies, it is only the PD-L1 IHC 22C3 pharmDx assay that has obtained regulatory status as a companion diagnostic. We performed a literature search in PubMed for studies using the 22C3-PD-L1 assay published before Mar 1, 2017 , 15, 26 Recent studies, including ours, have investigated the prognostic impact of PD-L1 expression in NSCLC. However, the results are conflicting . DiffereA framework was previously proposed to stratify the tumor microenvironment into different types based on the presence or absence of TILs and PD-L1 expression , 27, 28.In conclusion, incidence of PD-L1 expression (by 22C3) in patients with resectable NSCLC is relatively lower than in patients with advanced NSCLC. Our findings do not support PD-L1 expression, TILs, or the combination of both as a significant prognostic factor for resectable NSCLC.th TNM staging [Primary tumor samples were from the archive of patients with surgically resectable NSCLC and pathologically confirmed adenocarcinoma or squamous cell carcinoma at the Fujian Cancer Hospital in China between January 2008 and December 2010. None of the patients had prior anti-PD-1/PD-L1 therapies, neo-adjuvant chemotherapy or EGFR/ALK-targeted therapy. The clinicopathologic information of patients was collected from the clinical records and pathology reports. The pathological TNM stage was reassigned according to the 8 staging  and lung staging . PatientWe conduct the study on PD-L1 expression at the Chinese University of Hong Kong, using automated staining by the Autostainer Link 48 with the murine 22C3 anti-human PD-L1 antibody  according to the manufacturer's protocol. Each staining run contained positive and negative controls along with a negative isotype-matched antibody control for each sample. Two board-certified pathologists  independently evaluated all stained slides for PD-L1 membrane staining. All areas in a tissue section were observed to appropriately evaluate the expression of PD-L1 on tumor cells. Following the standard recommendation as per prior publications, PD-L1 expression was determined by the TPS and classified into TPS <1%, TPS 1 to 49% and TPS ≥50%. . Using PThe independent scoring of TILs was performed in hematoxylin & eosin stained formalin fixed paraffin embedded tissue sections by two pathologists . Due to the lack of a standardized methodology for evaluating TILs in lung cancer, we adopted a recently reported system for standardized evaluation of TILs in breast cancer, which is also adopted in lung cancer . BrieflyBased on PD-L1 expression and TILs status, we classified the tumor immune microenvironment into four categories: 1) PD-L1 positive expression and TILs present (PD-L1+TILs+), 2) PD-L1 positive expression and TILs absent (PD-L1+TILs-), 3) PD-L1 negative and TILs present (PD-L1-TILs+), 4) PD-L1 negative and TILs absent (PD-L1-TILs-).Using PD-L1 TPS ≥1% as the cut off, PD-L1 expression were classified into positive (TPS ≥1%) and negative (TPS <1%) groups. The level of PD-L1 positive expression was compared in subgroups based on age (≤60yr or >60yr), gender , smoking status (never smoker or former/current smoker), primary tumor location , histology (adenocarcinoma or squamous carcinoma), TNM stage  and TILs (absence or presence) using the binary logistic analysis. Adjustment was made for age, gender, smoking status, primary tumor location, histology, TNM stage and TILs in multivariate binary logistic analysis.Overall survival (OS) was defined as the time from the date of diagnosis to the date of death or the last follow-up. The Kaplan-Meier method and a log-rank test were used for univariate survival analysis. Survival rate correlation of PD-L1 expression with, age, gender, smoking status, histology, TNM stage, PD-L1 expression, TILs and interaction between PD-L1 expression and TILs were estimated by the Kaplan-Meier method and survival curves were compared with the log-rank test. Cox proportional hazard models were used for multivariate survival analysis that controlled for the above factors and the hazard ratio (HR) and 95% CI were estimated.p<0.05.Statistical analyses were performed using SPSS16.0 software. All tests were two-sided. Statistical significance was set at


Staphylococcus aureus (S.aureus), and acceptable antibacterial ability against Escherichia coli (E. coli). Meanwhile, antibacterial efficiency closely correlates with coverage area of graphene film, and larger graphene coverage always leads to better antibacterial performance. The underlying mechanism is thought to be the integrative action of phospholipids disturbance and electron extraction at the interface between graphene and biomembrane. Meanwhile, the electron extraction action would further lead to the activation of platelet. This study might provide some new insights into the relationship between antibacterial ability and hemocompatibility based on graphene functionalized biomedical device.Germanium (Ge), as an elemental semiconductor material, has been an attractive candidate for manufacturing semiconductor microelectronic device. In the present investigation, to improve the biocompatibility of Ge-based device, graphene film is directly deposited on the Ge surface with different coverage area by controlling the growth time. Compared to bare Ge, the presence of graphene film entitles Ge with satisfactory antibacterial ability against  The rapid developments of biotechnology, biomaterials and tissue engineering have resulted in advances in medical diagnostics. Of particular significance are the bio-electronic devices . Nevertheless, the biocompatibility is always a crucial issue which limits the feasibility and longevity of the implantable bio-electronic device235Considering the sensibility of physiological environment, biomaterials and biochips remain subjected to the host immune response against foreign bodies342 hybridized carbon atoms packed in two-dimensional honeycomb structure, has been one of the most fascinating nano-materials ascribed to its exceptional physical and chemical performance1314151516171819Graphene, as a single-atom-thick sheet of spOur previous works have proved that relatively perfect monolayer graphene films can be directly deposited on Ge surface by CVD2021−1), the 2D peak (~2700 cm−1) and the defect related D peak (1350 cm−1), is shown in 232D/IG) above 3 is observed in 3-hybridization peak located at 285.1 eV, which comes from natural carbon contamination2-hybridization peak at 284.6 eV as well as a slight decrease in the intensity of the sp3 peak can be observed in 2 hybridization of the C 1 s spectra dominates (The graphene films are deposited on the Ge(110) substrate by atmospheric pressure chemical vapor deposition (APCVD). ominates , suggestCorrosion resistance is one of the significant factors to be considered in course of practical application of biomedical devices. In order to evaluate the impact of the graphene coverage area on the corrosion resistance of the underlying Ge, the dynamic potential curve test was conducted in the physiological saline (0.9 wt% NaCl solution). As depicted in Staphylococcus aureus (S.aureus) and Gram-negative Escherichia coli (E. coli) are used as model bacterium to inoculate on the surface of graphene coated Ge substrate. In order to better visualize the interactive behaviors of bacteria when contacting with graphene covered Ge substrate, Live/Dead Kit is utilized for bacteria fluorescent staining. As shown in S.aureus significantly. Meanwhile, the damaged bacteria display even more remarkable tendency, and observation vision is filled with intense red fluorescence derived from the listless and dead bacteria due to the damaged cell membrane, as depicted in the middle panel. However, the obvious increase in the proportion of the dead bacteria is observed on the Half-Gr where the surface is covered with graphene films, although not completely covered. Furthermore, massive damaged bacteria fill up the whole surface of Full-Gr. However, in case of E. coli, the red spots which refer to the dead bacteria (in web version) on the None-Gr are barely observed, while the surface is occupied by green spots, which refer to the living bacteria (in web version), indicating the inability of None-Gr to inactivate E. coli. The dead bacteria are hard to be observed on Half-Gr either , and only appear on Full-Gr with trace amount. Therefore, compared to E. coli, S.aureus is more susceptible to the presence of graphene coverage on Ge substrate.Gram-positive S.aureus and E. coli which are detached from the surface of the None-Gr are observed on the agar plate. However, when the bacteria contact with the Half-Gr surface, the number of S.aureus colonies reduces significantly, while the number of E. coli colonies remains high. In addition, the number of S.aureus colonies continues to decrease on the Full-Gr. Meanwhile, the number of E. coli colonies which are detached from Full-Gr ones also present a partially decrease compared to that from the Half-Gr ones. The result of reduction of Alamar blue in In addition, the bacterium solution is re-inoculated on the agar plate after detaching from the surface of incubated specimen to further incubation for 16 h, and the typical photographs is depicted in S.aureus on the None-Gr surface basically maintains original spherical appearance whereas those on the Half-Gr surface are disintegrated with severely distorted shape. Furthermore, the glomerate bacteria on the Full-Gr surface exhibit apparently deathlike state due to the crack of membrane. Meanwhile, the evident residue of exudative cytoplasm appears around the bacteria swarm. The similar manner is also observed for E. coli. On the None-Gr surface, the bacteria keep the typical rod-like shape. When the bacteria contact with Half-Gr surface, a minority of bacteria behaves with disrupted shape and loses the integrity of cell membrane while the majority of bacteria still remain the intrinsic shape. Nevertheless, the bacteria on the Full-Gr show irregular appearance characteristics, some bacteria have totally lost the cytoplasm ascribe to the fracture of membrane, which indicates poor living state. And, some robust bacteria with intact membrane still exist as well.To further confirm the influence of the graphene coverage on the bacteria phenotype, SEM is utilized to inspect the membrane integrity and morphology of the two distinct bacteria, as displayed in Infaust interface interaction between red blood cells and biomaterials in the physiological saline has the opportunity to give rise to the leakage of intercellular hemoglobin, which indicates the occurrence of hemolysis. According to the ISO 10993-4, biomaterials with the hemolysis rate less than 5% can be considered as feasible blood-contacting materials. As shown in S.aureus and E. coli at the same conditions, this could be due to the bacteria structure difference where the outer film of E. coli protects itself against extracellular perturbation, whereas S.aureus has no outer filmFor the past few years, the antibacterial mechanism of graphene and its derivates proposed by previous works mainly focus on physical damage caused by piercing through the bacteria membrane due to the sharp edge structure of graphene272920I-V characteristic curve . After heating to the desired temperature (910 °C), methane was introduced to deposit graphene films for various durations ranging from 100 min to 200 min. After deposition, the supply of methane was switched off and the furnace was cooled down to room temperature under the mixture of flowing H2 and Ar gas.The graphene films were deposited on the Ge (110) substrate by atmospheric pressure chemical vapor deposition (APCVD). Ge substrate was cut into small pieces and put in an inner quartz processing tube (50 nm in diameter) located at the center of horizontal quartz tube. Then the quartz tube was evacuated to approximately 0.01 Pa and followed by filling with gas mixture comprised of 200 standard cubic cm per min (sccm) argon  and 50 sccm hydrogen (HThe surface morphology was characterized by atomic force microscope (AFM Bruker Multimode 8 system) in tapping mode and scanning electronic microscopy (SEM Zeiss Supra 55) with an acceleration voltage of 3 keV. Raman spectroscopy (HORIBA Jobin Yvon HR800) was utilized to evaluate the quality and uniformity of the CVD-grown graphene film. The analysis of the X-ray photoelectron spectroscopy (XPS) was carried out by using a Gaussian–Lorentzian peak shape after removing background spectra by Shirley algorithm.A typical system consisting of three electrodes was used to conduct the electrochemical test. The graphite rod, saturated calomel electrode and the test sample were used as a counter electrode, reference electrode and working electrode, respectively. The physiological saline (0.9 wt% NaCl solution) was used as the electrolyte during the electrochemical test. The scanning parameter of the electrochemical workstation (CHI760C) was controlled at 1 mV/s from the cathode to the anode direction.®BaclightTM Bacterial Viability Kit  was used to implement fluorescence staining to reveal the viability of bacteria on the samples, and then the fluorescence staining results were observed by fluorescence microscopy (Olympus GX71). This kit contains a mixture of a SYTO®9 green fluorescence nucleic acid stain and red fluorescence nucleic acid stain, propidium iodide (PI).A LIVE/DEAD7 CFU mL−1 was seed on the sample surface, incubated at 37 °C for 24 h, fixed by 2.5 v% glutaraldehyde solution for night at 4 °C and then dehydrated by graded ethanol  for 10 min each. Subsequently, samples were finally dried by hexamethyldisilazane ethanol solution series followed by coating platinum before SEM observation.For scanning electron microscope (SEM) observation, 60 μL bacterium solution with the concentration of 10To evaluate the hemolysis rate (HR) of the samples, fresh human whole blood was obtained from the healthy donors. The blood collecting procedures were performed in compliance with institutional guidelines approved by Zhongshan Hospital, Shanghai, and the experiments were conducted according to National standard of China GB/T 16886.4-2003 protocol under the condition of donors’ consent. All samples were sterilized by 75 v/v% ethanol solution and blow-dried. Then samples were rinsed with physiological saline and then immersed in physiological saline at 37 °C for 30 min. Meanwhile, untreated physiological saline and ultrapure water were utilized as the negative and positive controls respectively. Subsequently, diluted human whole blood was add to each well and incubated at 37 °C for another 1 h. Afterward, the mixture solution were centrifuged for 5 min at 3000 rpm. Finally, supernatant were measured at 545 nm by Multimode Reader (Biotech Cytation 5), and HR can be acquired by the equation as follows545, N545 and P545. respectively.the absorbance for samples, negative control and positive control were represented by SPlatelet rich plasma (PRP) was obtained by centrifugation of human whole blood for 15 min at 1500 rpm. The samples was sterilized and dried as mentioned above, and then incubated for 30 min. Soon afterward, 50 μL PRP was seeded on each surface of the samples and incubated for another 2 h. After rinsed with PBS , samples were fixed by immersing in 2.5 v% glutaraldehyde solution for night at 4 °C. In order to observe the microstructure morphology, the platelets on the samples were dehydrated by the same processing conditions as mentioned in section of Antibacterial ability assessment.How to cite this article: Geng, H. et al. Antibacterial ability and hemocompatibility of graphene functionalized germanium. Sci. Rep.6, 37474; doi: 10.1038/srep37474 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.