The vacuum environment ensures that the graphene is not oxidized by air during hot pressing molding, which is essential for achieving conductive property in the graphene/PEKK composite; The hot pressing temperature of each graphene/PEKK composite powder is higher than glass transition temperature but lower than melting temperature, which ensures the graphene/PEKK composite powders is fully compacted and then graphene is fully lapped in the composite sheet. In addition, the graphene/PEKK composite sheet shows conductive property when the graphene content increases to 3wt%, and then the conductivity of the composites increases and then decreases with a peak value at 5wt% with increasing graphene content. By comparing the mechanical properties and microstructure morphology of the graphene/PEKK composite sheets, it was obtained that graphene content has an obvious effect on the mechanical properties of the composites, e.g., the mechanical properties will be increased as the graphene content increasing when graphene content is more than 3%. The graphene distribution law of the composite material with different graphene contents is analysed using a scanning electron microscope (SEM).Polymer composites are gradually replacing traditional metal materials in the fields of aviation, aerospace, automotive and medicine due to their corrosion resistance, light weight and high strength. Moulding technology and organization morphology of polymer composite are key elements affecting the quality of products and their application, so a vacuum hot pressing process for graphene The different elements, structures, combinations and forming methods of polymer composite materials determine the mechanical, physical and chemical properties of polymer composite products. According to this feature, researchers have modified the polymer composite materials to obtain different properties and realize different applications. The polyaryletherketone3 group of polymer products is a widely used product in aerospace and oilfield, usually forming polymer composites with metals and organic and inorganic substances. Polyaryletherketone matrix composites have been developed to meet the demands of various applications4.Recently, with the rapid development of materials science, computer science, mathematics and physics, polymer composites are gradually replacing some traditional metal materials to be applied in aviation, aerospace, automotive and medical applications because of their unique mechanical and chemical properties8. PEEK is more popular than PEKK both in field of scientific research and the real engineering as polymer matrix of composite. While, in addition to similar mechanical properties and crystalline behaviour to PEEK, PEKK received considerable attention because of its low price, low melting point and stable process, which results in better molding performance for PEKK composite and more applications9 as compared to PEEK composite. However, PEKK is insulator, which limited its application in many fields, such as electronic engineering field. Given its resistance to corrosion, conductivity, lightweight, versatility and simple process, graphene is used as a filler material, which is dispersed into the PEKK matrix to produce a new composite material and replace traditional metal materials, and then its application range has been further expanded10.The Poly(ether ketone ketone) (PEKK) and Poly(ether ketone ketone) PEEK are the well-known members of the PAEK family11. Injection moulding can achieve complex shapes for thermoplastic composite material, and it has good material flowability, high production efficiency and good dimensional accuracy. While, it is limited to high fluidity of liquid, which is hard for PEKK matrix, even in a high temperature above its melting temperature. Extrusion injection moulding12 is a highly efficient, continuous, low-cost forming technology for polymeric composites, but the cost of equipment and moulds is high. With the development of additive manufacturing14, 3D printing technology16 has been developed to a large extent, and it is gradually applied to the medical field, printing human organs, etc. While the formed materials are poor in density, in addition to demanding requirements to the size of row material powder particles, which greatly limits the application of 3D printing technology for polymeric composites.Although the preparation of polymer composite materials is a prerequisite for a good product, the moulding process is an indispensable condition for a good product. Moulding determines the quality and additional performance of the product. For the thermoplastic composite material, traditional forming technology includs injection moulding, hot pressing moulding and extrusion injection moulding. Injection moulding is an important polymer processing method19. Donadei revealed that residual stresses formed during hot pressing process for carbon fiber-reinforced PEKK powder determined most of the defects during heating, and an annealing treatment was proposed to reduced the formation of these defects20. Alsadon processed PEKK polymer using a standard ceramic pressing furnace and aimed to optimize the hot pressing parameters in order to achieve optical and mechanical properties of pressed samples21. Fujihara prepared CF/PEEK composite by hot pressing process with a molding temperature around melting temperature of PEEK, and the experimental results indicated higher hot pressing temperature led to decomposition of the PEKK matrix22. It is obvious that the design molding parameters according to raw material powder and the final performance is crucial for hot pressing molding. However, there is relative less research on GR/PEKK composite process, especially on its hot pressing molding23.In constrast to above forming methods, hot pressing process is a simple and widely used processing method in the plastic processing industry, especially for thermoplastic polymer matrix composites. The factors that affect the material state during hot pressing process are mainly the composition of the polymer composite, the pressure on the moulding, the ambient temperature and atmosphere environment, etcx/PEKK  composite powders in order to achieve conductive graphenex/PEKK composite sheet with good mechanical properties with a focus on design of molding parameters. In addition, the effect of graphene content on the conductivity, mechanical performances and corresponding microscopic morphology of the composite sheets were investigated.In this paper, we aimed to explore a vacuum hot pressing process for molding of graphene23. Table Given the poor dispersibility of graphene in the PEKK matrix based on the traditional solution dispersion and melt blending in the preparation of polymer composite materials, graphene/PEKK conductive composite powder was prepared by situ polymerization to make graphene nano particles uniformly dispersed in the PEKK matrix, which has been presented in our previous workMG1/PEKK, GRMG3/PEKK, GRMG4/PEKK and XF001w/PEKK composite powders presented a granular distribution. GRMG3 and GRMG4 composite powders were relatively regular, and the particle size distribution was relatively concentrated, which is an important factor in hot pressing moulding. The particle size distribution of GRMG2/PEKK composite powder was dispersed, and significant aggregation occurred, which is an unfavourable factor for the moulding of graphene/PEKK composite powder. The agglomeration of the composite powder also indicated that the remaining oxygen-containing groups on GRMG2 were relatively high, which caused the agglomeration of graphene.Figure MG3 powder will be selected as the experimental materials to prepare graphenex/PEKK composite materials. Figure Based on the above-mentioned discussion, GRx/PEKK composite material is in contact with air during forming, and this condition affects the electrical conductivity of the graphenex/PEKK composite products. Thus, vacuum environment during hot pressing  was carried out between 0 and 450 °C at a heating/cooling rate of 10 ◦C/min for graphene/PEKK composite powders. Electronic universal testing machine  was used to investigate mechanical properties of the graphene/PEKK composite sheet, in addition, the conductivity of the composite sheet was characterized by Four-probe tester . The microtopography of the graphene/PEKK composite sheet was investigated with scanning electron microscopy (SEM) with S-3004N, Hitachi Ltd, Japan.We agree to publish this article.The above-mentioned composite materials were processed and formed into electronic products and conductive part. The later sintering temperature of graphene/PEKK composite powders under hot pressing are designed according to their respective transition temperatures according to DSC test. The DSC curves, glass transition temperature and melting transition temperature of pure PEKK powder and graphene/PEKK composite powders with different contents are shown in Fig. Tg refers to the glass transition temperature, which determines the softening temperature of the material. Graphene in the graphene/PEKK polymer composite caused Tg of the composite material to increase and decrease around Tg of the PEKK matrix. The graphene content increased to more than 3%, and Tg of the composite material changed around Tg of the matrix as the graphene content increased with the increase of Tg. Graphene has high thermal stability, which improves Tg and Tm of the composite material. In addition, graphene was used as a conductive filler. With increasing graphene content, the damaging effect of graphene on the dense distribution of the matrix molecular segments increased (increased the movement ability of the chain segment). Therefore, graphene was the main factor in reducing Tg and Tm of composite materials.The downward absorption peak in the DSC curve is called the melting peak. Tm is a key parameter that determines the powder moulding temperature: graphene makes Tm of the composite material close to that of PEKK, but overall Tm of the matrix material is slightly lower. The addition of graphene increased the distance between molecular chains to a certain extent, improved the high mobility of the matrix segment, and destroyed the crystallization behaviour of the PEKK macromolecular segment, thereby lowering the melting point. Based on the above-mentioned experimental results, these different contents of graphenex/PEKK composite powders were used to obtain composite sheets using hot pressing pressure.x/PEKK composite sheets molded by hot pressing. In addition, this study compared the microstructure morphology with the mechanical performance of graphenex/PEKK composite materials to determine the cause of the difference in mechanical behaviour. In this paper, the mechanical performance of PEKK and graphene /PEKK composites sheets obtained from hot pressing process was also discussed. Table Tm), glass transition temperature (Tg) and sintering temperature. The conductivity of the composite material is necessary.Graphene/PEKK composite powders with different graphene contents were used as raw materials to obtain graphene-conductive samples by hot pressing. This paper focused on the mechanical properties of conductive grapheneTm and Tg of graphene/PEKK composite powders, the range of sintering temperature of hot pressing for these composite powders can be determined. During hot pressing, the powder in the mould is heated to the sintering temperature, and the composite powder presents an approximate fluid state, and finally cools and solidifies into the desired shape.Sintering temperature is an important factor affecting hot pressing moulding quality. A low sintering temperature cannot easily melt the composite powder, whereas a high temperature can easily cause decomposition of the PEKK matrix. On the basis of the x/PEKK  were moulded by vacuum hot pressing . As shown in Fig. x/PEKK composite material, the tensile strength ranges from 116 to 186 MPa, the elastic modulus ranges from 2.6 to 3.6 GPa, and the value range of elongation at break is from 4 to 6%. It should be noted the mechanical properties of graphenex/PEKK composite with highest graphene content (6%), which shows a strength of 177 MPa, an elastic modulus of 2.8 GPa, and an elongation at break of 6%.Figure PEKK is a semi-crystalline polymer, and its high strength depends on the crystalline structure inside the material to a large extent. Graphene destroys the regular arrangement of PEKK molecular chain segments to a certain extent and reduces its crystallinity. Thus, the strength of the prepared composite material is reduced. In addition, given the lamellar microstructure and powdery macroscopic state of highly rigid graphene in the prepared composite material, the network structure of the material is destroyed to a certain extent after it enters the PEKK material matrix, which directly leads to the material. The brittleness is increased, and stress concentration easily occurs, thereby damaging the material and decreasing tensile strength. Graphene improves the elastic modulus of the composite material probably because of its high modulus. It enters the network voids of the PEKK polymer matrix and serves as a skeleton, which can still be used to a certain extent. The elastic modulus of the material is also improved.x/PEKK composites prepared by situ polymerization and molded by hot pressing in this paper have a higher elastic modulus and tensile strength of approximately 40 MPa compared with the 10% nano-graphene composites discussed in previous literature24. However, previous studies and the present study clearly show a decrease in toughness of the composites with the addition of graphene, thereby indicating that with the increase of carbon, the toughness of the material decreases, but the stiffness increases.The strength results show that grapheneFigures Compared with (b), (c), (d) and (e), the graphene sheet shown in Fig. x/PEKK composite powder with different graphene contents, pressing moulding of graphenex/PEKK composite powders was explored by using vacuum hot pressing equipment to realize conductive property and good mechanical properties of the graphene/PEKK composites sheets with thickness of 1.25 mm and diameter of 80 mm.Based on situ polymerization of grapheneTg and Tm of each graphene/PEKK composite according to DSC tests, which are crucial for the quality of the molded product, especially for mechanical properties.The vacuum environment and molding temperature were particularly designed in order to obtain high conductive and good mechanical properties for the graphene/PEKK composite sheet. The vacuum environment ensure that the composite powder is not oxidized and then lose electrical conductive property; the designed molding temperature was between x/PEKK composite was higher than 50%, although its toughness was less than 10%. The composite sheet with a graphene content equal to or above 3wt% showed good conductive property. The SEM microscopic morphology of graphenex/PEKK composite sheets with different graphene contents showed that graphene were satisfactory dispersed in the PEKK matrix. This experimental study provided a practical solutions for molding of graphene/PEKK composite powder for achieving high conductive properties and good mechanical properties.In addition, compared with the elastic modulus of the PEKK polymer sheet, the elastic modulus of the graphene


The morphology and the composition of the sensing layers are investigated through scanning electron microscopy (SEM) and RAMAN spectroscopy. The RH sensing capability of each carbonaceous nanocomposite-based thin film was analyzed by applying a current between the two electrodes and by measuring the voltage difference when varying the RH from 0% to 100% in humid nitrogen. The sensors have a room temperature response comparable to that of a commercial humidity sensor and are characterized by a rapid response, excellent linearity, good sensitivity, and recovery time. The manufactured sensing devices’ transfer functions were established, and we extracted the response and recovery times. While the structures with GO/CNHox/PVP at 1/1/1 ratio (w/w/w) had the best performance in terms of relative sensibility, response time, and recovery time, the sensors employing the GO/CNHox/PVP nanocomposite at the 1/2/1 ratio (w/w/w) had the best linearity. Moreover, the ternary mixture proved to have much better sensing properties compared to CNHox and CNHox-PVP-based sensing layers in terms of sensitivity and linearity. Each component of the ternary nanocomposites’ functional role is explained based on their physical and chemical properties. We analyzed the potential mechanism associated with the sensors’ response; among these, the effect of the p-type semiconductor behavior of CNHox and GO, correlated with swelling of the PVP, was dominant and led to increased resistance of the sensing layer.This paper presents the relative humidity (RH) sensing response of a resistive sensor employing sensing layers based on a ternary nanocomposite comprising graphene oxide-oxidized carbon nanohorns-polyvinylpyrrolidone (GO-CNHox–PVP), at 1/1/1, 1/2/1, and 1/3/1 w/w/w mass ratios. The sensing structure is composed of a silicon substrate, a SiO In the last decades, relative humidity (RH) sensors have gained increasing interest due to their relevance in a wide variety of industrial, commercial, and residential applications such as building ventilation control, the medical field , food/beverage processing, chemical industry , pharmaceutical processing , agriculture (soil moisture control during irrigation), nuclear power reactors, meteorology, mining industry, robotics, textiles, paper industry, etc. ,2.Consequently, through the years, a plethora of principles and technologies have been improved or developed, each exhibiting both advantages and drawbacks in terms of sensitivity, response time, accuracy, drift, detection limit, cross-sensitivity, price, and so forth .Among the types of devices used in the control and monitoring of humidity-capacitive , gravimeHowever, the resistive humidity sensors’ performance, in terms of aspects such as sensitivity, response time, and recovery time, is strongly related to the sensing materials’ properties . TherefoCarbon-based materials are intensively utilized as sensing layers in the manufacturing of humidity sensors. Among these materials, we can mention the following: carbon nanotubes and their nanocomposites ,19, nanoIn the last years, two interesting nanocarbonic materials have received increasing attention regarding their utility as sensing layers in resistive humidity sensors: carbon nanohorns and graphene oxide.2 hybridized carbon atoms that form a conical molecular architecture of 2–5 nm in diameter and 30–50 nm in length [3 induced the decrease of the resistance carbonaceous sensing layer, whereas the adsorption of NH3 increased the resistance of the nanocarbonic thin film [Single-walled carbon nanohorns (SWCNHs), also known in the literature as nanocones, are carbon nanostructures with a particular morphology and were first reported by Iijima and co-workers in 1998 . SWCNHs n length ,34. Thesn length . The SWCn length , carriern length , the desn length , rechargn length , gas ston length , and so hin film . Suehirohin film .Different nanocomposites and nanohybrids based on oxidized carbon nanohorns (CNHox) were also used in the design and manufacturing of chemiresistive sensors for ethanol vapor detection ,44.Oxidized carbon nanohorns (CNHox), a hydrophilic type of functionalized carbon nanohorns, were recently introduced as a key sensing element in the design of resistive humidity sensors. The structure exhibited good RH sensitivity when varying RH from 0% up to 90%, either in humid nitrogen or in moist air ,46.CNHox’s nanocomposites with hydrophilic polymers, such as poly(ethylene glycol)-blockpoly(propylene glycol)-block-poly(ethylene glycol) (PEG-PPG-PEG) and polyvinylpyrrolidone (PVP), are also used as sensing layers for resistive monitoring of humidity. The resistive sensors based on these sensing films are characterized by a rapid response time, excellent stability, and good sensitivity, comparable to that of a commercially capacitive relative humidity sensor ,48.2-PVP and CNHox-ZnO-PVP were used as sensing layers in resistive humidity sensor designs, using a metallic interdigitated transducer (IDT) structure deposited on Si/SiO2 structure. For both nanohybrid configurations investigated, the sensing layer’s conductivity decreases while the RH level increases. Two types of sensing mechanisms are identified and discussed [Nanohybrids such as CNHox-SnOiscussed ,50.Graphene oxide (GO), one of the most studied derivatives of graphene, has received increasing attention as a sensing layer for humidity detection ,52,53,54This paper presents the RH sensing response of a resistive sensor employing a sensing layer based on a ternary nanocomposite comprising oxidized carbon nanohorns GO-CNHox-PVP at different w/w/w ratios. The experimental data were compared with the sensing data of the sensitive layers based on CNHox and PVP-CNHox binary mixture previously reported in the literature ,47.For the first time to our knowledge, the results demonstrate the relative humidity sensing capabilities of a GO-CNHox-hydrophilic polymer nanocomposite at different ratios when used in a resistive humidity sensor operating at a room temperature.2/g (BET). CNHox contains 10% graphite and has no metal contamination.Powder of CNHox . For surface visualization, a field emission gun scanning electron microscope/FEG-SEM-Nova NanoSEM 630  (FEI), with superior low voltage resolution and high surface sensitivity imaging, was used.−1 Raman line of a silicon substrate which correspond to the longitudinal optical-transverse optical (LO-TO) phonon. The spectrometer scanning data collection and processing were carried out by a dedicated computer using WiTec Project Five software .The Raman spectra were collected at room temperature with a Witec Raman spectrometer  using 532 nm as an excitation. The 532-nm diode-pumped solid-state laser has a maximum power 145 mW. The incident laser beam with a spot-size of about 1.0 µm was focused onto the sample with a 100× long-working distance microscope objective. The Raman spectra were recorded using a 20 s exposure time; the scattered light was collected by the same objective in back-scattering geometry with 600 grooves/mm grating. The calibration of the Raman systems was carried out using the 512 cmFor the investigation of the relative humidity sensing capabilities of the nanocarbonic materials-based nanocomposites, the following chemical compositions of the sensing films were designed and tested: GO-CNHox-PVP (1/1/1), GO-CNHox-PVP (1/2/1), and GO-CNHox-PVP (1/3/1), all mass ratios (w/w/w).We developed and followed a unique synthesis procedure for all three ternary compositions mentioned above. For example, the synthesis of the solid-state sensing films based on GO/CNHox/PVP = 1/1/1 as a mass ratio (w/w/w) is described below.CNHox (0.1 g) was dispersed in 20 mL isopropanol and subjected to stirring in an ultrasonic bath for three hours at room temperature. PVP (0.1 g) was added to this dispersion, followed by stirring in the ultrasonic bath for two hours at room temperature. An amount of 0.1 g GO dispersion was added to the previous suspension, and continuous stirring was performed in the ultrasound bath for three hours at room temperature .−  heating for 20 h at 80 ℃ under low pressure (2 mbar);− heating for 90 h at 110 ℃ under low pressure (2 mbar).Finally, a two-step annealing process was performed as follows: The sensing layer was obtained by depositing—using the drop-casting method—the dispersion over the IDT sensing structure, with the contact area being masked.2 layer (1 µm thickness)  dual-comb structure fabricated on a Si substrate (470 µm thickness), covered by a SiOickness) . The metThe reference humidity sensor used during the experiments was an industrial grade capacitive sensor, with a typical accuracy of ± 2% in the humidity range 0–100% RH and with a working temperature domain of 0–900 ℃ (for temperature).3) was changed by mixing dry nitrogen passing through the containers with deionized water in different ratios. In the mixing chamber  only. Thus, experimentally, an answer for the relative humidity according to the Equation (1) was achieved.A Keithley 6620 current source , ensuring a current variation between 0.01–0.1 A was employed; the data were collected and analyzed with a PicoLog data logger . All of the measurements were recorded at constant room temperature.Test conditions for the proposed experimental setup were not always identical. We tried to make identical jumps of humidity in the testing room, but there were small differences as shown by the commercial sensor (reference sensor). This was the reason for using both sensors in the testing room, the commercial one, whose answer was accepted as a reference answer and the investigated sensor. The idea of the experimental setup was to be able to track the response of the investigated sensor with the reference sensor in order to find out if the sensor is suitable as a resistive humidity sensor.The surface morphology of the deposited films was relatively homogenous in all cases . The hydAt the same time, the interaction between the two nanocarbon materials with each other and with PVP was proven using Raman spectroscopy. −1), G (~1584.1 cm−1), D’ (1601 cm−1), and second-order bands of 2D and D + D’ band localized at 2655.5 and 2915.9 cm−1 which are characteristic for carbon nanomaterials. The Raman band from about 3202.7 cm−1 may be associated with the so-called 2D’ band and this may come from the vibration spectrum of graphite, the most important impurity from oxidized carbon nanohorns [The recorded spectrum exhibits the typical D (~1338.8 cmanohorns .−1 is associated with the silicon substrate [−1) [−1, which is not assigned in the spectrum of the carbonaceous nanocomposite, may come from the overlapping of the Raman bands of the silicon substrate and PVP [The very high intensity of the peak located at ≅ 520 cmubstrate . Moreoveate [−1)  overlapp and PVP . Some of and PVP  are a coSensor 111—resisitive sensor which employed sensing layer based on GO/CNHox/PVP at 1/1/1 ratio (w/w/w);Sensor 121—resisitive sensor which employed sensing layer based on GO/CNHox/PVP at 1/2/1 ratio (w/w/w);Sensor 131—resisitive sensor which employed sensing layer based on GO/CNHox/PVP at 1/3/1 ratio (w/w/w);The relative humidity monitoring capability of each carbonaceous nanocomposite-based thin film was explored by applying a current between the two electrodes and measuring the voltage difference when varying the RH from 0% to 100%. For the simplicity of our description, we use the folowing abbreviations:An important characteristic of these devices is low power consumption, below 2 mW. The commercial sensor used in our experiments had a power consumption of up to 5 mW.The behavior of all three manufactured sensors is presented below.The resistance of the ternary nanocarbonic materials-based sensing layer increased when RH increased in the range from 0% to 100% RH and was measured with 10% RH steps.From The most plausible explanation is related to the presence of mass ratio graphene oxide-CNHox-PVP in the ternary nanocomposite. Due to a strong hydrophilic character, an increased content of GO in the composition of the ternary nanocomposite could yield to the generation of water clusters, with detrimental effects from the perspective of drift. An increased content of CNHox (more hydrophobic than GO and PVP) could avoid this drawback. On the other hand, decreasing the content of GO in the ternary nanocomposite, the bundles of oxidized carbon nanohorns cannot be dispersed in some extent to avoid capillary condensation of water.Moreover, it was observed that the resistance of the sensitive layer for the RH value “0” decreased with the increase of the CNHox content in the nanocomposite. This is a natural behavior because CNHox is a nanocarbonic material with high conductivity, and increasing its concentration leads to a decrease of the electrical resistance of the nanocomposite. humidR and dry nitrogenR are the measured resistances exposed to humid conditions and dry nitrogen, respectively. The response of the sensors with the composite sensing layer when exposed to different RH levels at room temperature is plotted in The sensor response is defined as:r) to relative humidity variations (RH) was defined. For the comparison of the sensing performances of the RH resistive sensors, with different initial resistance values, a relative sensitivity . R0 is the estimated resistance from the graph calibration line resistance = f (relative humidity) by extrapolation for the value at RH 0%. The comparison is summarized in The sensitivity of the sensor was defined as follows:CNHox and CNHox-PVP-based sensing layers were proven suitable for RH resistive monitoring, but the latter proved to have a superior performance. Thus, the beneficial effect of the PVP addition in terms of increasing the sensitivity of the sensor for almost the entire relative humidity range was demonstrated ,47. Hower. The most plausible explanation is related to the presence of graphene oxide. Carbon nanohorns and their functional derivatives generate large aggregates (a possible structure is presented in The results presented in For a sensor operation with the sensing film kept at room temperature (RT), it is important to evaluate the response and recovery time. Both adsorption and desorption of water molecules to and from the polymer matrix may be very slow under such RT circumstances. R(t) is the response of the device in time, the R(t) reaches 90% and 10%, respectively, from the total variation of the sensor’s resistance as a result of a change in the value of the RH [The response time . All these features make CNHox a promising candidate for monitoring the RH at room temperature.Each component of the ternary nanocomposites used for the resistive monitoring of RH had a well-defined role. CNHox exhibited outstanding properties such as increased conductivity (p-type semiconductor), high uniformity, hydrophilicity high purity , and a large surface area  is a hydrophilic polymer with excellent binder properties. It is important to mention that PVP is a hygroscopic, electrically insulating polymer, absorbing up to 25% moisture at 75%. Therefore, water is adsorbed into the bulk of the sensing layer, not only at the surface of the film.Given the features of the above-mentioned materials, three distinct sensing mechanisms can be discussed.+ and OH− ions. The protons generated by the water dissociation process may tunnel from one water molecule to another through hydrogen bonding, increasing the overall electrical conductivity of the sensitive film [The first approach takes into account the dissociation of water. The adsorbed water molecules on GO and CNHox hydrophilic surface may dissociate to Hive film .The second mechanism considers the fact that GO and CNHox are p-type semiconducting materials. When interacting, the water molecules donate their electron pairs, thus decreasing the number of holes in both nanocarbonic materials. Therefore, the humidity sensing layer based on the ternary nanocomposite becomes more resistive. Several results reported in the literature are in agreement with this statement .Last but not least, PVP is a dielectric polymer with hydrophilic properties, swells due to interaction with water molecules, and has a negligible change in resistance in response to variation in RH as shown for the presented composites .As a consequence, the distance between GO and CNHox moieties increases, and electrically percolating pathways are reduced. Again, the sensing layer becomes more resistive .As we can see, the interaction of the sensing layer with moisture has two opposing effects on resistance. We presume that the effect of the p-type semiconductor behavior of CNHox and GO correlated with the swelling of PVP dominates and leads to the overall increasing resistance of the sensing layer.In fact, the overall increasing resistance of the ternary nanocomposite-based sensing layer are in line with the previous results regarding the RH resistive sensing capabilities of CNHox alone and CNHox-PVP nanocomposite. Both sensing layers are suitable for RH resistive monitoring, but the latter proved to have a superior performance. Thus, the beneficial effect of the PVP addition in terms of increasing the sensitivity of the sensor for almost the entire relative humidity range was demonstrated ,47. All This paper reports the RH sensing response of a resistive sensor employing a sensing layer based on a ternary nanocomposite comprising graphene oxide-oxidized carbon nanohorns-polyvinylpirrolidone (GO-CNHox-PVP), at 1/1/1, 1/2/1, and 1/3/1 w/w/w ratio. The sensors have a room temperature response comparable to that of a commercially humidity sensor, characterized by a rapid response, excellent linearity, good sensitivity, and a good recovery time. A detailed analysis in order to obtain the transfer functions, response, and recovery times of all manufactured sensing devices was performed. While the structures with GO/CNHox/PVP at 1/1/1 ratio (w/w/w) had the best performance in terms of relative sensibility, response time, and recovery time, structures with GO/CNHox/PVP at 1/2/1 ratio (w/w/w) had the best linearity. The key role of each component of the ternary nanocomposites was explained based on their chemical structure and their physical and chemical properties. Special attention was devoted to the complex interaction (π-π stacking and hydrogen bonding) between GO and CNHox which seems to be responsible for the better performance of the sensor. Three sensing mechanisms were considered and analyzed. The effect of the p-type semiconductor behavior of CNHox and GO correlated with swelling of PVP dominated and led to the overall increasing resistance of the sensing layer.The low power consumption of these devices, below 2 mW, and the sensing performances at room temperature, as well as the simplicity of the sensor manufacturing are the most important advantages of presented devices.Ternary nanocomposite for the relative humidity resistive sensor and method for its manufacture, Patent Application No. A/00585, 23.09.2019, OSIM, Romania, Bogdan-Catalin Serban, Octavian Buiu, Viorel Avramescu, Cornel Cobianu, Roxana Marinescu.


The system consists of 16 PGSPSs that were used to analyze different gait signals, including walking, running, and jumping, to verify its daily application range. After comparing the test results with a high-precision digital multimeter, the system is proven to be more portable and suitable for daily use, and the accuracy of the waveform meets the judgment requirements. The system can play an important role in monitoring the safety of the elderly, which is very helpful in today’s society with an increasingly aging population. Furthermore, an intelligent gait diagnosis algorithm can be added to realize a smart gait monitoring system.Monitoring gait patterns in daily life will provide a lot of biological information related to human health. At present, common gait pressure analysis systems, such as pressure platforms and in-shoe systems, adopt rigid sensors and are wired and uncomfortable. In this paper, a biomimetic porous graphene–SBR (styrene-butadiene rubber) pressure sensor (PGSPS) with high flexibility, sensitivity (1.05 kPa Gait is the behavioral characteristic of human walking, which can reflect people’s problems in physiology, athletic ability, and psychology . In clinThe traditional gait recognition system is mainly carpet-type ,22,23. ICompared to sensors applied to other parts of the body ,31, wearIn this paper, we realized a low-cost, portable, and wireless in-shoe real-time gait monitoring system based on a biomimetic flexible porous graphene–SBR (styrene-butadiene rubber) pressure sensor (PGSPS). Compared with the sensors mentioned above ,31, the To achieve low-cost preparation of the PGSPS and realize sustainable manufacturing, the main materials, including the graphene suspension, SBR foam, and silver paste, used in the fabrication process are all achieved by utilizing standardized production, ensuring that the cost is low and stable. In the process of the experiment, since excessive graphene dispersion was collected during the preparation of PGSPS, we improved material utilization through redissolution and ultrasonic dispersion, reducing the average consumption of graphene dispersion per PGSPS to 0.9 mL. In the preparation process, apart from the ultrasonic treatment to further reduce the graphene sheet diameter (in order to achieve uniform dispersion), there is no need for other high-energy-consumption and high-cost processes. The low-cost and low-energy-consumption preparation of PGSPS has been preliminary realized in the laboratory, which provides the possibility for subsequent large-scale manufacturing.Furthermore, in the application of gait monitoring, multiple PGSPSs with the same performance are used at the same time, which requires performance stability and uniformity of the PGSPS. We found solutions in the preparation process: Firstly, the graphene sheet diameter was reduced by ultrasound to facilitate its uniform adhesion to the porous structure of the SBR foam. Second, for SBR foams that have been cut to a uniform size, they need to be repeatedly extruded after being immersed in the graphene suspension to ensure that the graphene is fully absorbed into the foam. Finally, the not-tightly-attached graphene should be flushed with water.A miniaturized system was designed to realize long-term real-time monitoring of foot pressure during walking. A) by calculating the resistance change in a PGSPS by the formula VA = Rref/(Rref + Rsensor) VCC. Then, the MCP3008 changes the VA to a digital signal (VD) by the formula VD = (210 VA)/Vref. After that, the signal processing module . In the final in-shoe real-time monitoring system based on the PGSPS, the gait signals of an adult were monitored real-time, gait information was transmitted wirelessly and displayed on a smartphone.The micromorphology and structure of the PGSPS were characterized by a field emission electron scanning microscope (FESEM) (JSM-7001F) . Raman characterization was performed using LabRAM HR Evolution . The mechanical properties of the PGSPS were observed and recorded in real-time with a mechanical tester (SHIMADZU AGS-X/EZ-X)  and a digital multimeter (RIGOLDM3068) . The preliminary gait test that was performed using the insole consisted of sixteen PGSPSs with the help of a DMM7510 7Nature has always inspired designs, especially bionic structures for wearable applications. For vertebrates, bones are the hard organs that make up the endoskeleton, which supports and protects the body. The soft organs and tissues attached to bones undertake functions such as systemic circulation and physiological information transmission. Their softness ensures that they change with body shape and follow the skeleton’s movement. The design idea of the PGSPS is similar to vertebrates’ bodies, consisting of an SBR foam skeleton and a graphene functional layer. The porous structure of SBR foam not only provides plenty of attachment points for graphene but also supports the PGSPS. The graphene layer that attaches to it forms a dense, three-dimensional, and flexible conductive network, which is suitable for electrical signal transmission under deformation conditions. Besides, the magnificent flexibility of graphene  ensures −1, ~1580 cm−1, and ~2700 cm−1 in the Raman spectrum, corresponding to the D, G, and 2D characteristic peaks of graphene, respectively. I2D/IG < 1, which proves that the multilayer structure of the graphene. The D peak is a low-strength peak and the 2D peak at ~1620 cm−1 is extremely weak (ID/IG = 0.64), indicating that graphene only has few micro-defects.0)/δP, where ΔR is the change in relative resistance, R0 is the initial resistance of the sensor when no pressure is applied, and P is the applied pressure value. Considering the thickness of the commonly used insole, PGSPSs with 5 mm thickness were tested. The ground reaction force at first touch is generally equal to the combination of body weight and acceleration, which is usually 120–140% of body weight at a normal pace [−1, but decreases when the pressure exceeds 22 kPa, with the sensitivity decreasing. This is because under low pressure, the increasing of the horizontal spacing of conductive paths exceeds the effect that the vertical conductive path density increases. Moreover, the graphene sheets attached to the three-dimensional skeleton structure produce cracks under the action of low pressure, leading to an increase in resistance. Subsequently, as the pressure gradually increases, the increase in the vertical conductive path density exceeds the effect that the horizontal spacing of conductive paths increases, which causes the resistance to decrease rapidly and reach saturation after 150 kPa . The sensitivity of the PGSPS is reduced to its lowest (1.05 kPa−1) at the maximum operating pressure of 150 kPa. Because the gait monitoring application is designed to be used in the range of 0–150 kPa, we take the lowest sensitivity within this range as the device sensitivity. The linearity of the fitting curve (R2) is 0.990–0.998, which reflects the accuracy of calculating sensitivity. To verify the stability of the pressure sensor, cyclic tests of the PGSPS for in-shoe application conditions were conducted , a raspberry PI system (RPI), a data acquisition (DAQ) circuit, and data processing and displaying software (on a smartphone) was designed and fabricated. As shown in To verify that the performance of the portable insole system is suitable, a waveform comparison between the RPI system and a DM6510 high-precision digital multimeter was carried out. The sensing results of all sixteen PGSPSs using the RPI system and DM6510 as well as the measurement error are shown in In addition to the gait monitoring, the portable insole system can also monitor running, standing, squatting, and jumping. For jumps, the signal is more intense. Points four , six (metatarsus), and ten (midfoot) are suitable for jump monitoring and were selected for analysis . CompareIn summary, it has been verified that a flexible, portable, and wireless in-shoe system based on the PGSPS can satisfy monitoring multiple gaits in daily life and reflect the characteristics of the corresponding gait clearly. Besides, the system integrates the sensing module, the signal processing unit, the wireless transmission module, the display software, and the data processing module. The miniaturized system can be worn with shoes and paired with a smartphone for daily gait monitoring. The SBR foam and graphene nanoplate paste used in this work are low cost, and the uniform and stable performance of the PGSPS is suitable for large-scale production. Moreover, a raspberry PI can be changed into a dedicated signal processing circuit, which will greatly reduce the cost and size of the processing unit, making it suitable for in-shoe applications. In addition to the current IOS app, software suitable for other systems can be designed so that users can conduct daily gait monitoring on their smartphones. The system has potential for guiding gait training and detecting diseases, and can be used for the daily tracking of motion as well as rehabilitation training. However, in the following work, it is also necessary to solve various problems: (i) To further improve the signal quality. The contact problem between the PGSPS insole and the sole should be solved, and the PGSPS structure should be iteratively optimized. (ii) To improve the ability of the system by adding gait analysis for non-healthy states, and increasing the system’s monitoring stability in running and jumping. (iii) A better man–machine interaction can be achieved by analyzing the impact of customization design on performance, and giving the software abnormal gait judgment ability through the application of intelligent algorithms, which will help to realize a smart gait monitoring system.To optimize our device and system according to actual requirements, we are currently working with a hospital to find solutions for the diagnosis of cerebral palsy through gait monitoring. As a neurological disease, cerebral palsy can be directly reflected in the patient’s gait . TraditiIn this work, a low-cost, portable, and wireless in-shoe system based on flexible porous graphene–SBR foam was designed and fabricated. A PGSPS with a biomimetic structure based on vertebrates’ skeleton and attached tissues was designed, which realized a large-range and high-sensitivity porous graphene–SBR pressure sensor for gait monitoring that can be integrated into shoes. The system has the complete ability to collect, transmit, process, and display gait information, which can realize real-time monitoring of a variety of peoples’ gait through a smartphone. Compared to a high-precision digital multimeter, it is more portable, suitable for daily use, and the accuracy of the waveform meets the judgment requirements. The system has potential in monitoring the safety of the elderly, which is very helpful in today’s society with an increasingly aging population. It can also guide gait training and detect diseases, as well as be used for the daily tracking of motion and rehabilitation training. In follow-up work, an intelligent diagnosis algorithm can be developed to realize a smart gait monitoring system.


In this paper, we theoretically designed and numerically analyzed an ultrabroadband meta-absorber with near unity absorptivity that works in terahertz spectrum. A wideband meta-absorber composed of bilayer patterned graphene and dielectric cylinder array with high symmetry was proposed. The wideband absorption mechanism benefited from two aspects. The first one was enhanced surface plasmons based on bilayer patterned graphene. And the second one was the coupling of continuous resonant modes within Fabry-Perot cavities to the enhanced surface plasmons in the graphene. An ultrawide bandwidth with absorptivity over  Electromagnetic wave absorber is a device that converts electromagnetic wave into ohmic heat or other forms of energy by interacting with the electric field or magnetic field of the incident electromagnetic wave . ElectroTraditionally, the absorption was realized using absorbing materials, by absorbing electromagnetic energy efficiently and reducing the reflection. Since Landy first proposed the metamaterial perfect absorber in 2008 ,4,5,6. FMany tunable absorbers with materials consisting graphene ,14, blacHowever, the absorption bandwidth relied on resonances of surface plasmon polaritons of graphene is too narrow to satisfy certain potential applications ,25. MuchIn this letter, we propose a polarization insensitive broadband meta-absorber based on patterned graphene in terahertz spectrum. We utilize the enhanced surface plasmons with dielectric cylinders loaded patterned bilayer graphene structure and merge varied resonances to realize the increased absorption within an ultra-wide bandwidth. Due to simultaneously generated electric and magnetic resonances of both plasmon resonance and Fabry-Perot (FP) resonance, the proposed broadband absorber can have tunable absorption spectrum from The schematic unit cell of the proposed absorption structure is depicted in model as : ϵ(ω)=ϵ∞T is the temperature in Kelvin, e is the electron charge, and ℏ is the reduced Planck’s constant, In the above configuration, the periodic silicon cylinder arrays loaded with patterned graphene can also supply local resonant reflection, forming an asymmetric FP cavity. When the incoming electromagnetic wave propagates in the multilayer medium, the electric field and magnetic field will be modulated, and the propagation state of the electromagnetic wave will change complex. Different from the traditional medium, graphene is a monolayer of carbon with a specific conductivity governed by the Kubo formula . In the ector as ,38: (2)kThe surface plasmons of graphene can be achieved when the wave vector matching condition is met as:Then we can conclude that our structure can produce multiple discrete plasmon resonances, what’s more, for each mode, a continuous resonances exist for the reason of continuously variable y. Then, the multi-band continuous resonances can be adjusted through parameter scanning to locate within the spectrum of two absorption peaks caused by the patterned graphene. In the silicon cylinders, we define R, the plasmon resonance can be adjusted within the expected spectrum. Then, we will have a wideband property with near-unity absorption under certain conditions.Equation  implies When the uniform electromagnetic wave is incident into the multilayer medium, part of the energy is reflected and part is transmitted. The final absorption rate in this paper can be expressed as Theoretical research is developed using the commercially available finite element simulator HFSS. To asure the computing accuracy and high efficiency, the term Maximum Number is set to 15 and the term Maximum Delta S is set to Before analyzing the absorption mechanism of the whole structure, the adoption of bilayer patterned graphene is clarified below. The comparison of normalized absorption spectra of three kinds of absorber with monolayer graphene, bilayer graphene and tri-layer graphene are plotted respectively, as shown in Then, no matter how to tune the loading voltage, the absorption in a wide band can not be improved only based on the bilayer structure. Once the periodic silicon cylinders with high permitivity are attached to the top, as mentioned above, the simulation result shows that we indeed have improved the absorption rate within the wide spectrum, as shown in To further validate the absorption mechanism, simulated electric field distribution, surface current density of the first graphene layer, the second graphene layer and the backed Au ground and the profile magnetic field distribution of structure at In For the second absorption peak of 6 THz, strong electric field appears in the edge of the graphene and the junction of graphene and cylindrical dielectric, as shown in Then in order to give the verification of the mechanism derived above further, the main parameters that affect the main absorption peaks are analyzed and presented in Even if the structural parameters are fixed, the tunability of the absorption is still achievable benefited from the controllable eletromagnetic characteristics of graphene. Through adjusting the biasing voltage from 0.1 eV to 0.97 eV, as shown in x-axis and electric vector of plane wave. We have investigated the absorption consistency of variational trend under the changes of the polarization angle ranging from Next, we will investigate the property of polarization independence under different incident angles. In the above mentioned analysis, we discussed the absorption characteristics only under normal incidence scenario with angle Besides, the absorption spectrum under varied oblique incidence in both the TE and TM modes are studied, as depicted in Finally, we obtain a polarization insensitive and wide angle independence broadband meta-absorber with absorption larger than In summary, a bilayer graphene-based absorption structure with Au cylinder arrays on parylene substrate is proposed and numerically simulated at THz wavelengths. We use the patterned bilayer graphene to produce localized surface plasmon polaritons and delocalized surface plasmon polaritons to obtain dual frequency absorption, then additional silicon cylinder arrays on the top of the patterned graphene produce a series of discrete plasmon resonances located within the dual frequency interval, at the same time, enhancing the absorption rate through improving the matching condition. Ultra-wide bandwidth is realized when the guided surface plasmon resonance is enhanced by tuning the parameter of silicon cylinders, promoting field confinement on the surface of the structure. The absorption rate and bandwidth of the structure can be independently tuned by varying the chemical potential of biasing voltage on both of the graphene layers. Simulation results demonstrate that the absorption of the proposed structure can be as high as more than 90% from


Notably, high molecular weight thiophene-containing polymers are successfully synthesized by Suzuki–Miyaura polycondensation of 2,5-thiophene bis(boronic acid) derivatives with different dibromo- and triple bromo-substituted aromatics in 5–15 min.To address the limitations of conventional Pd catalysts in the polymerization of thiophene-containing conjugated polymers, an active catalyst system based on Pd (0) and a phosphine-based bulky ligand, L1, is explored systematically in Suzuki–Miyaura polymerizations using thiophene boronic acid pinacol ester as one of the monomers. This active catalyst is found very efficient in synthesizing a series of thiophene-containing linear and hyperbranched conjugated polymers. First, as a model example, coupling reactions between electron-rich/moderately hindered aryl or thienyl halides and thiophene boronic acid pinacol ester give excellent yields with lower catalyst loading and can be completed in a shorter reaction time relative to Pd(PPh In part3)4 as the catalyst precursor 4, the catalyst Pd2(dba)3/L1 showed higher performance in generating TON and TOF. Furthermore, this catalyst system can be conducted for Suzuki polycondensation of polymers based on 2,5-thiophenebis (boronic acid pinacol ester), and high molar mass polymers can be easily gained within 15 min.A palladium complex containing a bulky electron-rich ligand facilitates the oxidative addition of the aryl halide . The che3)4 was prepared according to the literature (2 and monitored by thin-layer chromatography. Column chromatography was conducted on silica gel (200–300 mesh). 1H NMR was performed in CDCl3 on a Bruker DM 300, AV 400, or AV 600 spectrometer. The gel permeation chromatography (GPC) measurements were performed on a Waters chromatography system connected to a Shimadzu LC-20AD differential refractometer with THF as an eluent or at 150°C with 1,2,4-trichlorobenzene as an eluent and calibration with polystyrene standards.All chemicals were obtained from commercial suppliers and applied without purification. Solvents were disposed according to the standard process. 5-Bromothiophene-2-carbaldenhyde and L1 were gained according to a previous literature procedure . The catterature . All rea-1 halide), water, the base (5 equiv), Pd2(dba)3, and L1 was mixed under nitrogen and refluxed. CH2Cl2 was then poured into the mixture, and the organic layer was separated and dried with MgSO4. The crude product was purified on silica gel eluting with petroleum ether (60–90°C)/acetate ester to provide the title compound.A mixture of aryl halides or thienyl halides, thiophene boronic ester, THF , water, the base (5 equiv), Pd2(dba)3, and L1 was mixed under nitrogen and refluxed. Water was then added, and the organic layer was separated and precipitated into methanol. The crude product was purified to provide the polymers.A mixture of aryl halides or thienyl halides, thiophene boronic ester, THF (5 L mol3 h−1) /L1 have been screened in nditions  /L1 (1:3) within 30 min as a test case (3)4, ligand L1 gave better yields in reactions of aryl halide and 2-thiopheneboronic ester under the above conditions. For example, the coupling reaction of bromobenzene or benzyl bromide and aromatic boronic acid pinacol ester with 0.1% Pd (0) (Pd: L1 = 1:3) within 15 min gave excellent isolated yields of the corresponding products, wherein TOFs of 3,840 h−1 and 3,800 h−1 were obtained, respectively (−1 within 30 min (3)4 furnished the target products with a 24% isolated yield and only a 6 h−1 TOF after 2 h. Notably, the Suzuki cross-coupling reactions of 2,5-thiophenebis (boronic ester)s and ortho-substituted bromobenzene Pd (0)/L1 provided the target products in an isolated yield of 90% with a value of TOF of 900 within 30 min, whereas under the similar conditions, Pd(PPh3)4 gained the isolated yield of 18% with only a 4.5 h−1 TOF after 2 h 4 gained the isolated yields of 31% and 48%, with TOFs of 15.5 h−1 and 24 h−1 after 2 h, respectively (3)4, the catalyst system Pd2(dba)3/L1 showed higher performance in yield and TOF. The general mechanism for the Pd-catalytic cross-coupling reaction is divided into three steps, including oxidative addition, transmetalation, and reductive elimination 3 (Pd/L1 = 1/3) was tested for the synthesis of hyperbranched polymers based 2,5-thiophenebis (boronic acid pinacol ester)s (2(dba)3 (Pd/L1 = 1/3) and Pd(PPh3)4 were also applied for the same polymerization as catalyst precursors. Suzuki polycondensation of 2,5-thiophenebis (boronic acid pinacolester)s (M2) with tris(4-bromophenyl)amine (M1) or tris(4-bromophenyl)amine (M4) and 2,7-dibromo-9,9-dioctyl-9H-fluorene (M3) was carried out in a biphasic mixture of THF and aqueous K2CO3 with freshly prepared Pd2(dba)3/L1 or Pd(PPh3)4 as the catalyst precursor in 15 min. The results of polymerization are displayed in 2(dba)3/L1 as the catalyst precursor, the polycondensation proceeded very rapidly, and large amounts of precipitation were observed in the reaction flask after 5 min. Polymers were not soluble in common reagents such as CHCl3 or THF even at the refluxed temperature, but little were soluble in 150°C 1,2,4-trichlorobenzene. The molecule molar masses of P1 and P2 were determined by GPC at 150°C with 1,2,4-trichlorobenzene as the eluent and calibration with polystyrene standards. The soluble fractions of polymers P1 and P2 had molecular weights Mw of 24,800 g mol−1 and 7,900 g mol−1, respectively. Using the representative palladium catalyst Pd(PPh3)4, Suzuki polycondensation of monomers (M1 or M4) and monomer M3 with 2,5-thiophenebis (boronic acid pinacolester)s (M2) provided only low molecule molar mass oligomers with an Mw less than 3,000 g mol−1.To test the performance and wide scope of the catalyst precursor Pd (0)/L1, the catalytic system with ligand L1 and Pd ester)s . For com2(dba)3/L1 as the catalyst precursor, polymer P3 was synthesized, which was completely soluble in THF at room temperature in 15 min. The GPC elution curve of P3 showed narrow molecular weight distribution. The number-average molecular weight and weight-average molecular weight of P3 were 32,000 g mol−1 and 11,2000 g mol−1, respectively (3)4 as the catalyst precursor. GPC data showed that polymer P3 was gained with a molecular weight Mw 8,400 g mol−1. The results are also displayed in To test the performance and obtain fully soluble polymers in the reaction solvents, we chose 4,9-dibromo-2,7-bis(2-octyldodecyl)benzo(lmn)phenanthroline-1,3,6,8-tetraone (M5) and 2,5-thiophenebis (boronic acid pinacolester)s (M2) as monomers. Using Pdectively . The pol3)4, this catalyst system also demonstrates higher activity in the Suzuki polymerization of aryl halide and 2,5-thiophenebis (boronic acid pinacol ester)s, resulting in various thiophene-containing conjugated polymers with good yields and high molecular weights within 15 min.In conclusion, the catalyst system based on Pd (0)/L1 was studied for the Suzuki–Miyaura cross-coupling reactions of thiophene-2-boronic ester with aryl bromides and unactivated thienyl bromides. The catalytic system is efficient in good to excellent yields and high TOFs with low catalyst loadings or shorter reaction times. In addition, relative to Pd(PPh


The in vitro findings were further assessed using in vivo mouse models. The expression of NEDD4L was down-regulated in lung tissues of IPF patients and mouse models. Overexpression of NEDD4L restricted the formation and progression of IPF in mice and attenuated the proliferative, invasive and differentiative abilities of LFs. Further, NEDD4L halted LFs activity by enhancing β-catenin ubiquitination and down-regulating the CTHRC1/HIF-1α axis. Also, in vivo experiments then validated that NEDD4L silencing repressed β-catenin ubiquitination and activated the CTHRC1/HIF-1α axis, thereby aggravating IPF in mice. NEDD4L may suppress the formation and progression of IPF through augmenting β-catenin ubiquitination and inhibiting the CTHRC1/HIF-1α axis.Interstitial pulmonary fibrosis (IPF) is a severe progressive lung disease with limited therapeutic options and poor prognosis. Initially, we found the downregulated level of neural precursor cell expressed developmentally down-regulated 4-like protein (NEDD4L) in IPF-related expression microarray dataset, and this study was thus performed to explore the molecular mechanism of NEDD4L in IPF. The expression of NEDD4L was subsequently validated in lung tissues of IPF patients and mouse models. Then, mouse primary lung fibroblasts (LFs) were collected for  Interstitial pulmonary fibrosis (IPF) represents a common type of IPF and is characterized by deposition of the extracellular matrix and aberrant fibrotic remodeling of the lung, leading to progressive loss of lung function and ultimately death Further, myofibroblasts have been recognized as the main cell type able to stimulate the accumulation of extracellular matrix in fibrotic diseases, and targeting the proliferation and differentiation of fibroblast has thereby been considered as a promising therapeutic strategy for IPF On the other hand, a previous report has pointed out that collagen triple helix repeat containing protein 1 (CTHRC1) could promote the development of pulmonary fibrosis and the level of CTHRC1 was elevated in lung tissues of IPF Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China. Detailed study aims as well as planned procedures were explained to all patients, who were subsequently provided with signed informed consent documentation. Animal experiments were approved by the Animal Care and Use Committee of Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China and performed in accordance with https://www.ncbi.nlm.nih.gov/gds), which contained 8 samples of lung tissues from IPF patients and 15 of normal lung tissues. The Limma R language package (http://www.bioconductor.org/packages/release/bioc/html/limma.html) was then utilized to identify differentially expressed genes in IPF samples, with “|log fold change (FC)| > 1, p value < 0.05” as the screening threshold.Pulmonary fibrosis-related GSE10667 expression microarrays were retrieved from the Gene Expression Omnibus (GEO) database , and normal lung tissues from normal organ donations . All patients met the American Thoracic Society/European Respiratory Society's diagnostic criteria for lung fibrosis. Apart from outpatient and telephone follow-ups, patients were required to have a lung biopsy every month for the first three months after discharge, then every three months, and then every six months two years after the operation. Part of the tissue samples were quickly frozen in liquid nitrogen and stored at -80°C, and the other part was made into paraffin sections for subsequent experiments.After dewaxing, hydration, washing, endogenous peroxidase neutralization and antigen recovery, the 4 μm-thick tissue sections were blocked with goat serum at 37°C for 1 h, and then incubated overnight with anti-β-catenin  and anti-CTHRC1 antibodies  at 4°C. On the second day, the sections were washed with phosphate buffered saline (PBS) and processed with a two-step assay kit . Subsequently, the sections were stained with DAB kit  and hematoxylin, dried at 65°C, and sealed, followed by observation using an optical microscope .A total of 60 C57BL/6 male mice  were housed with a 12-h light/dark cycle at 21 - 23℃. After one week of adaptation, the mice were randomly divided into 2 groups . IPF mice were anesthetized with 5% pentobarbital sodium and given Bleomycin by intratracheal suction ; and sham-operated mice was intratracheally administered the same amount of normal saline as controls. Two weeks later, Resistance and Compliance Plethysmographs  were adopted to evaluate lung functions.9 TU/mL, and the tail vein injection was performed on the 5th day of Bleomycin induction and then every 4 days. Three weeks later, the mice were euthanized to collect the lung tissues, which were then incubated with 4% paraformaldehyde (PFA) and embedded in paraffin for subsequent experiments.Further, 54 IPF mice were randomly divided into 9 groups and subjected to tail vein injection of different lentiviral (lv) particles: IPF , IPF + NC (2 mL lv-NC particles), IPF + NEDD4L (2 mL lv-NEDD4L particles), IPF + sh-NC (2 mL lv-sh-NC particles), IPF + sh-NEDD4L , IPF + DMSO (2 mL DMSO), IPF + PR-619 (2 mL deubiquitinase inhibitor PR-619), IPF + PR-619 + sh-NC , and IPF + PR-619 + sh-NEDD4L  groups. The titer of lentiviral particles  was 1×10Lung tissues were fixed with 4% PFA, embedded with paraffin, and sectioned with an automatic microtome . After deparaffinization and rehydration, the sections were stained following the instructions of H&E staining kit , and images were photographed during observation using an optical microscope . Then, two pathologists double-blindly observed the morphological changes of the tissue with an optical microscope, and the pathological changes of the tissue was based on the degree of edema, inflammatory cell infiltration, hemorrhage and necrosis, as previously described After deparaffinization and rehydration, the sections were stained utilizing the Masson staining kit  and observed using an optical microscope . Images were photographed and analyzed with image J software.Lung tissues was dried at 100°C for 24 h, hydrolyzed with 0.5 μM acetic acid and 0.1 mg/mL pepsin under hypoxia, and placed with vials of vacuum, followed by overnight incubation at 4°C. Then, the tissued were examined with the hydroxyproline kit  to measure the content of hydroxyproline.3, and treated with digestion buffer containing 30 mg collagenase IV, 1 g fetal bovine serum (FBS), 96 mL Hanks buffer Solution, and 4 mL 0.25% trypsin . Then, 1 h later, the tissue suspension was filtered through a cell strainer, and centrifuged to collect the precipitation, which was resuspended in DMEM (Sigma) and centrifuged again to collect the supernatant. After centrifugation, the obtained cells were identified as lung fibroblasts (LFs) utilizing Vimentin immunohistochemical staining and resuspended with DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. The cell resuspension was then incubated in a medium containing TGF-β1  at 37℃ and 5% CO2.The six to eight-week-old C57BL/6 mice were euthanized. Collected lung tissues were cut into pieces of 1 mm5 cells/well for 24-h culture, and treated with 10 μM MG132  upon the cell confluence reached 60-75%. Then, transient cell transfection was performed following the instructions of Lipofectamine 2000 . Cells were divided into several groups and transfected with plasmids expressing NEDD4L or carrying small interfering RNA (siRNA) targeting NEDD4L or overexpressing CTHRC1 or corresponding NCs, or treated with DMSO or β-catenin inhibitor or deubiquitinase inhibitor PR-619. The transfection plasmids were synthesized by and purchased from Genechem. The medium was renewed 6 h after the transfection, and the cells were collected after 48 h of culture for subsequent experiments.The LFs in logarithmic growth period were digested with 0.25% trypsin, seeded in a 6-well plate at 1×103 cells/well and incubated with 10 μL of the CCK-8 reagent  at 37°C for 1 h. Then, a Micro-plate Reader  was used to detect the absorbance of each well at 450 nm. Cell viability (%) = average absorbance of the treatment group/average absorbance of the control group × 100%.The LFs in the logarithmic growth phase were seeded in a 96-well plate at a density of 5×104 cells/cm2, and then seeded in the upper chamber of the Biocoat Invasion Chamber  covered with Matrigel gel (50 μL/well). The lower chamber was added with 800 μL of medium supplemented with 10% FBS. After 24-h incubation at 37°C and 5% CO2. the chamber was washed and stained with 0.1% crystal violet for 30 min, followed by PBS washing to remove non-invaded cells. Then, 10 fields of view were randomly selected for observation and cell counting under an inverted microscope .The LFs in the logarithmic growth phase were suspended in serum-free medium at a density of 5×10The LFs in the logarithmic growth period were fixed in 4% PFA at room temperature for 30 min, permeabilized, and then blocked, followed by incubation with anti-α-SMA antibody  at 4°C overnight. Next, the cells were incubated with Alexa Fluor® 555-coupled goat anti-rabbit antibody  in the dark, and the nucleus was stained with 4′6-diamidino-2-phenylindole  for 5 min. Then the cells were observed with a fluorescence microscope  and images were photographed.HEK293 cells  were lysed with protease-inhibitor cock-tail (Roche) supplemented with cell lysis buffer (Sigma). The lysate was centrifuged to collect the supernatant, which was then incubated with Protein A/G Sepharose beads  for 30 min at 4°C. Meanwhile, protein A/G sepharose beads were incubated for 10 min in PBS containing 0.1% Triton X-100 and protease-inhibitor with the following antibodies: NEDD4L , CTHRC1 , β-catenin , Ubiquitin , and IgG . Then, the treated supernatant was incubated with the antibody-treated beads at 4°C for 3 h to collect the precipitation by centrifugation. After washing with PBS containing 0.1% Triton X-100 and protease-inhibitor, the precipitation was resuspended in SDS buffer for Western blot assay.Based on the instructions of the Ion 550TM ChIP kit , cells of each group were harvested when the cell confluence reached 70-80% and incubated with 10 μM MG132 and 1% formaldehyde, followed by 10-min fixing at room temperature. Then, the cross-linking of internal DNA and protein was broken by ultrasonic treatment into fragments and centrifuged to collect the supernatant. The supernatant was divided and added into three tubes, respectively incubated overnight at 4°C with positive control antibody RNA polymerase II, negative control antibody normal human IgG, and rabbit anti-β-catenin . The mixtures were treated with Protein Agarose/Sepharose to precipitate the endogenous DNA-protein complex, followed by centrifugation to collect the precipitation, i.e. the non-specific complex, which was then de-cross-linked at 65°C overnight. The DNA fragment was obtained by phenol/chloroform extraction and purification, and RNA extraction and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to measure the expression of β-catenin.△△Ct = △Ct experimental group - △Ct control group, △Ct = Ct (target gene) - Ct .Total RNA was extracted by Trizol reagent  from tissues and cells. Subsequently, RNA was reverse-transcribed into cDNA utilizing the A3500 Reverse Transcription System , and the cDNA was amplified utilizing the Fast SYBR Green PCR Master Mix . The primers were listed in Total protein was extracted from cells and tissues utilizing RIPA lysis buffer , followed by the determination of total protein concentration using BCA detection kit . Then, 30 μg cell lysate was mixed with corresponding 5× loading buffer , denatured at 100°C for 10 min. The protein was then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electro-transferred to polyvinylidene fluoride (PVDF) membrane, and blocked with 5% skimmed milk powder in Tris buffered saline (TBS). Subsequently, the membrane was incubated overnight at 4°C with diluted rabbit primary antibodies, including anti-NEDD4L , anti-β-catenin , anti-CTHRC1 , anti-HIF-1α , anti-collagen 1 , anti-Fibronectin  and anti-β-actin . After washing, the membrane was further incubated for 1 h with horseradish peroxidase (HRP)-labeled IgG secondary antibody . The enhanced chemiluminescence detection kit was then utilized to visualize the protein bands, and the gray level of protein bands was quantified with the Image J 1.48 software .t test was applied for comparison between data of two groups; one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed for comparison among data of multiple groups. Pearson analysis was applied for the correlation between indicators. Moreover, the level of significance was p < 0.05.Data in this study were processed utilizing GraphPad Prism 8.0 . Measurement data were summarized as mean ± standard deviation. Independent sample Through bioinformatics analysis we identified the down-regulated expression of NEDD4L in lung tissue of IPF Figure A. ResultAfter identifying the inhibitory effect of NEDD4L on IPF, we then explored the underlying molecular mechanisms, especially the potential regulatory effect of NEDD4L on LFs. Through manipulating NEDD4L expression in mouse LFs, we identified si-NEDD4L-2 with the best silencing efficiency and thereby selected it for subsequent experiments  (Figure Further, with immunohistochemical staining we identified up-regulated level of β-catenin in lung tissue of IPF Figure C. PersonWhen silencing NEDD4L in LFs, the ubiquitination of β-catenin was repressed; while treatment of PR-619, a deubiquitinase inhibitor, led to increased β-catenin ubiquitination (Figure in vitro substantiation of the regulatory mechanism of NEDD4L, we then moved to in vivo experiments. Our data unraveled that NEDD4L silencing led to down-regulated level of NEDD4L in lung tissues of PR-619-treated mice (Figure in vivo.Following the e Figure A. Resulte Figure B. Moreove Figure C, represe Figure D, reducee Figure E, and ree Figure F, whereaIPF represents a severe progressive interstitial lung disease with limited therapeutic options and poor prognosis Our initial finding of the down-regulated expression of NEDD4L in IPF-related expression microarrays indicated the involvement of NEDD4L in the pathogenesis of IPF. Then, our data revealed that NEDD4L overexpression repressed inflammatory cell infiltration and protect lung functions, thereby restricting the progression of IPF. This finding corroborates a previous report that the conditional deletion of NEDD4L in lung epithelial cells of IPF mice could trigger pulmonary fibrosis and exacerbate IPF, which also indicated that NEDD4L was mainly distributed in epithelial cells lining the distal airways of IPF patients via stimulating β-catenin ubiquitination. This corroborates previous studies which indicated that β-catenin could bind to the promoter region of CTHRC1 in vivo experiments further validated that NEDD4L stimulated β-catenin ubiquitination, repressed the CTHRC1/HIF-1α axis, and thus alleviated IPF.Then, through a series of gain- and loss- of function experiments we found that NEDD4L inversely regulated the CTHRC1/HIF-1α axis to repress the proliferative, invasive, and differentiative abilities of LFs. In relation to this, it has been pointed out that CTHRC1 could up-regulate the expression of HIF-1α in gastric cancer via augmenting β-catenin ubiquitination and inhibiting the CTHRC1/HIF-1α axis. By inducing the ubiquitination of β-catenin, NEDD4L could repress the activation of the Wnt/β-catenin signaling pathway, and restrict the CTHRC1/HIF-1α axis due to the CTHRC1 expression-promoting effect of β-catenin; then, CTHRC1 and HIF-1α suppression could attenuate the viability and differentiation of LFs, thereby impeding the formation and progression of IPF. In summary, this study elucidated the regulatory network of NEDD4L in IPF, deepened our understanding of molecular mechanisms involved in the progression of IPF, and, more importantly, provided promising therapeutic targets for IPF treatment.Taken together, the evidence provided in this study has led us to a conclusion that NEDD4L could suppress the formation and progression of IPF Supplementary table.Click here for additional data file.