Performance of an electronic device relies heavily on the availability of a suitable functional material. One of the simple, easy, and cost-effective ways to obtain novel functional materials with improved properties for desired applications is to make composites of selected materials. In this work, a novel composite of transparent n-type zinc oxide (ZnO) with a wide bandgap and a unique structure of graphene in the form of a graphene flower (GrF) is synthesized and used as the functional layer of a humidity sensor. The (GrF/ZnO) composite was synthesized by a simple sol–gel method. Morphological, elemental, and structural characterizations of GrF/ZnO composite were performed by a field emission scanning electron microscope (FESEM), energy-dispersive spectroscopy (EDS), and an x-ray diffractometer (XRD), respectively, to fully understand the properties of this newly synthesized functional material. The proposed humidity sensor was tested in the relative humidity (RH) range of 15% RH% to 86% RH%. The demonstrated sensor illustrated a highly sensitive response to humidity with an average current change of 7.77 μA/RH%. Other prominent characteristics shown by this device include but were not limited to high stability, repeatable results, fast response, and quick recovery time. The proposed humidity sensor was highly sensitive to human breathing, thus making it a promising candidate for various applications related to health monitoring. Measurement of humidity is an essential factor for various industries including semiconductor fabs, pharmaceutical, food and beverage, agriculture, textile, refineries, paper and chemical industries, etc. . The sig2, Fe2O3, CuO, and TiO2 have been engaged in humidity sensing applications due to their unique electrochemical and electronic properties [Presently, researchers are primarily focused on improving the characteristics of the functional material in humidity sensors by experimenting with various advanced materials and composites of existing materials and, therefore, several types of materials, including ceramics, polymers, biowastes, metal oxides, 2D materials, and their composites have been employed in humidity sensors ,12,13,14operties ,16,17,18operties ,22,23,24operties ,27,28,29operties ,22,23,242, and WS2 illustrated great response to the change in humidity owing to their high surface to volume ratio, large surface area, and improved chemical and electrical properties [2 g−1, being optically transparent and having high chemical stability. Therefore, graphene and its derivative composites were widely investigated for the application of sensing change in humidity like SnO2/reduced graphene oxide (RGO) [2 [The composite of ZnO with different 2D materials like graphene, MoSoperties ,31,32. Ade (RGO) , methyl de (RGO) , black pde (RGO) , lignosude (RGO) , BP/grapde (RGO) , non-wovde (RGO) , orange de (RGO) , graphen(RGO) [2 , chitosa(RGO) [2 , etc. St(RGO) [2 , graphen(RGO) [2 , graphen(RGO) [2 , ZnO/ po(RGO) [2 , ZnO/GO (RGO) [2 , etc. ImHerein, we propose a simple approach to fabricate and characterize the humidity sensor based on the composite of ZnO with graphene in the form of graphene flowers, a type of graphene that is directly synthesized based on a high rate CVD method using a bottom-up system. The proposed sensor showed a swift response time of a few milliseconds and a small recovery time of only 3 s, excellent stability, and sensitivity to long-range relative humidity (RH)%. ZnO thin film was grown by the sol–gel method, and the thin film of graphene flowers was spray coated on ZnO. GrF/ZnO composite was characterized by a field emission scanning electron microscope (FESEM), energy-dispersive spectroscopy (EDS), and an X-ray diffractometer (XRD). The prepared sensor’s electrical response was measured by an LCR meter and a precision source measurement unit at different RH% levels. For the preparation of graphene flower (GF)–ZnO composite, zinc–acetate–dihydrate (ZAD), monoethanolamine (MEA), ethanol isopropyl–alcohol (IPA), acetone, and deionized water were purchased from Sigma-Aldrich, Seoul, Korea. Graphene flower (GF) solution in methyl-ethyl-ketone (MEK) (99.9 wt%) was purchased from InALA, Kobe, Japan. All materials were used without modification while glass substrate and silver paste for patterning electrodes were purchased from MTI Korea.3COOK, CaCl2, K2CO3, NaBr, CuCl2, NaCl, KCL, and K2SO4 were purchased from DAEJUNG Materials Korea to make supersaturated solutions for achieving different RH% levels. Nine types of different salts LiCl, CHThe entire process of device fabrication is illustrated in Morphology of ZnO thin film and GrF/ZnO composite was investigated by a field emission scanning electron microscope (FESEM)  while the gap between the deposited electrodes was visualized with the help of an optical microscope . 3COOK(23%), CaCl2 (32%), K2CO3 (46%), NaBr (60%), CuCl2 (67%), NaCl (72%), KCl (80%) and maximum RH% of 86% from K2SO4 super-saturated solution with an accuracy of ±2%. A commercially available humidity sensor, HTU21D, was used along with an Arduino board to calibrate the humidity level of supersaturated solutions. The device under test (DUT), i.e., the fabricated sensor, was placed in the desired RH% environment by placing it inside the jar of corresponding salt solution for electrical measurements with respect to humidity. The used experimental setup is shown in The experimental setup is shown in Change in the electrical properties of proposed humidity sensor with respect to change in RH% were measured with different electronic devices such as LCR meter (KEYSIGHT U1733C) which was used to measure impedance and capacitive response of sensors with respect to different RH% levels. A precision source measurement unit (KEYSIGHT 2911A) was used to measure current change with respect to RH%, response time, recovery time, stability, and different application tests of the proposed humidity sensor.An FESEM was used to examine the morphology of GrF, ZnO and GrF/ZnO thin films. For the uniform thin film of ZnO deposited by the sol–gel method, multiple spin coatings were required ,48. TherEDS analysis is usually used to confirm the elemental composition of the given samples; therefore, EDS analysis was performed to verify the successful synthesis of ZnO and GrF/ZnO thin films. Samples were made on fluorine tin oxide (FTO)-coated glass to perform EDS analysis. EDS of FTO glass, ZnO thin film, and GrF/ZnO is shown in XRD analysis was performed to investigate the crystal structure of prepared ZnO and GrF/ZnO thin films using the X-ray diffractometer (Empyrean) at a step size of 0.02° from 5° to 80°. XRD analysis of ZnO thin film exhibited the peaks at 31.7°, 34.4°, and 36.4° as shown in 2SO4, respectively. A biased voltage of 3 V was given to the fabricated sensors using the KEYSIGHT B2911A Precision Source/Measure Unit, and the current was measured by placing the sensor at different RH% levels. RHoI is the value of current at 15% RH and IRH is the value of current at exposure RH%.The experimental setup shown in o = 0.7 V at different test frequencies of 1 kHz, 10 kHz and 100 kHz). Similarly, as current change over RH% was measured, impedance and capacitance change with respect to RH% was also measured using the KEYSIGHT U1733C LCR meter  when the sensor is exposed to a given level of humidity. The recovery time is defined as the time needed to recover to 90% of the initial baseline after turning-off the humidity. Response and recovery time of the proposed sensor were measured by changing its position from one jar (low-level RH%) to the other jar (high-level RH%) which takes few minutes; therefore, response and recovery time measurement using this setup was not possible, so an alternate method was used. For measuring the response time, proposed sensor was moved from open air 35% RH to 85% RH jar and recovery time was measured by moving back the proposed sensor from 85% RH jar to open air. Stability is another key feature to evaluate the performance of a sensor; therefore, we measured the stability of our fabricated humidity sensor by placing it inside jars with different levels of RH% at an applied voltage of 3V and observed the response of resulting current continuously for 1 h as shown in We tested our fabricated sensor for the application of human health monitoring by exposing its surface to human breathing so that it can sense the water vapors. Our proposed sensor showed a fast response time with a quick recovery time and had the capability of measuring rapid and small RH% changes. The sensor response over normal breathing was tested. Graphene and its derivate composites with ZnO have been reported by many authors for humidity sensing applications. 2 [2 [To improve the humidity sensing properties of ZnO, its composition with other 2D materials like MoS2  and WS2 2 [2  etc., haFurthermore, graphene composites with different materials like semiconductors , 2D mateThis work demonstrated an easy, solution-processed, and cost-effective way to fabricate a humidity sensor based on the composite of graphene flower and ZnO. Characterizations by FESEM, EDS, and XRD were carried out to completely understand the properties of GrF/ZnO composite. It was observed that GrF/ZnO composite had a high surface area with a porous nanowire-like structure, making it a suitable candidate for humidity sensing. Multiple electrical characterizations of the prepared humidity sensors were performed at different RH% levels. The proposed humidity sensor showed an increasing current and a decreasing impedance response with respect to increase in RH% from 15% to 86%. The demonstrated sensor showed a fast response with a current change of 7.7 μA/RH%. Moreover, the sensor revealed a fast recovery time and offered good stability over a wide range of humidity. The proposed sensor showed a measurable current change in response to normal human breath, making it a compelling candidate for health monitoring applications. GrF/ZnO composite has excellent morphological, chemical, and electrical properties that make it a useful candidate for many applications.


Comparing with pristine PANI, the shifts of the characteristic peaks of PANI-based nanocomposites in UV-visible absorption spectra (UV-vis) and Fourier transform infrared spectroscopy (FT-IR) indicate the chemical interaction between the PANI matrix and the nanofillers, which is also confirmed by the scanning electron microscope (SEM) images. Corresponding coloration efficiencies were obtained for the WO3/PANI (40.42 cm2 C−1), graphene/PANI (78.64 cm2 C−1), and WO3/graphene/PANI (67.47 cm2 C−1) films, higher than that of the pristine PANI film (29.4 cm2 C−1), suggesting positive effects of the introduced nanofillers on the electrochromic performance. The areal capacitances of the films were observed to increase following the order as bare WO3 < WO3/graphene < pristine PANI < WO3/PANI < graphene/PANI < WO3/graphene/PANI films from both the cyclic voltammogram (CV) and galvanostatic charge-discharge (GCD) results. The enhanced energy storage and electrochromic performances of the PANI-based nanocomposite films can be attributed to the capacitance contributions of the introduced nanofillers, increased PANI amount, and the rougher morphology due to the embedment of the nanofillers into the PANI matrix. This extraordinary energy storage and electrochromic performances of the WO3/graphene/PANI film make it a promising candidate for combined electrochromic and energy storage applications.Different polyaniline (PANI)-based hybrid films were successfully prepared by electro-polymerizing aniline monomers onto pre-spin-coated indium tin oxide (ITO) glass slides with WO In contrast, metal oxides and conducting polymers as main materials for pseudo-capacitors always possess much larger capacitances but the shrinkage and swelling lead to much shorter cycling life. In order to overcome their corresponding deficiencies, it is proposed that the combination of EDLCs materials with pseudo-capacitive materials is an excellent choice since this approach can take advantage of both the long cycling life of EDLCs and high capacitance merits of metal oxides [Energy shortage and environmental pollution have been two major subjects of modern society, which urgently requires developing clean, efficient, and renewable sources of energy, as well as advanced technologies associated with energy storage and conversion ,2,3. As l oxides .Meanwhile, electrochromism (EC) has also attracted intense research attention over the last few decades, where the material color changes with electrochemical reactions ,9. The p3) as a typical electrochromic material with certain pseudocapacitive property have been widely investigated and demonstrated promising application potentials for electrochromic and supercapacitor electrodes [3 with PANI is expected to give multicolored and enhanced energy storage performances due to the combined electrochromic and pseudocapacitive properties as well as the resulted porous morphology. However, since PANI and WO3 exhibit bad cycling stabilities during the redox reaction [3 or graphene is expected to have positive effects on the electrochromic and supercapacitor performance of the pristine PANI films. Among all the materials for supercapacitor and electrochromic applications, conducting polymers belonging to both categories have received a lot of research interests because of their tunable electrical properties, flexibility, and high processability within solution . Among aectrodes ,28,29,30reaction ,32, carbreaction ,34,35,36reaction ,38,39. Ereaction ,41,42,433, graphene, and PANI films by employing combined spin coating and electro-polymerization method. The interactions between the specific components and the morphologies of the synthesized different hybrid films  have been successfully investigated by various characterization methods. Their energy storage and electrochromic performances were also studied. All the PANI-based hybrid films exhibit greatly enhanced energy storage abilities and coloration efficiencies compared to the pristine PANI, demonstrating positive effects of the nanofillers. The triple layer WO3/graphene/PANI film shows great potential for combined energy storage and electrochromic applications. To the best of our knowledge, comprehensive characterizations and studies of different hybrid WO3, graphene, and PANI films for energy storage and electrochromic applications have not been reported.In this work, we have successfully synthesized different hybrid WO6H7N, ≥99.0%), sulfuric acid , hydrogen peroxide solution (PERDROGEN® 30% H2O2 (w/w)), ethanol , and ammonium hydroxide  were all purchased from Fisher Scientific . Tungsten oxide  were purchased from Nanostructured & Amorphous Materials, Inc. . Graphene was provided by Celtig LLC . The microscope glass slides and indium tin oxide (ITO) coated glass slides were also obtained from Fisher Scientific . Before the usage of the ITO coated glass slides, they were first sonicated in ethanol for 10 min, and then immersed in an aqueous solution containing 4.0 mL NH4OH, 4.0 mL H2O2 and 20.0 mL deionized water for 10 min. Finally, the ITO glasses were sonicated in deionized water for another 10 min and dried naturally. Deionized water was used throughout the experiments.Aniline  on the synthesized WO3 film and maintained at 2000 rpm for another 20 s. The electro-polymerization of aniline onto the as-treated ITO glass or formed WO3, graphene, or WO3/graphene films were performed on an electrochemical working station VersaSTAT 4 potentiostat . A typical three electrode electrochemical cell was employed, in which a saturated calomel electrode (SCE) served as the reference electrode, a platinum (Pt) mesh served as the counter electrode and the spin coated ITO glass or bare ITO glass slide with an effective area of 6.0 cm2 served as the working electrode. A typical electrochemical polymerization was performed 10 cycles scanned back and forth from 0 to +1.2 V vs. SCE at a scan rate of 50 mV/s in 0.5 M H2SO4 aqueous solution containing 0.5 M aniline. All the films were dried naturally overnight.Six species of films as bare WO−1 at a resolution of 4 cm−1. The UV-vis of the films deposited on the ITO coated glass slide were observed in the range of 200–800 nm at room temperature using a UV-visible spectrophotometer . A long path length home-made spectro electrochemical cell with Teflon cell body with front and rear windows clapped with two steel plates was used where the ITO glass slide was used as the working electrode for energy storage and optical characterizations. In a standard three-electrode system, the electrochemical behaviors of the PANI-based nanocomposites films were investigated by CV scanned from −0.2 to 0.8 V vs. SCE at a series of scan rates and galvanostatic charge-discharge measurements from 0 to 0.8 V with different current densities. The electrochemical impedance spectroscopy (EIS) was carried out in the frequency range from 100,000 to 0.01 Hz at a 5 mV amplitude referring to the open circuit potential (OCP) on the same electrochemical working station VersaSTAT 4 potentiostat. The cycling stabilities of the PANI-based films were also investigated by evaluating the capacitance retentions by running 1000 galvanostatic charge-discharge cycles. The spectroelectrochemistry measurements were performed on a Jasco V-670 spectrophotometer coupled with the potentiostat for applying electrochemical potentials. The in situ chronocoulometry (CC) were conducted under a square-wave voltage of 0.8 and −0.2 V with a pulse width of 20 s using the electrochemical working station VersaSTAT 4 potentiostat. The morphologies of the thin films grown on the ITO glass slides were characterized by scanning electron microscope  and atomic force microscopy . The thickness of the thin films grown on the ITO glass slides were also determined by AFM. Imaging of AFM was done in acoustic ac mode (AAC) using a silicon tip with a force constant of 2.8 N/m and a resonance frequency of 70 kHz. The FT-IR spectrometer coupled with an ATR accessory  was used to characterize the surface functionality of the thin films grown on the ITO glass slides in the range of 2000 to 500 cm3-coated ITO, and (d) double layer WO3/graphene-coated ITO. The PANI film are deposited on the glass by sweeping the potential between 0 and 1.2 V at a scan rate of 50 mV/s in 0.5 M H2SO4 aqueous solution containing 0.5 M aniline. The film growth can be clearly verified by the continuously increased current with the CV cycles. Similar CV curves except different anodic current peaks are obtained for these PANI-based nanocomposites films. The anodic irreversible peaks start at around +0.9 V, corresponding to the oxidation of aniline monomers and the initiation of the PANI electro-polymerization. However, it is noted that the anodic peak current densities of graphene/PANI (2) and WO3/graphene/PANI (2) are much higher than that of pristine PANI (2), which can be attributed to the improved electrical conductivity caused by the introduced graphene. In contrast, WO3/PANI shows decreased anodic peak current density (2) compared to pristine PANI film because of the increased resistance of WO3 film on the ITO.m is the mass of PANI polymerized onto the substrate, gram (g); C is the total Faradic charges consumed in the electro-polymerization, coulomb (C); mM is the molecular mass of aniline monomers (93.13 g/mol); and F is Faraday constant . About 14.6, 28.6, 12.3, and 27.8 μg PANI polymers were calculated for the pristine PANI, WO3/PANI, graphene/PANI, and WO3/graphene/PANI films, respectively. The fewer and higher amount of PANI in the WO3/PANI and graphene/PANI films further confirm the increased and decreased resistance caused by the introduced WO3 and graphene, respectively.In addition, the mass of the electropolymerized PANI onto the substrate can be roughly estimated through the total Faradic charges consumed in the electro-polymerization assuming that 2.5 electrons for each aniline monomer in emeraldine, as the following Equation (1):3, double layer WO3/PANI, pristine PANI, double layer graphene/PANI, double layer WO3/PANI, and triple layer WO3/graphene/PANI. 3 and double layer WO3/graphene films compared with that of the PANI-based ones, indicating the successful deposition of PANI in the other four films. Furthermore, a clear shift of the characteristic peak is observed for all the PANI-based nanocomposites, indicating the existence of chemical bonds between the PANI matrix and WO3 particles. The nitrogen atoms in PANI are inferred to possibly form coordinated compounds with the exposed tungsten atoms on the surface of WO3. The strong coordination bonds between the nitrogen atom and the tungsten atom might cause the observed peak shifts. FT-IR as a strong technique probing the inherent composition and inner relationships of the PANI-based nanocomposites is carried out and shown in −1, corresponding to the characteristic peaks of PANI [3/graphene/PANI > WO3/PANI, which is consistent with the PANI amounts calculated above.In order to characterize the relationships between nanofillers and PANI matrix as well as the inherent composition of the nanocomposites, UV-Vis and FT-IR are conducted and shown in  of PANI ,22. More3/PANI, and (d) WO3/graphene/PANI films, respectively. The high magnifications of the corresponding SEM images are shown as the insets. For pristine PANI, 3/PANI, 3 nanoparticles and PANI matrix are also uniformly distributed on the ITO glass with clear interaction between WO3 particles and PANI fibers, which is also confirmed from the magnification of 3/graphene/PANI, 3 particles are clearly seen to distribute in the PANI matrix, the interactions are also confirmed by the close wrapping of PANI fibers on graphene sheets and WO3 particles shown as inset in 3 film, 3 nanoparticles. In contrast, other films have constant thickness values based on the corresponding profiles. The thickness of the synthesized PANI-based films follows an order as WO3/graphene/PANI (~33 nm) > WO3/PANI (~30 nm) > graphene/PANI (~22 nm) > pristine PANI (~20 nm), which is consistent with the composition of the synthesized films.The morphologies of the synthesized hybrid films have been characterized by SEM as shown in 3 film, (B) double layer WO3/graphene film, (C) pristine PANI film, (D) double layer graphene/PANI film, (E) double layer WO3/PANI film, and (F) triple layer WO3/graphene/PANI film at different potentials  in 0.5 M H2SO4, respectively. For all the PANI-based nanocomposites with applied potential from −0.5 to 0.8 V  because of the introduced graphene. However, the film also exhibits obvious decreased transmittance values and typical PANI peaks with the increasing potentials. to 0.8 V C–F simil3/PANI, and triple layer WO3/graphene/PANI nanocomposites films, respectively. Decreased transmittance modulations of graphene contained films confirm further the block effect of the introduced graphene.The coloration switching responses as an important parameter is vital to evaluate the electrochromic performance in terms of energy consumption and coloration switching. The coloration properties of all the PANI-based nanocomposites films were studied by applying potential steps of 0.8 and −0.2 V with a pulse width of 20 s. η) is a critical parameter of electrochromic materials for practical applications. It is defined as the change in the optical density (OD) per unit charge (Q) inserted into (or extracted from) the electrochromic films, that is, the amount of energy to achieve color change. The CE is calculated from Equations (2) and (3):OD is the change in the optical density, λ is the dominant wavelength for the material, dQ is the charge density (injected/ejected charges per unit electrode area), beleachedT refers to the transmittance of the film in the bleached state, and coloredT refers to the varying transmittance of the film during the coloring process. OD from the second cycle in the transmittance-time curve at 633 nm versus the corresponding inserted charge density obtained from charge density-time curve  at a high scan rate of 50 mV/s within a potential range of −0.2 to 0.8 V in 0.5 M H2SO4 solution from 0 to 0.8 V with a current density of 0.08 mA/cm2, as shown in 3 and graphene nanofillers, confirmed by the discharging time order as pristine PANI < WO3/PANI < graphene/PANI < WO3/graphene/PANI, which is also in good accordance with the area of CV results. In addition, the voltage drop at the initiation of the discharge curve is extremely small for WO3/graphene/PANI among all the films, indicating a very low equivalent series resistance (ESR) in the supercapacitor and also enables high-power operations. The specific capacitances of different films calculated from the CV at 50 mV/s and the GCD at 0.08 mA/cm2 are summarized in 3/graphene/PANI > graphene/PANI > WO3/PANI > pristine PANI > WO3/graphene > bare WO3, confirming the positive effects of graphene and WO3. Furthermore, the correlation between the specific capacitance and the current density for these four PANI-based films is also presented in 3/graphene/PANI has the highest capacity among all samples at all the same scan rates. For instance, the WO3/graphene/PANI achieves a specific capacitance of 11.267 mF/cm2 with 0.08 mA/cm2 current density, which is 1.3 and 2.6 times higher than that of graphene/PANI (8.709 mF/cm2) and WO3/PANI (4.312 mF/cm2), respectively. Notably, WO3/graphene/PANI shows capacitance three times larger than that of pristine PANI (3.693 mF/cm2), indicating a dramatic capacitance increasement when employing graphene and WO3 nanofillers. Furthermore, the triple layer WO3/graphene/PANI film shows a capacity of 12.27 and 7.017 F/g with 0.04 and 0.32 mA/cm2 current density, respectively, suggesting a considerable rate capability of WO3/graphene/PANI. The improvement in power density of WO3/graphene/PANI is further confirmed by the Ragone plots of these four PANI-based films as shown in 3/graphene/PANI > graphene/PANI > WO3/PANI > pristine PANI as a function of energy density. Furthermore, the as-developed WO3/graphene/PANI can outperform a lot of reported mass-based values in terms of capacitance and energy density [3/graphene/PANI film may also show considerable performances regarding PANI related practical applications like chemical vapor sensors, transparent conductors, and actuators.Galvanostatic charge-discharge (GCD) as a reliable approach to measure the specific capacitance of supercapacitor is also carried out in the same H density ,31,32. I2SO4. It is clearly seen that the resistance of bare WO3 film is larger than the others throughout the mostly frequency region due to the low conductivity of WO3. Meanwhile, the decreased resistance of WO3/PANI and WO3/graphene results from the introduced highly conductive PANI and graphene, respectively. However, no resistor-capacitor (RC) loops or semicircles appear in the high frequency region for PANI-based films, indicating negligible charge resistances and the better energy storage properties of the PANI-based films. The excellent energy storage performance of WO3/graphene/PANI can be clearly demonstrated as the tail approaching an almost vertical line compared with others. In addition, the equivalent series resistance (ESR) of each sample can be calculated from the Z′ values at Z″ = 0, which has been shown in 3/graphene/PANI < graphene/PANI < WO3/graphene < pristine PANI < WO3/PANI < WO3, demonstrating enhanced reaction kinetics because of the positive effects of graphene and WO3 nanofillers, which is also consist with the CV and GCD analysis. Electrochemical impedance spectroscopy (EIS) as a power technique probing the inherent reaction kinetics of the electrode was also employed as shown in 2. The cycling performances with specific capacitance retention are shown in 3/graphene and WO3/graphene/PANI electrodes have slight capacitance increases during the cycling process. The capacitance increases can be attributed to the introduced WO3 nanoparticles, which were increasingly accessed along with cycling. The WO3 particles used in this work have a nanoscale size around 40–50 nm, indicating a high specific surface area of the nanoparticles. Therefore, a combined electric double-layer capacitance from high surface area and pseudo-capacitor capacitance from redox reactions can be delivered by WO3 nanoparticles. Accompanied by the charge-discharge cycles, the activation and volume change of WO3 nanoparticles lead to higher pseudo-capacitor capacitance from more redox active sites, that is, enhanced capacitance during cycling. The cycling performance of the as-prepared PANI films follows the order as WO3/PANI > WO3/graphene/PANI > graphene/PANI > pristine PANI, which implies the WO3/graphene/PANI film is capable to serve as a promising supercapacitor material because of its much-enhanced capacitance and cycling performance. The above results clearly reveal the potential applications of this triple layer WO3/graphene/PANI for supercapacitors, while also highlighting again the positive effects of WO3 and graphene nanofillers on the PANI matrix. Since a long cycling performance is of the most important criteria for supercapacitors, the cycling performances of these four PANI-based films were conducted by GCD for 1200 cycles with a current density of 0.16 mA/cm3, graphene, and PANI films have been successfully prepared by electrodepositing PANI monomers onto pre-spincoated WO3, graphene, and WO3/graphene ITO glasses. Multi-color electrochromic phenomenon and supercapacitive performance of the different PANI films have been observed in this nanocomposite film owing to the dominant PANI component in the composites. Higher coloration efficiency and faster switching responses of the PANI-based nanocomposites films than those of the pristine PANI film are obtained from the inner interactions between the PANI matrix and the nanofillers, as well as the resulted rougher morphology. However, the introduced graphene can decease the light transmittance modulation of the PANI-based films because of its light block effect. The PANI-based nanocomposites films also exhibit enhanced areal capacitances compared to that of the pristine PANI film because of the capacitive role of nanofillers and the increased deposition amount of PANI materials. Largely enhanced cycling stabilities were also obtained because of the positive effects of graphene and WO3. In summary, this novel triple layer WO3/graphene/PANI nanocomposites film, incorporating the advantages of both graphene and WO3 in PANI matrix, has exhibited promising application potential in devices integrated the functions of both electrochromic and energy storage because of its considerable electrochromic and capacitive behaviors.Different hybrid WO


However, this binding promiscuity represents a major challenge for defining the biological contribution of each specific interaction. We developed a strategy for addressing these challenges by combining cell microarray and high-throughput FACS methods to screen for promiscuous binding events, map binding interfaces, and generate functionally selective reagents. Applying this approach to the interactions of mPD-L1 with its receptor mPD-1 and its ligand mB7-1, we identified the binding interface of mB7-1 on mPD-L1 and as a result generated mPD-L1 mutants with binding selectivity for mB7-1 or mPD-1. Next, using a panel of mB7-1 mutants, we mapped the binding sites of mCTLA-4, mCD28 and mPD-L1. Surprisingly, the mPD-L1 binding site mapped to the dimer interface surface of mB7-1, placing it distal from the CTLA-4/CD28 recognition surface. Using two independent approaches, we demonstrated that mPD-L1 and mB7-1 bind in cis, consistent with recent reports from Chaudhri A et al. and Sugiura D et al. We further provide evidence that while CTLA-4 and CD28 do not directly compete with PD-L1 for binding to B7-1, they can disrupt the cis PD-L1:B7-1 complex by reorganizing B7-1 on the cell surface. These observations offer new functional insights into the regulatory mechanisms associated with this group of B7 family proteins and provide new tools to elucidate their function  The immunoglobulin superfamily (IgSF) includes approximately 400 proteins that comprise co-stimulatory molecules , co-inhibitory molecules , cell adhesion molecules & cytoskeletal regulators  and cytokine receptors . The spatial and temporal mechanisms by which these proteins modulate the immune system remain to be fully defined. Among the best characterized of these proteins are members of the CD28 receptor family , a subset of the IgSF, which provides the principal signals for optimal T-cell function . These sRemarkably, even within the well-studied CD28/B7 families, additional interactions continue to be discovered. B7-1 has been demonstrated to bind PD-L1, resulting in context dependent bi-directional inhibitory signals that are not fully understood . ICOS-L in vitro and in vivo analyses. These reagents provide important opportunities for dissecting and discovering additional mechanisms that impact normal physiology and pathologies.Herein, we utilize two independent experimental approaches to confirm that mB7-1 and mPD-L1 interact in cis and additionally present data indicating that mCTLA-4 or mCD28 engagement alters the cell surface organization/presentation of mB7-1, which unexpectedly impacts the mPD-L1:mB7-1 cis interaction. Furthermore, we describe the application of two platforms, cell microarrays and high-throughput flow cytometry to identify the protein binding surfaces involved in the formation of the PD-L1:B7-1 complex. These studies demonstrate that PD-L1 utilizes overlapping, but non-identical recognition surfaces for the engagement of PD-1 and B7-1. We extend these findings by exploiting libraries of mB7-1 and mPD-L1 mutants to demonstrate that PD-L1 recognizes a surface overlapping with the B7-1 homodimer interface residing distal to the CTLA-4/CD28 binding surface. Importantly, these approaches supported the generation of set of unique PD-L1 and B7-1 variants with a range of biochemical properties, including selective receptor binding, for TM; BMS) and pembrolizumab  [Given their prominent role in regulating the immune response, it is not surprising that proteins within the CD28 family remain a primary focus for the development of targeted immunotherapies. Prime examples include ipilimumab , a function blocking mAb against the CTLA-4 inhibitory receptor , and the; MERCK) –24, whic; MERCK) –28. This2. For transfection, cells were pelleted at 500xg and resuspended in fresh media. For small-scale (1mL cells at 1x106/mL) transient transfections performed in 24-well non-treated tissue culture plates, 2 μg Polyethylenimine (PEI) was added to 0.5 μg diluted plasmid DNA in a final volume of 100 μL. For large-scale transfections (600mL cells at 1x106/mL) carried out in 2L baffled sterile shake flasks, 2 mg PEI was added to 400 μg diluted plasmid DNA.HEK 293 suspension cells were maintained in HEK Freestyle Media (Invitrogen) supplemented with Pen/Strep antibiotics and grown at 37 C in a humidified shaking platform incubator (Kuhner) with 5% COAll site-directed mutagenesis of mPD-L1 and mB7-1 was performed using high fidelity KOD polymerase, 2mM dNTPs and 4mM MgCl2. The template used for the mPD-L1 mutagenesis included the coding sequence for full-length mouse PD-L1 cloned between the SacI and BamHI sites of the Clontech N1 mCherry vector. For B7-1 the template used included the full-length native mouse mB7-1 coding sequence was cloned between the XhoI and SacII sites of the Clontech N1 mCherry vector by In Fusion (Clontech). Additional Information regarding the mutagenesis can be found in the supplemental information.Our cell microarray protocol was based on the method described by Sabatini and colleagues; the DNA plasmid concentration, percentage of gelatin, cell numbers plated, slides used and incubation times were all optimized for reproducibility in our system. A detailed description of our method can be found in the supplemental information.Transfected cell microarray slides were washed three times with 1x PBS. Fc-fusion proteins of hCTLA-4, hCD200R, mPD-L1, mPD-1 and mB7-1 were purchased from R&D Systems. For microarray queries, 0.6 μg of Fc-fusion protein and 2.0 μg of Alexa 647 labeled Goat anti-mouse secondary antibody were premixed in a final volume of 300 μL 1x PBS and 0.2% BSA and incubated on transfected slides at RT for 30 minutes. Additional details can be found in the supplemental information.PD-L1 mCherry mutant constructs were transiently transfected into HEK 293S cells and subsequently queried with protein A microbeads (Milltenyi) pre-saturated with a 4:1 mixture of PD-1 Fc-fusion and FITC-Fc protein. A Fc protein to bead ratio of 5μg protein:10μL microbeads was utilized on the basis of a previous report from Genentech . Please + T-cells isolated using mouse anti-CD4 microbeads (Milltenyi). The CD4+ T-cells were collected in complete RPMI media supplemented with 10% FBS, pen/strep antibiotics, 2mM L-glutamine and 0.1% BME. The cells (107 / mL) were stained with 2.5 uM CFSE (Invitrogen) for 10 min at 37°C, washed two times with cold complete media and recounted. On the same day, 75,000 cells were plated per well in a 96-well TC plates in complete RPMI media and either left inactivated, activated with 33.3 nM (~ 5 μg /mL) anti-CD3, or activated with 33.3 nM anti-CD3 in the presence of a ~5-fold molar excess (174.3 nM) of either control Fc, WT PD-L1-Fc or mutant PD-L1 Fc proteins. Four days post activation, proliferation was determined by analyzing CSFE dilution. The data from each experiment were normalized to the control Fc population and a total of three independent experiments were averaged.Spleens were harvested from C57BL/6 mice and CD4th the total amount of DNA for transfection  and 2 μg PEI. Transfection efficiencies were typically >60%. 100K cells from each transfection were incubated with either recombinant mPD-1 hIgG1 or mCTLA-4 hIgG1 protein (R&D systems) for 1 hour at room temperature. After binding, cells were washed 2X with PBS and 0.2% BSA and binding detected with anti-human (H+L) Alexa 647 secondary antibody (0.25 μg). Flow cytometric analysis was used to determine the percentage of mPD-L1 GFP expressing cells positive for Alexa 647 positive (PD-1 bound). Similarly, CTLA-4 binding was determined as Alexa 647 positive (CTLA-4 bound) as a percentage of mB7-1 mCherry expression (or mutant). PD-1 or CTLA-4 binding was normalized to that observed for cells expressing WT mPD-L1 alone or WT mB7-1 alone, respectively.HEK 293 suspension cells were transiently transfected with WT mB7-1 mCherry or mB7-1 mutants in combination with WT mPD-L1 GFP. WT mPD-L1 GFP was also transfected alone as a control. For 1 mL transfection, 50 ng of each expression plasmid was used representing 1/106 cells/ mL using Freestyle growth media (Invitrogen). For luminescence measurements, 50 μL of each normalized cell population were transferred to a 96-well half-well luminescence plate to which 12.5 μL of diluted Nano-Glo® live cell substrate (Promega) was added. Cells were incubated with substrate for 10 minutes and then immediately read on an Envision plate reader (Perkin Elmer) to detect luminescence using a 5 sec integration time. Details regarding the competition experiments using the split nanoluciferase can be found in the supplemental information.Split nanoluciferase constructs were generated by cloning full-length WT mB7-1, SER mB7-1, CYS mB7-1, WT mB7-2 and WT mPD-L1 into the NheI and XhoI of both the pBiT1.1-C (TK/LgBiT) and pBiT2.1-C (TK/SmBiT) vectors from Promega. For experiments, 1 mL of suspension HEK 293 cells were transfected with different combinations of SmBit and LgBit constructs (250 μg each) that were mixed and incubated with 2 μg PEI. Two days post transfection all transfected cell populations were counted twice and normalized to 1 x 10Additional methods are described in the Supplemental Methods.In vitro competition assays using recombinant proteins demonstrated that mPD-1 directly competes with mB7-1 for binding to mPD-L1, while mCTLA-4 and mCD28 do not directly compete with mPD-L1 for binding to mB7-1. In contrast, in cell-based experiments, we observed mCTLA-4- and mCD28-dependent reorganization of cell surface expressed mB7-1 and mB7-2 and demonstrate that this reorganization in turn affects cis mPD-L1:mB7-1 complex formation.We confirmed the previously reported mPD-L1:mB7-1 interaction using cell microarray- and high-throughput flow cytometric-based approaches, and employed these same strategies to systematically evaluate libraries of point mutants to define the residues contributing to the binding interface. Using full-length mouse B7-1 and PD-L1 constructs, complementary cell-based approaches involving reconstitution of split reporter proteins corroborated the recent report that the IgV domains of human B7-1 and PD-L1 engage in a cis-interaction on the plasma membrane of the same cell. To evaluate the murine PD-L1:B7-1 interaction, we first employed the cell microarray technology introduced by Sabatini and colleagues ,30. ThisLigation Independent Cloning (LIC)  was usedThe cell microarray platform was initially validated using the known murine PD-L1:PD-1, CTLA-4:B7-1 and CD200R:CD200 interactions. Live cell microarrays consisting of alternating rows of cells expressing mCherry fusions of murine PD-1, B7-1, CD200 or mCherry alone  the lower affinity of B7-1 for PD-L1 reduced the signal to noise compared to those queried with PD-1; 2) complete loss of binding was easily identified, but modest reductions in binding were often more variable; 3) inherent slide-to-slide variability associated with independently printed, transfected and treated slides resulted in signal to noise variations that precluded more quantitative comparisons.To generate more selective PD-L1 reagents, the X-ray structure of the PD-L1:PD-1 complex  was usedTo more quantitatively evaluate the binding characteristics of PD-L1 mutants, we implemented a high-throughput fluorescence activated cell sorting (FACS) assay, which enables the interrogation of 400 samples per hour. This FACs platform affords an enhanced dynamic range compared to cell microarrays and employs a modified strategy for presentation of the query protein. While bivalent Ig-fusions, as used in the microarray platform, are effective for the identification of interactions with high or moderate affinities, weaker interactions might be missed. To support detection of the wide range of apparent affinities anticipated in the library of PD-L1 mutants, we exploited the higher valency afforded by magnetic microbead capture and presentation . For exaBriefly, HEK293 cell lines were individually transfected with 55 different surface displayed mutant mPD-L1-mCherry fusions. These cells were probed by flow cytometry for their ability to bind FITC-decorated microbeads loaded with either wild type mPD-1 Ig-fusion or wild type mB7-1 Ig-fusion proteins . Importa50 and Bmax) (d) of the mPD-L1:mB7-1 interaction was previously reported to be 1.6 μM, which is about three times weaker than that of the mPD-L1:mPD-1 interaction (Kd of 0.59 μM) [As the output of FACS analysis is mean fluorescence intensity per event , relative binding affinities of individual panel members can be assessed by comparison to wild type. This approach was used to further evaluate the binding characteristics of a selected panel of mPD-L1 mutants with altered binding to mPD-1, mB7-1 or both. Transiently transfected HEK cells expressing cell surface presented mPD-L1 wild type or mutant proteins were titrated with soluble mPD-1 or mB7-1 Fc-fusion proteins and binding detected using a fixed concentration of anti-mouse IgG H + L Alexa 488 secondary antibody. Saturation binding curves were collected in triplicate, averaged, and fit to a single-site binding model , which rnd Bmax) . It is i0.59 μM) .Our mutagenesis data are consistent with overlapping binding surfaces for mPD-1 and mB7-1 on mPD-L1, suggesting that mPD-1 and mB7-1 should compete with one another for binding to PD-L1. This hypothesis was directly tested with a bead-based FACS competition assay utilizing mB7-1 and mPD-1 Fc-fusion proteins harboring two different Fc fusion isotypes. Beads coated with PD-L1 (mIgG2a) and were incubated with a fixed saturating concentration of mB7-1 (hIgG1), and titrated with mPD-1 (mIgG2a) or mIgG2a. The concentration of mB7-1 required to saturate the beads was determined empirically by incubating a fixed amount of mPD-L1 coated beads with increasing concentrations of mB7-1. Competition was monitored using an anti-human Alexa 488 secondary antibody. We optimized high-throughput transient transfection of HEK 293 cells in 24-well suspension tissue culture plates for the production of recombinant secreted Fc-fusion proteins in amounts consistent with screening. Utilizing this method, we purified Fc-fusion proteins representing a subset of the mPD-L1 mutants with altered binding characteristics . Followiin vitro T-cell activation assay involving plate-bound anti-CD3 antibody to simulate activation of T-cells via the T-cell receptor. Anti-CD3 was co-plated in the presence of IgG control, wild-type mPD-L1 or mPD-L1 mutants and the activation of CSFE-labeled primary CD4+ mouse T-cells was measured. In the context of anti-CD3-mediated CD4+ T-cell activation, wild type mPD-L1 inhibits activation compared to isotype control  and the murine B7-1 IgV domain (4RWH), we identified solvent accessible residues on mB7-1 . From this list, we generated a set of 152 mB7-1 mutants covering both the IgV and IgC domains. Expression validation in HEK 293 cells confirmed that greater than 95% of the mB7-1 mutants showed expression levels and localization comparable to wild type . This paOur mapping results show mPD-L1 binding to the homodimer interface of mB7-1, opposite the binding sites for mCTLA-4 and mCD28. Given this binding arrangement, it would be expected that mCTLA-4 and mCD28 do not directly compete with mPD-L1 for binding to mB7-1. This prediction was tested using the bead competition-binding assay and indeed in the presence of saturating mPD-L1 hIgG1, neither mCTLA-4 nor mCD28 competed with PD-L1 for binding to mB7-1 decorated beads , S15 FigNext we examined whether mB7-1 and mPD-L1 could bind in cis using a co-transfection competition experiment. The hypothesis was that if mB7-1 and mPD-L1 bind in cis then binding of mPD-1 protein would be reduced (compared to no mB7-1 present), as our competition data demonstrate that mPD-1 directly competes with mB7-1 for binding to mPD-L1. We further hypothesized that mCTLA-4 binding would not be affected as our competition data indicated its binding was mPD-L1-independent in the bead-based assay. For this assay, HEK cells were transiently transfected to co-express mB7-1 mCherry (or mutant) and mPD-L1 GFP, and were subsequently queried for binding to mCTLA-4 hIgG1 or mPD-1 hIgG1 protein . Our resFreeman and colleagues recently reported the use of a split nanoluciferase assay (Promega) to demonstrate that hPD-L1 and hB7-1 could participate in a cis interaction on the same plasma membrane . To confTo evaluate whether the observed luminescence was specifically due to cis binding between mPD-L1 and mB7-1, we co-transfected in a third construct along with the SmBit mB7-1 and LgBit mPD-L1 . AdditioWe also examined mutants of mB7-1 identified in the mapping experiment that showed reduced mPD-L1 binding but are located in the CTLA-4/CD28 binding site not at the dimer interface of mB7-1. In our mB7-1 mapping experiments using soluble mPD-L1 Fc-fusion protein, mB7-1 mutants, S118D and K123D, showed less than 20% mPD-L1 binding , while tB7-1<0.001, PB7-2 <0.01, CTLA-4 Cells; PB7-1<0.001, PB7-2<0.01). However, the fold change for mB7-1 was ~3 times higher than that of mB7-2. Soluble monomeric CTLA-4 was not capable of inducing increases in luminescence signals, demonstrating the requirement for a dimeric effector. Addition of soluble mCD28 hIgG1 did not cause any enhancement in luminescent signal; however, addition of mCD28 GFP expressing cells did cause a significant increase in luminescence for both mB7-1 and mB7-2 , but these increases were 2.3-fold less than that observed with mCTLA-4 expressing cells. Addition of either mPD-1 hIgG1 protein or mPD-1 GFP-expressing cells did not have a significant impact on the luminescence of any of the SmBit/LgBit pairs examined in Using the split nanoluciferase system, we examined the effect of mCTLA-4, mCD28 and mPD-1 receptor binding on the organization and behavior of ligands on the cell surface. Adding either soluble recombinant mCTLA-4 hIgG1 protein  or mCTLACTLA-4<0.01 (for both comparisons), PCD28<0.01,and 0.05 for comparison to cells expressing B7-1 SmBit/B7-2 LgBit and B7-1 LgBit /B7-2 SmBit respectively), suggesting the possibility that in addition to crosslinking , both receptors may be capable of enhancing mB7-1 dimer formation .To examine the relative contribution of “crosslinking” and homodimer formation to the increase in luminescence observed for mB7-1 and mB7-2 in the presence of CTLA-4 and CD28, we co-transfected SmBit mB7-1 with LgBit mB7-2 and vice versa . As mB7-protein<0.001, Pcells <0.01)  . No signBinding promiscuity is increasingly appreciated as a major mechanistic feature of the processes underlying multi-cellularity and is particularly important to a range of innate and adaptive immune responses. We implemented high-throughput cell microarray- and FACS-based platforms to examine the promiscuous binding interactions of mPD-L1 with mPD-1 and mB7-1, define the respective protein recognition interfaces on mPD-L1 and mB7-1, and to generate selective mPD-L1 and mB7-1 mutants with distinct biochemical and functional properties. The cell microarray platform provided confirmation that both mPD-1 and mB7-1 are ligands for mPD-L1 and identified a series of mPD-L1 point mutants that are highly selective for binding only mB7-1 , only mPD-1 , or which are defective in binding both mB7-1 and mPD-1 . While the cell microarray platform benefits from high-density and rapid analysis, it is limited with respect to its dynamic range. As the panel of mPD-L1 mutants was expected to display a wide range of affinities, we adopted a high-throughput FACS approach in which the mPD-L1 mutants were expressed and presented on cells, and queried with multivalent microbeads decorated with either mPD-1 or mB7-1. mPD-L1 mutants evaluated in this fashion exhibited behavior consistent with the initial microarray experiments and afforded a more faithful assessment of the relative binding propensities. We also examined mPD-L1 variants harboring mutations in the IgC domain and identified only one mutant with a strong effect on binding (T185D). Although it is possible mB7-1 forms a direct contact with this IgC residue, it is more likely this mutant impacts overall stability or orientation of the IgC or IgV domain, which impacts mB7-1 binding.For a subset of the mPD-L1 mutants, purified Ig-fusion proteins were used in FACS-monitored titrations to estimate binding relative to the wild type protein. As illustrated in + T-cell activation assay, those mPD-L1 mutants unable to bind mPD-1 could no longer support inhibition of T-cell activation, while those defective in mB7-1 binding did support inhibition comparable to wild type mPD-L1. These data indicate that, in the context of this particular assay format, engagement of PD-1 is the predominant PD-L1-driven inhibitory pathway, suggesting that the activities of the PD-1 and B7-1 specific PD-L1 pathways are distinct. Robust cell-surface expression of PD-L1 as well as B7-1 and PD-1 has been observed on mouse CD4+ T-cells after activation[in vivo studies utilizing an αPD-L1 monoclonal antibody that specifically blocks B7-1 binding, but not PD-1 binding, demonstrated a significant role for the PD-L1:B7-1 complex in protecting against transplant rejection and developing sustained immune tolerance [reg maturation and inhibition of self-reactive T-cells suggesting specific roles for this complex in autoimmunity, as well as in immune evasion in many cancers [In a conventional αCD3-driven CD4ctivation. Therefoolerance ,12. Howe cancers ,35. Thusin vitro T-cell activation assays where B7-1 or PD-L1 are co-plated with activating antibodies. The development of novel assays designed to look specifically at the impact of cis-interacting PD-L1 and B7-1 will be important for elucidating the functional consequences of this interaction. It is our hope that the mPD-L1 and mB7-1 selectivity mutants generated herein will contribute to this effort. More generally, the demonstration of similar interactions involving the human and murine orthologs suggests the PD-L1:B7-1 cis interaction is conserved among mammals.Using two different methods we complemented recent work of Freeman and colleagues showing that hPD-L1 and hB7-1 interact in cis on the same cell surface . These rThe PD-L1 ectodomain is composed of consecutive N-terminal membrane distal IgV and membrane proximal IgC domains. The IgV domain, which is primarily responsible for binding PD-1 and B7-1, exhibits typical IgV domain topology, with front β-sheet (strands GFCC’C”) and back β-sheet (strands ABED) forming a two layered β-sandwich. The PD-L1:PD-1 crystal structures demonstrate that the binding interface is formed predominately by residues contributed from the front faces  of PD-1 and PD-L1 ,36, and in vitro competition data. However, our detailed mapping suggests there are modest differences in the contacting residues in the CTLA-4 complex versus the CD28 complex, as two mB7-1 mutants (Q71A and V120A) only exhibited lost binding to CD28. Most interesting were the results obtained from querying the mB7-1 mutants with mPD-L1, in which a very different set of residues were identified as being important for binding . Mapping these residues to the crystal structure of the CTLA-4:B7-1 complex shows the majority of these residues lie on the surface contributing to the B7-1 homodimer interface near the hinge region between the IgV and IgC domains. We also identified a small number of residues  located near the CTLA-4/CD28 binding interface; however, experiments examining the cis B7-1 and PD-L1 interaction  reorganizes B7-1 and B7-2 on the cell surface. The increase in luminescence observed was due, at least in part, to crosslinking because soluble monomeric CTLA-4 was unable to exhibit the same effect for either ligand. Interestingly, we observed an ~3-fold higher increase in luminescence for B7-1 (dimeric) compared to B7-2 (monomeric) suggesting that the valency of the ligand also contributes to or effects the luminescence signal. To further explore the relative contributions of ligand valency and crosslinking, we compared the effect of each receptor on cells expressing only B7-1 (SmBit + LgBit) or cells expressing B7-1 and B7-2 (SmBit + LgBit). As B7-1 and B7-2 do not heterodimerize, the increase in luminescence observed must be due solely to reconstitution of the SmBit and LgBit fragments between crosslinked B7 molecules. The luminescence observed was much higher for CTLA-4 than for CD28, providing further support (though not proof) for the hypothesis that although CD28 is dimeric it binds monovalently to B7 ligands and does not efficiently crosslink B7 ligands ,44. In ain vitro, yet we do not know whether these complexes can form in the context of cell-cell contacts associated with engagement of T-cells and APC cells, how stable they might be or how effectively PD-L1 cis binding is reduced. Our experiments do show that addition of mCTLA-4 results in stronger dissociation of the murine PD-L1:B7-1 cis complex than mCD28, which may reflect the differences we observed in the effect each receptor had on the cell surface organization of mB7-1, perhaps as the consequence of their different valencies. Also notable is the inability of soluble mPD-1 and relatively weak ability of cell-surface expressed mPD-1 to compete with mB7-1 for cis bound mPD-L1. This result was surprising given the reported affinity for PD-1 binding to PD-L1 is ~2 times higher than that of B7-1 [Most surprising was the dramatic effect mCTLA-4 binding had on the cis interaction between mB7-1 and mPD-L1. Although our data and previously reported data  clearly Together our results suggest a model in which engagement of mB7-1 by mCTLA-4, and to a lesser extent mCD28, results in a reorganization of cell surface expressed mB7-1 in a fashion that negatively impacts the ability of mPD-L1 to bind to mB7-1 in cis . To furtOur model also has implications for the mechanistic interpretation of CTLA-4 based therapeutics. Abatacept  and Belatacept  are two FDA approved biologics, consisting of the extracellular domain of CTLA-4 fused to the Fc domain from human Ig, used to treat autoimmune diseases and kidney transplant rejection, respectively –48. Thesin vivo roles of these intersecting signaling pathways, and afford the opportunity to develop biologics with modified selectivities and/or affinities, which could translate into more efficacious treatments with new indications and fewer deleterious side effects. By expanding our expression library to include other key cell surface proteins  this approach can be used in the future to identify and characterize novel or promiscuous protein interactions.Our approach successfully identified selectivity mutants for mPD-L1 and mB7-1 and is readily applicable to other major immune regulators. The reagents generated provide the foundation for the creation of new murine model systems to examine the S1 Method(PDF)Click here for additional data file.S1 FigThe top panel shows 24 representative images of HEK 293 cells expressing different Ig superfamily targets in our Type I mCherry vector. All images were acquired on a EVOS inverted benchtop florescence microscope. Cytosolic mCherry was transfected as a negative control for membrane localization (Top Left rectangle) and the bottom right most rectangle shows untransfected cells. The bottom panel shows a 10X zoomed images of select constructs depicting the difference observed between cytosolic and membrane localized constructs.(PDF)Click here for additional data file.S2 FigA) We employed ligation independent cloning (LIC) methods for the design of custom vectors in which the SacB killer gene is replaced by the full-length ectodomain of a gene of interest flanked by on the N-terminus by the leader sequence from the human erythropoietin gene and flanked on the C-terminus by the transmembrane domain from mouse PD-L1 followed by mCherry fluorescent protein. B) The same LIC sites (and therefore the same PCR products) can be used to clone into a separate vector for the expression of Fc fusion proteins for downstream validation experiments.(PDF)Click here for additional data file.S3 FigTOP Graph shows the %mCherry positive HEK 293 cells transfected with wild-type PD-L1, mutant PD-L1 or mCherry empty vector control. Data is the average from three independent transfections with error bars showing the standard deviation. BOTTOM One-way ANOVA analysis was performed to determine statistically significant differences between each mutant compared to WT PD-L1. To aid in visualizing the results of this analysis, the graph shows the fold change in average expression for each mutant compared to wild-type PD-L1 . All of the mutants shown in BLUE were not statistically different from wild-type, those in GREEN were significantly different but showed higher expression than WT, those in RED were significantly different and showed ~25% less expression than WT.(PDF)Click here for additional data file.S4 FigTOP HEK 293 suspension cells were transiently transfected with either wild type or mutant mPD-L1 or mB7-1 as indicated in 24-well suspension plates. Two days post transfection cells were imaged for mCherry expression using an EVOS inverted benchtop florescence microscope. BOTTOM HEK 293 suspension cells were transiently transfected with either wild type or mutant mPD-L1 or mB7-1 as indicated. Two days post-transfections, 100,000 cells from each transfection were incubated with 0.5ug of each monoclonal antibody (R&D Systems MAB90783 (anti-mPD-L1) and R&D Systems MAB740 (anti-mB7-1) for 1 hour with shaking at room temperature. Cells were subsequently washed three times with 1X PBS with 0.2% BSA and incubated with secondary antibodies (anti-Rabbit 647 (PD-L1) and anti-Rat 647 (B7-1). Cells were analyzed by flow cytometry and data presented as the GeoMean of 647 (bound).(PDF)Click here for additional data file.S5 FigData shows a representative set of FACS scatter plots obtained from the microbead binding experiment. Microbeads coated with either control, PD-1 or B7-1 Fc-fusion protein were used to challenge cells expressing wild-type PD-L1 or mutants. The E60A mutant did not affect binding of PD-L1 to either PD-1 or B7-1. G119D and G120D lost binding to B7-1 but maintained binding to PD-1. The A121R mutant does not bind either PD-1 or B7-1. The D122A, Y123R and R125A mutants all maintained binding to B7-1 but lost binding to PD-1.(PDF)Click here for additional data file.S6 Fig360 degree rotation of a space filling representation of the PD-1:PD-L1 crystal structure (PDB: 3SBW). Residues are color coded the same as previously described . Most of the PD-1 specific residues are buried at the interface within the complex and therefore not visible. In contrast, many of the B7-1 residues remain exposed in the space fill model demonstrating that these positions are not involved in and do not impact the PD-1 binding interface.(PDF)Click here for additional data file.S7 FigA) A panel of 65 PD-L1 IgC mutants were examined for binding to mPD-1 (Blue Bars) and mB7-1 (Red Bars) using the microbead binding assay described in the main text. Gray bars depict the %mCherry expression for each mutant normalized to wild-type. All data represents two independent experiments with error bars showing the standard deviation. B) Mapping of the IgC mutants onto the structure of PD-L1 (PDB: 3SBW). In the IgV domain the color coding is the same as the main text, green = PD-1 binding affected, red = B7-1 binding affected, gray = both PD-1 and B7-1 binding affected. For the IgC domain T185D showed the most significant effect on B7-1 binding, highlighted red while the other mutants identified showed more modest effects, highlighted yellow. C) Table showing the normalized average binding of PD-1 and B7-1 to these select mutants.(PDF)Click here for additional data file.S8 FigA) Cartoon depiction of the competition assay. Briefly, protein A beads were saturated with mPD-L1 mIgG2a protein and subsequently incubated with 20nM mB7-1 hIgG1 and an increasing concentration of either mIgG2a, mPD-1 mIgG2a. Binding of mB7-1 hIgG1 was determined using an anti-human Alexa 488 antibody. B) Heat map showing results from one representative experiment. In the presence of control mIgG2a no loss of mB7-1 hIgG1 binding was observed. The graph shows the average and standard deviation for data from three independent experiments. This data was fit using a one-site competition model equation in the software Prism and the calculated EC50 was 8.3 ± 1.5 nM.(PDF)Click here for additional data file.S9 FigTop panel: A coomassie stained SDS/PAGE gel showing the recombinant Fc-fusion proteins that were purified over nickel affinity resin and gel filtration. Lower panel: FACS scatter plots for a protein binding experiment. Cells transiently expressing either mPD-1 or mB7-1 as GFP fusions were challenged with purified recombinant protein as shown. Protein binding was detected using an anti-mouse Alexa 594 secondary antibody followed by FACS analysis. These data demonstrate that the purified recombinant proteins maintain the same binding phenotype.(PDF)Click here for additional data file.S10 FigA) Left Analytical gel filtration traces for small-scale purified recombinant WT PD-L1-mIgG2a, PD-1-mIgG2a and B7-1-mIgG2a proteins. Right Analytical gel filtration traces for select PD-L1 mutants used for the T-cell activation experiments. B) S200 size exclusion chromatography of nickel affinity purified WT PD-L1-mIgG2a from a large-scale 600mL HEK 293 suspension culture. C) Same as B) but for a subset of PD-L1 mutants with selective binding for either PD-1 (G119D and G120D) or B7-1 (D122A and R125A).(PDF)Click here for additional data file.S11 FigA) Analytical Size exclusion for fresh preparations of WT PD-L1, Y56A, G119D, G120D, D122A and R125A (inset of gel shows eluates off His60 resin) from 150mL of HEK suspension cells. VPA was added at 24hours, culture supernatants were collected on day 7 and purified over His60 resin. Nickel eluates were concentrated to 500uL and loaded onto the Superose 6 Increase column 10/300. The void for this column runs at 8.5mL. The peak containing dimer Fc-fusion is found ~14.8mL and was collected. There was also a small peak at ~11mL and a shoulder peak at ~ 13mL. B) Analytical Size exclusion was re-run 2 weeks later for WT PD-L1, Y56A, G119D, G120D, D122A and R125A. Eluates collected from the initial size exclusion run were stored at 4C for 2 weeks, concentrated 2X and 100uL was loaded onto the same analytical column. The predominant peak for all of the proteins remains around 14.8mL. There is no longer a peak at 11mL though a more defined peak is observed at ~13mL (previously where the shoulder peak was). This data suggests the PD-L1 Fc fusion protein is reasonably stable and the mutants show some deviation but behave very similarly to wild-type protein.(PDF)Click here for additional data file.S12 Fig+ T-cells were isolated as described in the methods and plated in wells of a 96-well plates in the absence (TOP) or presence (BOTTOM) of 5ug anti-CD3. After 4-days cells were washed and incubated either with anti-human 647 secondary antibody alone (BLUE) or with primary anti-mPD-1 or anti-mB7-1 antibodies (human IgG1 from R&D Systems) as indicated. After staining cells were analyzed by flow cytometry and live 647 (APC) positive cells were gated based on the secondary antibody alone controls for each condition.CD(PDF)Click here for additional data file.S13 FigGraph shows the %mCherry positive HEK 293 cells transfected with wild-type mB7-1, mutant B7-1 or mCherry empty vector control. Data is the average from three independent transfections with error bars showing the standard deviation.(PDF)Click here for additional data file.S14 FigGraph highlighting the panel of 40 B7-1 IgC mutants examined for binding to mPD-L1 (Red Bars) and mCTLA-4 (Gray Bars) as described in the main text. Data shows percent bound from three independent experiments with error bars representing the standard deviation.(PDF)Click here for additional data file.S15 FigData shows saturation binding curves used to determine the lowest concentration of either mPD-L1 hIgG1 or mCD28 hIgG1 protein to saturate protein A beads loaded with mB7-1 mIgG2a. For competition experiments, 5nM was added to B7-1 loaded beads in the presence of increasing concentrations of the competing proteins .(PDF)Click here for additional data file.S16 FigA) Representative scatter plots from cell-cell conjugation experiment. HEK 293 suspension cells expressing full-length mPD-1 and mB7-1 mCherry fusions were mixed 1:1 with cells expressing either mCTLA-4 GFP, mPD-L1 GFP or GFP control. Positive binding between the populations of cells is observed as an increase in the percentage of events in Q2 (upper right). B) Quantification of three independent cell-cell conjugation experiments. Data shows the average and standard deviation.(PDF)Click here for additional data file.S17 FigA) This experiment was setup similarly to that shown in (PDF)Click here for additional data file.S1 Table+” signifies a fluorescent intensity in the 647 channel (bound protein) comparable to WT, “−”signifies no detectable fluorescence and “R” signifies reduced fluorescent intensity compared to WT PD-L1. Scores reflect observations made from three independent experiments. Mutants labeled BLUE lost binding to both PD-1 and B7-1, those in RED lost binding only to B7-1 and those in GREEN lost binding only to PD-1.The table shows scores for binding from the analysis of cell microarrays printed with PD-L1 mutants as shown in (PDF)Click here for additional data file.S2 TableRED lost binding only to B7-1 and those in GREEN lost binding only to PD-1. Mutants left black showed a reduction in both PD-1 and B7-1 binding.The table shows the analysis of PD-1 and B7-1 binding to a subset of PD-L1 mutants as determined by flow cytometry as shown in (PDF)Click here for additional data file.S3 TableRED show >20% binding comparable to WT B7-1 and boxes highlighted YELLOW show 20–50% binding comparable to WT B7-1.The table shows the analysis of B7-1 mutant binding to PD-L1, CTLA-4 and CD28 as determined by flow cytometry in (PDF)Click here for additional data file.


Synthesis of graphene materials in a plasma stream from an up to 40 kW direct current (DC) plasma torch is investigated. These materials are created by means of the conversion of hydrocarbons under the pressure 350–710 Torr without using catalysts, without additional processes of inter-substrate transfer and the elimination of impurities. Helium and argon are used as plasma-forming gas, propane, butane, methane, and acetylene are used as carbon precursors. Electron microscopy and Raman imaging show that synthesis products represent an assembly of flakes varying in the thickness and the level of deformity. An occurrence of hydrogen in the graphene flakes is discovered by X-ray photoelectron spectroscopy, thermal analysis, and express-gravimetry. Its quantity depends on the type of carrier gas. Quasi-one-dimensional approach under the local thermodynamic equilibrium was used to investigate the evolution of the composition of helium and argon plasma jets with hydrocarbon addition. Hydrogen atoms appear in the hydrogen-rich argon jet under higher temperature. This shows that solid particles live longer in the hydrogen-rich environment compared with the helium case providing some enlargement of graphene with less hydrogen in its structure. In conclusion, graphene in flakes appears because of the volumetric synthesis in the hydrogen environment. The most promising directions of the practical use of graphеne flakes are apparently related to structural ceramics. Its properties are similar to those of the graphene created by means of chemical vapor deposition (CVD); existing peculiarities are due to type of precursor [According to the Union of Pure and Applied Chemistry (UPAC) definition graphene layer is a single carbon layer of the graphite structure; its nature being an analogy to a polycyclic aromatic hydrocarbon of quasi-infinite size . In contrecursor . Graphenrecursor . In orderecursor , to makerecursor  or to marecursor . Nowadayrecursor  have useGraphene synthesis in the plasma jets is a fast process without catalysts, there is no need of intermediate processes, in outer heating of substrate. Regardless of the type of plasma generator there are several advantages. First, high temperature in the plasma reactor provides nearly full dissociation of injected reagents which helps to form high temperature materials in the vaporized state. Second, high energy density of thermal plasma provides a high productivity even in small reactors. Finally, in the plasma process a fast quenching is possible, allowing the high degree of overcooling and supersaturation, which is favorable for the formation of nanoparticles ,12,13.Shortcoming of plasma processes is existence of cool boundary layers and non-uniformity of process parameters. Girshick et al.  have devGraphene synthesized by means of microwave plasma, high-frequency induction plasma, or DC plasma torch, as a rule, has a unique structure—flakes with number of sheets from 1 up to 10 and more ,18,19. OThis paper discusses the influence of the DC plasma composition on the graphene morphology. According to references ,26 the tLayout of the setup for the graphene synthesis is shown in We measured the temperature in the zone of carbon vapor condensation and formation of graphene and graphane by the chromel-alumel thermocouple. Depending on the experimental conditions, the temperature on the collector surface varied within the range of 700–1200 К. This temperature range is optimal to obtain the hydrogenated graphene ,31.−8 g/cm3 and are investigated at the initial state after gathering in the cooled reactor. For the immediate registration of dimensional parameters and image data a scanning electron microscopy (SEM) with a Hitachi S5500 microscope  in the DF-STEM, BF-STEM, and SE modes was used. Samples were identified by means of a Raman spectroscopy study , a laser with a wavelength of 532 nm). In order to quantitatively investigate the elemental composition and the electron state of the atoms, we used the X-ray photoelectron spectroscopy (XPS), the measurements were performed at room temperature by means of the PHOIBOS 150 hemispherical energy analyzer ; we took the Mg Kα  emission without using the system of the surface charging neutralization. The elemental composition was determined by express-gravimetry (vario MICRO cube). The method is based on the pyrolytic burning of the substance in an empty tube in the scope of the quartz container with hanging washed by oxygen flow. After the burning, the compound with the element under consideration appears whose mass is to be measured. The phase composition of the synthesized samples was investigated by thermal methods , under the linear heating with the rate of 10 K/min, in the argon ambient, by means of the thermo-analyzer STA 409PC Luxx with quadrupole mass spectrometer QMS 403 C Aeolos .Samples are obtained as black powder with apparent density up to 10The X-ray structure analysis was performed using the standard technique and the DRON-2(Russia) facility (CuKα-emission). We obtained the X-ray diffraction spectra from the basic surfaces of the samples.2 occurs in the temperature range 820–920 K. It is worth to note that peak of the differentially scanning calorimetry (DSC) curve is produced not only by CО2, but also with output of other gases, among them may be pure hydrogen and hydrocarbon radicals like CH3, C2Hx, and C3Hx [−1, 1569 cm−1, and 2682 cm−1, respectively . But the hydrogenated graphene sp2-system is heavily disordered with considerable inclusion of sp3-system. Probably G-band is a superposition of G-band and D’-band. Besides the sample is an ensemble of randomly placed graphene flakes. As a result of all this there are more carbon atoms with sp2-hibridization around defects. Series of experiments were performed with the decomposition of propane and butane, methane, and acetylene in the helium plasma under the pressure 350, 500, and 710 Torr. Helium flow rate was kept constant at the level 0.75 g/s. The precursor flow rates are shown in and C3Hx . Resultsectively . Peaks DBy means of the direct express-gravimetry method, we determine the C:H content ratio in the samples. At the propane-butane mixture decomposition with the flow rate of 0.37 g/s in the helium plasma (710 Torr), we have obtained the hydrogen to carbon molar ratio of 1:4.Minimal output of graphene material occurred in the case of methane with flow rate 0.2 g/min. Its injection into the helium plasma jet leads to the formation of flake-like structures with lateral dimension 200–1000 nm a–c. FiguX-ray analysis shows that spectra of the samples contain 2 weak halos. The first one is in the range of Bragg angles 2ϴ = 11÷12°. Maximum intensity of this halo corresponds to the interlayer gap d = 0.7694 nm. Second halo is in the range of angles 2ϴ = 24÷28°. Maximum intensity of this halo corresponds to the interlayer gap d = 0.3401 nm.−1, the G band at 1586 cm−1, and the 2D band at 2693 cm−1. Ratio ID/IG is equal to 0.86.Analysis of the C1s carbon spectra shows that there are three ground states of carbon with the binding energies of 284.5 eV, 285.0 eV, and 291 eV. The spectra for the three states coincide within the domain of low binding energies and differ in the high-energy domain. These facts might be explained by surface shift of the C1s level and by the presence of defects in the surface sample site. The spectra differ in the width of the main peak because of the specimen non-uniformity caused by the defects and the different sizes of particles. The difference in the spectra within the lower binding energy region indicates different conductivity caused, possibly, by the presence of the C–H bonds. This fact is in agreement with .The minimal quantity of the hydrogen atoms per a single carbon atom equals to 0.017.D/IG, which is equal to 0.96, clearly demonstrates the deformities of graphene flakes. Experiments with the graphene synthesis by means of addition of acetylene into helium plasma jet show that in the jet volume flakes are forming under the pressure 350–500 Torr a,b. LateThus, experiments with the decomposition of propane, butane, methane, and acetylene show that typical morphology of graphene structures synthesized in the plasma jet is the shape of flake with bent edges. D/IG is equal to 0.87. It is somewhat greater than in the case with helium.In order to assess the influence of carrier gas on the shape of graphene, experiments with argon were carried out. Repeated technological conditions were used in the case of helium, but argon flow rate was 3.5 g/s, and arc current was 350 A. These values are chosen on the increasing part of the current-voltage characteristics, the same as for helium. C1s spectra have a conventional aspect with the main asymmetric peak at 284.4 eV and a satellite generated by excitation of π-π* transitions and π-plasmon (π-satellite), shifted from the main peak by 6.1 eV . SpectraWe investigated an influence of the addition of graphene flakes on the physical-mechanical properties of the structural ceramics based on the cubic boron nitride. Fabrication of industrial charge needed ~5 g of nanomaterials. This amount of pure graphene is obtainable only using the plasma chemical method of synthesis. The graphene with largest lateral size synthesized using argon/acetylene plasma was chosen. Ceramics were produced by means of the hot pressing  and reinforced with carbon nanotubes, as one can see in Thus, experiments with argon demonstrated as well that the formation of flake-like shape is related to the existence of hydrogen, but here particle dimensions are greater, and their dispersion is greater too.One-dimensional approach to the plasma flow is an attempt to determine the processes initiating the formation of flakes during the synthesis of graphene in the volume of helium and argon plasma jets.Common initial data for the comparison of helium and argon cases are the following: pressure 350 Torr, precursor – propane-butane mixture with flow rate 0.104 g/s.Flow rates of helium and argon are 0.75 and 3.5 g/s correspondingly.There are some motivations under the chosen figures. Helium flow rate is an optimum found experimentally; by the way it provides the carbon mass fraction in the plasma 0.1. Propane-butane mixture provides an intermediate value of H/C-ratio between methane and acetylene.Differences in the cases of helium and argon are investigated using equilibrium plasma composition as a function of temperature in the range 2000–4000 K. As a matter of fact just in this range the precursor turns into molecular hydrogen and condensed phase of carbon, identified in the database IVTANTERMO  as graph2H, C2H2, C2H3, C2H4, C4H, C4H2, C3H) is presented with two items: bound carbon (CnH) and bound hydrogen (HCn). They are complement to the list of conventional components: C – carbon atoms, Cgr – condensed carbon, Cn – carbon molecules , H – hydrogen atoms, H2 – molecular hydrogen.For the sake of simplicity the components with insignificant mass fractions are ignored in the composition of plasma, and ensemble of hydrocarbons (CMass fraction of ignored hydrocarbons in the considered temperature range is less than 1%.2. The difference in the HCn content leads to the difference in the synthesized graphene particles. In 2, C3, C5, C2H, C2H2, C4H2, and C3H. It can be assumed that hydrogen from C3H remains in the C-C structure and influences the shape of the future graphene structure.In In order to assess the effect of hydrogen on the shape of graphene, the experiments were selected producing graphene structures without impurities. Graphene quality was evaluated using the 5-point scale , with poThus, the presence of hydrogen in the graphene flakes demonstrates its key role in graphene synthesis, which is in agreement with a number of researches ,44,45. PVolumetric synthesis of graphene materials under the pressure 350–710 Torr in a plasma stream from a high-power DC plasma torch is demonstrated. For various carbon precursors like propane, butane, methane, and acetylene and for helium and argon as carrier gas the graphene is synthesized usually in the form of flakes regardless of the varying input and pressure. It is found that during the heating of the graphene flakes synthesized in the plasma of any composition output gas always contains hydrogen. Results of Raman spectroscopy show a high content of defects in samples. Apparently, hydrogen is responsible for the bent form of graphene. Hydrogen content in precursor and the type of carrier-gas are shown to influence the lateral dimensions of graphene and its purity. Greatest output of pure graphene occurs when propane-butane mixture is added to the helium plasma jet, and maximal graphene dimensions are obtained using methane and argon. Thus, it is possible to control graphene properties by means of the plasma forming gas. Variation of temperature profile in reactor is in direct correspondence with the properties of plasma forming gas. At every instance, different plasma components are formed according to the temperature profile. Thermodynamic equilibrium calculations show that in the temperature range 2500–3500 K hydrocarbon radicals appear with varying C:H relation in correspondence with the falling plasma temperature, and there always is hydrogen. In the case of argon, the residence time of particles in the hydrogen-enriched atmosphere is greater, hence the lateral dimensions of flakes are also greater. In conclusion, it can be stated that hydrogen is responsible for the deformation of graphene layer to flake which is a distinctive feature of graphene synthesized in a plasma stream. The addition of synthesized flakes to nitride bore ceramics increased its mechanical properties. Therefore, the most promising directions of the practical use of graphene flakes are apparently related to structural ceramics.


A significant difference is noticed on their glass transition and melting temperatures by DSC. The controlled formation and structural features of the polymer-functionalized-graphene is characterized by Raman, FT-IR, UV-Vis spectroscopy, NMR, and zeta potential measurements. The wettability measurements of the novel G-graft-PMMA on leather surface were found to be better in hydrophobic nature with a water contact angle of 109 ± 1°.Polymer nanocomposites have been synthesized by the covalent addition of bromide-functionalized graphene (Graphene-Br) through the single electron transfer-living radical polymerization technique (SET-LRP). Graphite functionalized with bromide for the first time via an efficient route using mild reagents has been designed to develop a graphene based radical initiator. The efficiency of sacrificial initiator (ethyl α-bromoisobutyrate) has also been compared with a graphene based initiator towards monitoring their Cu(0) mediated controlled molecular weight and morphological structures through mass spectroscopy (MOLDI-TOF) and field emission scanning electron microscopy (FE-SEM) analysis, respectively. The enhancement in thermal stability is observed for graphene-grafted-poly(methyl methacrylate) (G- Another 100 mL two necked round bottom was charged with NaNO2]n-graphene and sonication for 30 min. After the sonication, the reaction mixture was stirred at 55 °C for 6 h. Further, 20 mL of methanol was added to the reaction mixture at ambient temperature to precipitate the crude black solid product. The crude block solid was washed several times with deionized water to remove any alkaline residues and dried under vacuum at 50 °C for 6 h to obtain hydroxyl functionalized graphene (40 mg yield).Briefly, NaOH solution (3 N) added drop by drop into 40 mg of n-graphene (25 mg yield).Briefly, 30 mg of [OH]II/PMDETA complex and unreacted monomer. Finally, a pale gray polymeric solid was obtained after vacuum-drying for 30 h (yield 0.35 g).Briefly, 20 mg of graphene-initiator is dispersed in 1 mL of DMF and 5 mL of deoxygenated MMA was added to the reaction mixture and degassed by four freeze-pump-thaw cycles. Then, 3 mg of copper (5–50 nm) and 5 mL of PMDETA was added after degassed and purged with nitrogen gas and stirred in a thermostatic oil bath at 60 °C. After 24 h, the SET-LRP reaction was terminated by adding 100 mL of hexane. The resulting polymeric precipitate was filtered, redispersed in 50 mL of THF and again precipitated to remove the Cup-SWCNT via the addition of nitro radical towards synthesizing the polyurethane nanocomposites via covalent method n-graphene by the addition of nitro radical, which was produced from the mixture of sodium nitrite and conc. HNO3, followed by alkaline and bromine treatments. This afforded an enhanced yield of [OH]n-Graphene and [Br]n-Graphene as given in n-Graphene was covalently added in trace loadings to deoxygenated MMA monomer in the presence of catalysts such as Cu(0) and N,N,IN,IIN,IIIN-Pentamethyl diethylene triamine (PMDETA), which essentially to make the graphene to act as radical initiator itself to afford the G-graft-PMMA without compromising their unique properties, as shown in graft-PMMA(S) using sacrificial initiator.Graphite was rehabilitated into . Additionally, the highest molecular weight is also noticed at 8594.54 m/z, which indicated that the MMA monomer units are well polymerized onto the graphene surface n-Graphene is found to be stable up to 790 °C with weight loss around 6.52% and there is no decomposition due to strong π-π interaction. However, the [OH]n-graphene started to decompose slightly at 250 °C due to the presence of oxygen-containing groups that usually decomposed between 250–300 °C, whereas, the [Br]n-Graphene decomposition started at 150 °C and remained stable up to 677 °C with weight loss of 24.7%. Such variation is essentially occurred due to the presence of alkyl bromide moiety on graphene. The thermal stability of graphene-g-PMMA(S) and graphene-g-PMMA can be well-compared. From the results, two stages of degradation can be observed for graphene-g-PMMA(S). Accordingly, the first stage onset decomposition at 224 °C (43.65% residue) and second degradation at 348 °C (23.28% residue), which may have occurred due to the removal of polymeric unit, which is found to be stable up to 435 °C. In the case of graphene-g-PMMA, it has two stages of decompositions and stable up to 445 °C with weight loss residue of 1.9% as shown in g-PMMA shows a considerable shift of the weight loss towards higher temperature with a thermal stabilization up to 10 °C, which is higher than that of the G-g-PMMA(S) that may be due to the controlled and covalent addition of graphene. Apparently, considering the amount of oxygen-containing hydroxyl and bromide groups, the amount of 3sp hybridized carbon decreased with increasing amounts of 2sp hybridized carbon, which suggested that the conjugated graphene structure has been fully grafted with polymer chain through SET-LRP  and G-g-PMMA(S) , which revealed that the glass transition is lowered up to 10 °C. Further, the two-stage melting behaviour is also observed, where the first and second melting temperature is increased from 13 °C to 91 °C for G-g-PMMA (see Tg and Tm1 and Tm2) have also been enhanced upon the covalent addition of [Br]n-graphene into the polymer matrix, which may be due to a restriction in the main chain mobility and the confinement effect of nanofillers (graphene).It should be noted that the observed higher polydispersityvalue implies that there is some uncontrolled end chain happened during the polymerization reaction, which may be mediated through the sacrificial initiator. On the other hand, the G- SET-LRP ,37,38,39PMMA see . In addig-PMMA(S) material due to uncontrolled polymerization on the graphene surface, which is in good agreement with the FE-SEM and photographic images as shown in Generally, GO possesses the hydrophilic functional groups such as –OH, –COOH and epoxide, which can promote the intercalation of water molecules into graphene gallery, thus produce highly dispersible GO in aqueous medium. In this reaction, the alkyl initiating groups are introduced onto the graphene surfaces by esterification reaction between hydroxyl groups and bromoisobutyryl bromide, where it further reacted with methyl methacrylate monomer via SET-LRP and produced highly dispersed GO, where it is found to be soluble in polar and non-polar solvents with a colour change from brown to black during the radical polymerization. This essentially revealed that some structural changes occurred in the graphene surfaces. Hence, the polymer end chain is rapidly grown on graphene surface with excellent dispersion, where it appeared like the polymer macromolecules are wrapping on their surfaces see a–c. In ag-PMMA with the enhanced dewettingbehaviour (contact angle (CA) @109±1 (30 min) with respect to the water droplet on the leather surface showing better hydrophobic properties. Notably, materials with such characteristics can be pertinent to different applications, including the smart hydrophobic leather accessories. Further, water contact angle on the ‘finished’ control leather has been observed to be CA@75±1 (30 min) and graphene-g-PMMA(S) CA@90±1 (30 min). From these results, lower wetting behaviour is realized for graphene-g-PMMA(S) as compared to the graphene-g-PMMA (see In this context, we have also developed the graphene-PMMA see . This esIn conclusion, the effect of the sacrificial initiator and graphene based radical initiator was successfully surface polymerized using a controlled SET-LRP. The presence of bromoisobutyryl bromide based sacrificial initiator that generally produced the uncontrolled polymerization was evident by the cross-linking behavior as observed in FE-SEM images, and it also clearly confirmed the negative effect of sacrificial initiator during polymerization. Further, the presence of polymer backbone chains onto the graphene surface through complete grafting was confirmed via FT-IR, UV-vis spectroscopy, NMR, Raman, MALDI-TOF, DSC and TGA techniques. Furthermore, the functionalization of graphene through polymer was found to endow the better dispersion and improved zeta potentials, as evident by the dispersion measurements. The synthesized polymeric materials were coated on the leather surfaces and showed enhanced hydrophobic properties. Thereby, this novel nanocomposite material synthesized in the present work can be used for various damping and coating applications.