Blocking the interaction between Programmed Death Ligand 1 (PD-L1) and its receptor, PD-1, is an effective method of treating many types of cancers. Certain tumors overexpress PD-L1, causing host immune cells that express PD-1 to bind PD-L1 and cease killing the tumor. Inhibition of PD-L1 and PD-1 binding can restore host immunity towards tumor killing, and many new drugs have been developed to target this interaction. Current methods of PD-L1 diagnosis have shown to vary based on the antibody, detection kit brand, antigen retrieval method, and clinically defined methods by the FDA. To refine detection of PD-L1, we have identified a peptide, RK-10, and used it to detect PD-L1 expressing tumors with immunohistochemistry or flow cytometry. Flow cytometry was performed on cell lines and patient tissues using a fluorescent peptide (RK-10-Cy5). Immunohistochemistry using a biotin-modified peptide (RK-10-Biotin) was tested against the FDA-approved SP263 clone on biopsied patient tissues. For this study, we evaluated specificity of RK-10 using IHC in over 200 patient tissues, including NSCLC and Hodgkin’s Lymphoma. RK-10 shows staining in the tumor regions of FFPE tissues where the SP263 kit does not. RK-10-Cy5 peptide also demonstrates PD-L1 detection in NSCLC, breast, squamous cell carcinoma, and melanoma. By inhibiting immune checkpoints host immune response recover from tumor evasion. The innate immune response can potentially negate the tumor’s ability to resist targeted therapy, eliminating the need for continuous lines of therapy2. There are numerous drugs either approved or in the pipeline that target dominant immune checkpoints such as PD-L1 or CTLA44. One immune checkpoint of particular interest in human cancers is the interaction between Programmed Cell Death Receptor 1 (PD-1) and its ligand, Programmed Cell Death Ligand 1 (PD-L1)5. Overexpression of PD-L1 has been reported in many different tumor types, such as melanoma (40–100%), Non-Small Cell Lung Carcinoma (NSCLC) (35%-95%), Glioblastoma (100%), ovarian cancer (33–80%), and colorectal adenocarcinoma (53%)6. PD-L1 expression is characteristic of immune checkpoint evasion, allowing tumor cells to go unrecognized by immune T-cells as foreign. When an activated T-cell recognizes an antigen through binding of T-cell receptor to major histocompatibility complex, other checkpoints such as PD-1:PD-L1 are checked before the T-cell can recognize the cancer cell as foreign. When PD-1 on the T-cell surface and PD-L1 on the tumor surface are allowed to interact, the T-cell will be inhibited from destroying the foreign cell7  diagnostic agents for detecting PD-L1 in patients’ tissues suffer from three serious limitations. First, IHC agents for PD-L1 are based on antibodies raised against different clones of PD-L1; even though these IHC agents target the same marker they identify different parts of the marker. Therefore, these agents give different staining pattern based on the clone used. Second, the antibody used for detecting the primary IHC agent bound to the tissue would also be different in these assays resulting in varying performance based on the assay used for diagnosis. Third, the IHC agents were designed and developed by different companies and they would require the use of their own staining equipment and scoring algorithm. For example, Dako’s IHC agents used for selecting patients for nivolumab and pembrolizumab, utilize Dako IHC autostainer and their own scoring algorithm. In a similar fashion, for selecting patients for treating with drugs such as Atezolizumab and Durvalumab, Ventana diagnostics utilize Ventana automated IHC platforms and their own scoring algorithm. The data comparing these IHC agents for patients’ response, the Blueprint Project –a collaboration of 6 major pharmaceutical companies focused on comparing these tests with patient’s response data, is still ongoing. It is worth to mention here that factors such as tumor heterogeneity would not play a role in predicting patient response, as this factor is common in both PD-L1 positive and PD-L1 negative patients. Furthermore, running a different test for each drug evaluated is impractical due to limited tissue from biopsy, turnaround time, and cost. Potential harm to patients can result if inappropriate tests or cutoff levels are used to make treatment decisions16. Among all, the PD-L1 marker itself is also somewhat labile and must be evaluated soon after the biopsy17.Immune checkpoint inhibition has become an important modality for treating cancers, and has demonstrated significant success in recent yearsIn this paper, we report the specificity of RK-10 in binding PD-L1 in cells, patient blood samples, and tissues. We have demonstrated that RK-10 binds to PD-L1 positive tumor cells -breast, retinoblastoma, and lung cancer cell lines using flow cytometry. Flow cytometry using RK-10 was also performed in whole blood spiked with PD-L1 expressing cells, and squamous cell carcinoma and metastatic melanoma obtained from patients. The specificity of RK-10 has been evaluated using IHC methods in over 200 different patient tissues. We used gold standard Ventana SP263 PD-L1 antibody for comparison.All experimental protocols under Evaluation of Molecular Mutations in Lung Cancer, IRB #2004603, were approved by the University of Missouri Institutional Review Board. All methods were carried out in accordance with relevant guidelines and regulations. All patient specimens were obtained from the tissue core and the samples were collected previously with informed consent.The RCSB protein data bank was searched for the complex of PD-1 and PD-L1. Out of the results, the structure corresponding to the PDB ID “4ZQK” was selected for analysis because it represents the structure of the complex of human programmed death-1 (PD-1) and its ligand PD-L1 in its non-mutated form with an X-ray resolution of 2.45 Å. The selected structure was visually examined using the open-source program PyMOL Molecular Graphics System Version 1.8.20. A proprietary Fortran program was developed and used to analyze interactions between residues within the binding region. If distance between two residues in the binding region was less than or equal to 1.2 times the sum of the Van der Waal’s radii of the two atoms, it was regarded to be a contact and the residue-residue contact count was updated to +1. Number of occurrences for each sequence was calculated and used to identify the peptide sequences used in this study.6 cells per mL. Cy5-conjugated peptide solution was then added to the tubes to make the desired concentration of peptide in 200 µL. Eppendorf tubes were then placed in the incubator for 1 hour and vortexed at the 30-minute mark. After 1 hour, cell lines were analyzed on a BD FACS Canto II, a 3-laser, 8-color flow cytometer  using Diva 8.0 acquisition and analysis software . The cells of interest were gated using Forward and Side scatter (FSC/SSC) and positive antibody expression. 10,000 singlet events were collected for each specimen.Cell lines MDA-MB-231, Y79, and MCF-7 were purchased from ATCC, thawed, and grown in culture to confluency. When confluent, adherent cells were removed from the flask by scraping gently with a cell scraper and media removed using centrifugation. Suspension cells were pipetted from the flask and centrifuged to remove media. Cell lines were resuspended in Eppendorf tubes in 100 µL PBS at a concentration of 5 × 10Cases were evaluated using flow cytometry for suspected hematopoietic neoplasms. A portion of each fresh specimen was collected into RPMI. Each sample was prepared to create cell suspensions which were combined with neat amounts of the following antibodies : CD15 FITC, CD34 PE, CD33 PerCP-Cy5-5, CD13 PE-Cy7, CD11B APC, HLA-DR APC-H7, CD16 V450, CD45 V500C, Kappa FITC, Lambda PE, CD5 PerCP-Cy5-5, CD19 PE-Cy7, CD23 APC, CD20 APC-H7, CD10 BV421 V450, CD4 FITC, CD8 PE, CD2 PE-Cy7, CD56 APC, CD3 APC-H7, CD7 V450, CD38 PerCP-Cy5-5, CD10 APC, CD5 BV421 V450, CD23 PE, CD8 PE-Cy7, CD200 APC, and CD138 PerCP-Cy5-5  and incubated for 15 minutes in the dark. Any erythrocytes within the specimens were lysed with BD PharmLyse  and the specimens were washed with BD Staining Buffer with BSA . Each sample was evaluated using BD FACSCanto II, a three laser, eight-color flow cytometer  within 24 hours of collection. 50,000 events were collected for each sample. The expression data were analyzed using BD FACSDiva software, version 8.0 . Cases diagnosed as non-hematopoietic tumors were further subjected to evaluation with PDL-1 peptide if material was available combined with CK to identify the epithelial component. The cell suspensions were stained with 10 µl of BD Cytokeratin FITC (clone CAM5.2), 20 µl of Cy5-conjugated peptide solution, and 10 µl of BD Pharmingen CD274 PE (clone MIH1), incubated in the dark for 30 minutes, washed with BD Stain Buffer with BSA, and reconstituted to 500 µl with Stain Buffer with BSA in 500 ml polystyrene tubes for analysis. The specimens were analyzed on the FACS Canto II using the same panel template, gating strategy, and collection events as the cell line specimens.To detect PD-L1 in FFPE tissues we employed manual IHC techniques with a biotin-conjugated version of peptide RK-10-Cy5 [Cy5-GSGSGSTYLCGAISLAPKAQIKESL] and compared with Ventana PD-L1 (SP263) Rabbit monoclonal Primary Antibody stained on a Roche Benchmark Ultra autostainer. Seven PD-L1 expressing NSCLC patient tissues were obtained from the MU OneHealth tissue bank and de-identified according to IRB protocols. Paraffin-embedded patient tissue slides were baked overnight, then de-waxed and rehydrated according to standard protocols. Tissue sections were then subjected to antigen retrieval in EDTA at 95 °C for 20 minutes in EDTA (pH 0.9). The solution is then cooled for an additional 20 minutes on the bench top prior to buffer rinse. Tissues were then incubated with 15 µM biotinylated peptide for 2 hours in a humid chamber at RT. After 2 hours, slides were washed with buffer and treated with Pierce™ High Sensitivity Streptavidin-HRP (1:200 dilution) (Sigma) for 30 minutes at RT in a humid chamber. Once this was complete, slides were again washed in buffer then treated with DAB (Sigma) for 10 minutes. Slides were again washed in buffer, then dehydrated using graded alcohol and xylene and counterstained with hematoxylin. Slides were then imaged using bright-field microscopy on a Leica DM5500.We investigated PDL1 expression in the same seven FFPE tissues and 192 lung cancer cases on a microarray using our peptide conjugated with Cy5 fluorophore. A lung cancer tissue microarray (TMA) was purchased from U.S. Biomax that contains 192 separate cases of various types of lung cancers . In addition, seven NSCLC patient tissues were obtained from the Mizzou OneHealth tissue bank and de-identified according to IRB protocols. Paraffin-embedded tissue slides were baked overnight, then de-waxed and rehydrated according to standard protocols. Tissue sections were then subjected to antigen retrieval in EDTA at 95 °C for 20 minutes in EDTA (pH 0.9). The solution is then cooled for an additional 20 minutes on the bench top prior to buffer rinse. Tissues were then incubated with 15 µM Cy5-conjugated peptide for 2 hours in a humid chamber in the dark at RT. After 2 hours, slides were washed with buffer. The slides were then mounted using nucleus-specific DAPI counterstain and cover slipped. Slides were then imaged using fluorescence microscopy on a Leica DM5500 and compared to the same sections which had been stained with the Ventana antibody. For fluorescent analysis, DAPI channels and Cy5 channels were overlaid to image cell nuclei and PD-L1 expression, respectively.To test specificity of the PD-L1 peptide, we first blocked the PD-L1 receptors on the tissue with RK-10 peptide for 1 hour prior to autostaining the tissue with SP263 antibody using the Roche autostainer with the Ventana PD-L1 kit according to Roche’s specifications. We also investigated blocking of the RK-10 peptide using the SP263 antibody by first treating the tissue with SP263 antibody for 30 minutes prior to treating the tissue with peptide according to the previously mentioned protocol.We focused our initial research on understanding the interaction of PD-1 and PD-L1 based on X-ray crystal structure data (S1) with the goal of identifying the peptide sequence that is selectively mediating the interactions. After the crystal structure of each protein was identified, a proprietary Fortran program was used to analyze which amino acid sequences interact most closely between the two proteins. Number of occurrences for each sequence was calculated and used to identify the peptide sequences used in this study. Fig. . The cal18, along with retinoblastoma line Y79 and breast cancer line MCF-7, which show no meaningful PD-L1 expression19. To set our conditions for flow cytometry, we first examined titrations of PD-L1 peptide using all three cell lines. In each of the samples double-positives were selected by analyzing expression of both cytokeratin (FITC channel) and PD-L1 (Cy5 channel) (S2). All three cell lines showed a decrease in mean fluorescence intensity (MFI) as the concentration of peptide decreased  rabbit monoclonal primary antibody stained on a Roche Benchmark Ultra. In this study the peptide was conjugated with biotin, which was used to bind a secondary treatment of streptavidin-HRP. Formalin-Fixed Paraffin Embedded (FFPE) placenta tissue was used as the positive control, since PDL1 is expressed in placental trophoblastssue Figs . The SP2sue Figs . PlacentOnce confidence in PD-L1 staining was established in repeated placenta tissues, we compared staining in seven NSCLC patient tissues (patients ‘A’ through ‘G’) with the Ventana SP263 antibody and RK-10-Biotin . To stain with the fluorescent PD-L1 peptide, antigen retrieval was performed and tissue slides were treated with 15 µM fluorescent peptide in a dark, humid chamber for 2 hours, washed with buffer, then counter-stained and mounted with DAPI nucleus stain. Peptide-stained slides were imaged on a Leica DM5500 using channels specific for DAPI or Cy5, and channels were overlaid to examine PD-L1 expression. To confirm the data from the IHC stained tissues, the same seven patient tissues A-G were stained with RK-10-Cy5 Figs . The Cy522, to the point of PD-L1 being a diagnostic indicator of RS cells. Due to the characteristic expression of PD-L1 in RS cells, we examined PD-L1 levels in the four identified Hodgkin’s lymphoma patient samples using the fluorescent RK-10-Cy5 peptide and compared with the SP263 antibody , or fluorescent molecules for fluorescentanalysis (RK-10-Cy5). As mentioned above, antibody based IHC agents recognizes different epitopes in PD-L1; in sharp contrast, the identified peptide sequence recognizes the unique binding site between PD-1 and PD-L1. Additionally, the peptide based assay developed in this study is standalone, that is secondary antibody is not necessary for staining.RK-10 Peptide attached with biotin or fluorescent dye enables easy detection of PD-L1 biomarker in tissues and cell lines. The data presented in this study utilizes manual staining of RK-10 in human tissues; therefore, the need for autostainer specific for this agent is unnecessary. Additional advantages include that the peptide is relatively inexpensive, easy to synthesize, and can be mass produced in higher quantities.The PD-L1 targeting peptide RK-10-Cy5 was identified through structural analysis of PD-1:PD-L1 binding pocket structure. PD-L1 specific peptide RK-10 has shown high sensitivity and specificity for tumor cells in over 200 different cases of tissue – 192 lung cancer cases on a TMA, seven patient lung cancers, one placenta tissue, and four Hodgkin’s lymphoma cases. Patient tissues stained specifically and reproducibly within the tumor and PD-L1 expressing immune cells using either a biotin-conjugated peptide for IHC, or a Cy5 fluorophore-labelled peptide for fluorescent microscopy. RK-10-Cy5 staining showed a positive correlation with Ventana’s FDA-approved PD-L1 diagnostic (SP263) where the SP263 kit stained tumor positively for PD-L1 expression. While there were some cases that were negative using both SP263 and RK-10-Cy5, there were a large number of cases where RK-10-Cy5 showed very specific tumor staining that were not stained by the SP263 antibody. This could either be due to higher sensitivity of RK-10-Cy5 or due to a lower titration of SP263 to only detect PD-L1 above a clinical cutoff, since the SP263 kit is meant for clinical diagnosis for use with its companion therapeutic drug Durvalumab.In the Hodgkin’s lymphoma cases, PD-L1 expression as measured by the RK-10-Cy5 peptide matched up well with the IHC staining shown by the SP263 antibody, especially in the Reed-Sternberg cells. Since pembrolizumab was recently fast-tracked by the FDA to treat Hodgkin’s lymphoma cases, RK-10-Cy5 will need to be compared with the pembrolizumab companion diagnostic 22C3. Due to the multinucleate characteristics of the RS cells, it would be easy to detect and quantify the number of RS cells in a given Hodgkin’s tissue based on PD-L1 expression and nuclei. Since RK-10-Cy5 shows such specificity for tumor, it could detect a wide range of PD-L1 expression and inform more precise diagnostic levels for treatment. It has been shown that there is an urgent need for a PD-L1 diagnostic that can precisely detect PD-L1 protein irrespective of the drug intended to be used – a sensitive assay such as RK-10-Cy5 could be used to achieve this. Detection of PD-L1 expression in whole blood and metastatic melanoma suggests that RK-10-Cy5 could also potentially be used to detect low amounts of circulating tumor cells that express PD-L1.in vivo imaging methods, but upon excision for IHC no PD-L1 was detected23. Tumor mutations over a given period of treatment may lead to fluctuating PD-L1 levels, and as such may need to be monitored routinely.Recent debates about the diagnosis of PD-L1 in patients highlight the need for refined methods of determining PD-L1 levels in the patient. By utilizing a peptide-based approach, we can detect all levels of PD-L1 with a high sensitivity and specificity. In a heterogeneous tumor, identification of PD-L1 expression using traditional methods may not be an accurate way of determining a binary IHC cutoff, but would rather require a wider range of diagnostic levels to determine optimal therapy. Recent studies have also shown tumors that express PD-L1 according to Supplementary Information


Photocatalysts have attracted increasing attention for their widespread environmental applications. Here, a classic and simple electrospun technique is used to directly fabricate a porous a tungsten oxide nanoframework with graphene film as a photocatalyst for degradation of pollutants. The as‐synthesized film simultaneously possesses substantial adsorptivity of aromatic molecules, extensive light absorption range, significant light trapping, and efficient charge carrier separation properties, which remarkably enhance photocatalytic activity. In the photodegradation of Rhodamine B, a significant photocatalytic enhancement in the reaction rate is observed, which has superior photocatalytic activity compared to other bare WO In this regard, it is still an imperative and challenging issue to develop new and ultra‐efficient visible‐light‐sensitive photocatalysts base on WO3.For the sustainable development of human society, the development of both pollution‐free technologies for environmental remediation and alternative clean energy supplies is an urgent task.TableIn previous research, nanomaterials using as photocatalysts are powder, which resulted in improved photocatalytic activity Table1. Thus3 nano‐framework, and which can completely show the superiority of the graphene. The GWF showed superior performance in the photocatalytic properties under visible light irradiation . The reason for the high photocatalytic activity was also investigated.In response to this call, we developed a simple, electrospun technique for synthesizing porous tungsten oxide nano‐framework with graphene film (GWF). As expected, graphene film can be exposed in the porous WO26 and polyvinyl pyrrolidone (PVP) solution as the precursor for electrospun. After annealing in air, the final porous nanoframework film was formed, which evolve from the PVP molecules volatilized. Figure6/graphene and PVP/WCl6 respectively, which as prepared by the electrospun technique. It is apparent from Figure 3 nanofibers in hybrid materials can be used as the nanoframework to support the integrality of architecture and act as pillars  and effectively separate graphene nanosheets. Specially, plentiful graphene nanosheets can be exposed in the GWF resulting from the WO3 nanofibers as the nanoframework. Compared with GWF, we synthesized the WO3 nanofibers without graphene nanosheets as shown in Figure S1 (Supporting Information). The morphology of the pure WO3 is uniform nanofibers with a diameter of around 100 nm.Graphene was introduced into WClFigure3 nanofibers increase the number of micro‐ and mesopores to enhance the specific surface area. It is not obviously observe the graphene nanosheets in this TEM magnification. Figure 3 was further verified by measuring the nitrogen adsorption/desorption isotherms . The GWF samples have a higher surface area comparing with the bare WO3. Typically, the Brunauer–Emmett–Teller (BET) surface area of GWF was 56.4 m2 g−1, total pore volume was 0.69 cm3 g−1, mesopore volume was 0.56 cm3 g−1, mesopore to total pore‐volume ratio was 0.81, and an average pore diameter was 8.2 nm . The surface area of GWF is higher than pure WO3 nanofibers result of graphene nanosheets and porous structure. The responding density functional theory (DFT) pore size distribution  exhibits a hierarchical pore structure that includes micro‐, meso‐, and macropores (>50 nm). The results are consistent with that of the TEM data. The pore size distribution of GWF samples is wide, indicating unordered mesopores. Clearly, the original unordered mesopores maintain their structure by heat treatment. The most remarkable features of the as‐synthesized GWF are the high volume of mesopores and the high specific surface area, as well as the high photocatalytic activity under visible light. To understand the growth mechanism and substantial factors of porous film, precursor were annealing at different temperatures (350–550°C) and their scanning electron microscopy (SEM) images and growth mechanism have been given in Figure S4 (Supporting Information). In the precursor, the graphene nanosheets were curled up in the nanofibers. When annealing at high temperature, the curled graphene nanosheets can be extended with the PVP molecular decomposite. Meanwhile, the WO3 nanofibers form stable the framework on the surface of graphene nanosheets. When the annealing temperature is lower 350 °C, the PVP molecular cannot be decomposite completely. As the annealing temperature is higher than 450 °C in air, the graphene nanosheets can be oxidized, as shown in Figure S4 (Supporting Information).It is interesting to see from the transmission electronic micrograph (TEM) image Figure2a of a3. The crystal structure of the WO3 and GWF was further characterized by X‐ray diffraction (XRD), as shown in Figure3 [200], [002], [020] and [202] planes (JCPDS no. 71‐0131) were detected along with the characteristic features of graphene. In addition, it is clear observed that the [002] in GWF was the characteristic peak of graphene. From the XRD patterns of bare WO3, no peaks associated with other compounds were detected. The GWF was calculated from the TGA in air . The sample showed a significant weight loss between 520 °C and 620 °C due to the decomposition of graphene, and the composite contains ≈10% graphene. Meanwhile, we fabricated different graphene concentrations from 5% to 15% in mass ratio in WO3 nanoframework structure. We found the higher and lower graphene concentration in hybrid materials cannot be form the stable porous film.Figure −1 assigned to the stretching vibration of tungsten atom with neighboring oxygen atoms [υ (OWO)] ].−1), which are assigned to the local defects/disorders and the 2sp graphitized structure, respectively. This means that greater ID/IG peak intensity ratio can be attributed to higher defects/disorders in a graphitized structure such as graphene. From the shape of peaks at ≈1350 and 1585 cm−1, it is determined that the graphene is not exposed to the surface of the film.The Raman scattering spectrum also confirms the formation of the GWF, showing the two strongest peaks at 673.3 and 800.6 cm−1; 50 mL) under visible‐light irradiation as a probe reaction, and the result is shown in FigureC/C0), the instantaneous concentration of RhB during the photodegradation normalized to the initial concentration C0, was proportional to the normalized maximum absorbance (A/A0), which was measured at λ = 554 nm at regular time intervals. It can be seen that only after 3 min irradiation 44.5%, 21.8%, and 4.6% of the initial dye was decomposed by GWF, bare WO3 nanofibers, and P25 as the photocatalyst, respectively. After 6 min of irradiation, only 18.2% of the initial dye remained in the solution for GWF used as the photocatalyst . In contrast, nearly 65.5% and 90.8% of the initial dye still remained for bare WO3 nanofibers and P25 used as the photocatalyst after 6 min irradiation. It could be observed that GWF framework structures exhibited more prominent visible‐light photocatalytic activity. Continued irradiation, after total 15 min irradiation, almost 100% of the RhB molecules were decomposed, but 48.6 and 82.3% RhB molecules were intact when bare WO3 nanofibers and P25 were used as the photocatalyst  showed poorer activity in the photodegradation compared to the GWF . Accordingly, the exposed graphene plays an important role in the photodegradation, and the adsorption of RhB molecules contributed to the enhanced photoactivity.The photocatalytic activities of GWF (50 mg) were evaluated using the photodegradation of Rhodamine B  = kt, where k is the apparent rate constant and t is the irradiation time.k for GWF and bare WO3 were 0.2846 and 0.071 min−1, respectively. Furthermore, we note that the activity of GWF was ≈32 times those of P25 (k = 0.0089 min−1) and higher than those of other reported TiO2 and TiO2/graphene composites.The kinetics of the degradation reaction were fitted to a pseudo‐first‐order rate law at low dye concentration using the equation ln , the typical electrochemical impedance spectra were presented as Nyquist plots. It is observed that the semicircle of GWF in the plot is shorter than bare WO3. This result is indicated a decrease in the solid state interface layer resistance and the charge transfer resistance on the surface of GWF. Overall, both the multiple reflection effect on visible light in porous structures and electron‐accepting and ‐transporting properties of graphene in the composite films could contribute to the considerable improvement in photodegradation.We proposed 33 nanoframework with graphene film photocatalyst with high performance has been successfully and directly produced via the electrospun method. As demonstrated in the present case study, in the GWF, i) plentiful exposed graphene nanosheets can great adsorb RhB molecules via π–π stacking, ii) ultra‐efficient utilization rate of visible‐light irradiation resulting from a multi reflection effect of porous structures, iii) the reduced recombination rate of electrons and holes, and iv) 2D film structure can suitable for adsorption of RhB molecules and charge carriers transportation. A significant photocatalytic enhancement in the reaction rate was observed with GWF, which has superior photocatalytic activity to other bare WO3 and TiO2 materials under visible light irradiation. This remarkable result is anticipated to open new possibilities in the application of WO3 and graphene composites as the photocatalysts in environment remediation. And the preparation methods can be directly used in industrialized production to solve environmental problems occurring nowadays.In summary, highly porous WO4All solvents and chemicals were of analytical grade, purchased from Beijing Chemical Reagent Factory, and used without further purification.Synthesis of Highly Porous Graphene/Tungsten Oxide Nanoframework: Graphene has been prepared by micromechanical cleavage in which highly oriented pyrolitic graphite is peeled using Scotch tape. In the first step, 0.3 g WCl6  was dissolved in 2.3 g ethanol, and this mixed solution was stirred for 30 min. Then 0.003 g graphene was added to above solution and ultrasonic dispersed for 20 min. Second, 0.4 g polyvinylpyrrolidone (PVP)  was dissolved in 2.2 g N,N‐dimethylformamide (DMF)  and stirred for 30 min. Then the two prepared solutions were mixed together and stirred for 2 h. Thus, a viscous gel of PVP/graphene/WCl6 composite solution was obtained. As for typical electrospun process, the spinneret had an inner diameter of 0.6 mm. Grounded aluminum strips (260 cm in width) with parallel gaps of about 1 cm were used as the collectors. A distance of 15 cm and a direct current voltage of 20 kV were maintained between the tip of the spinneret and the collector. After electrospinning, the fibers were heated from room temperature to 450 °C at a rate of 2.5 °C min−1, and then held at 450 °C for 2 h in air.3 NanofibersSynthesis of Bare WO: In the first step, 0.3 g WCl6 was dissolved in 2.2 g ethanol, and this mixed solution was stirred for 30 min. Second, 0.4 g PVP was dissolved in 2 g DMF and stirred for 30 min. Then the two prepared solutions were mixed together and stirred for 2 h. Thus, a viscous gel of PVP/WCl6 composite solution was obtained. As for typical electrospinning process, the spinneret had an inner diameter of 0.6 mm. A distance of 15 cm and a direct current voltage of 20 kV were maintained between the tip of the spinneret and the collector. After electrospinning, the fibers were heated from room temperature to 450 °C at a rate of 2.5 °C min−1, and then held at 450 °C for 2 h in air.Characterization: The samples were characterized using X‐ray diffraction , scanning electron microscopy , and transmission electronic micrograph , nitrogen adsorption/desorption . The Brunauer‐Emmett‐Teller (BET) method was utilized to calculate the specific surface area using the relative pressure from 0.05 to 0.3. The pore volume (VP) and pore diameter distribution (DP) were derived from the adsorption branches of the isotherms by the Barrett‐Joyner‐Halenda (BJH) model. Thermogravimetric analysis (TGA) were carried out on a DTG‐60H thermogravimetric analyzer with a heating speed of 10 °C/min under air in a flow of 50 mL min−1. Raman spectroscopy (JY‐HR800 micro‐Raman). EIS measurements were performed by applying an AC voltage with 5 mV amplitude in a frequency range from 1 to 1 MHz .Catalytic Activity Testing: Photocatalytic decomposition of Rhodamine B (RhB) was carried out in a beaker containing a suspension of 50 mg sample in 50 mL RhB solution (20 mg L−1) under visible light irradiation. The visible light was taken from xenon lamp with the intensity at wavelength of 420 nm (250 W). During the photodegradation, the temperature was kept at 0 °C. Before the irradiation, the suspensions were kept in the dark for 60 min to ensure the adsorption/desorption equilibrium. At given time intervals (90 s), 3 mL aliquots were sampled and filtered to remove the catalysts. The filtrates were analyzed by recording the variations of the absorption band maximum (554 nm) in the UV–vis spectrum of RhB using a TU‐1901 spectrophotometer.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


MET proto-oncogene (MET) gene amplification.Cancer cells can achieve immune evasion by expressing the programmed death receptor 1 ligand (PD-L1) on the cell surface. Blockade of the receptor (PD-1) can avert this evasion. Here we aim at investigating PD-L1 expression in erlotinib-resistant lung cancer cells with MET gene amplification. PD-L1 mRNA (qPCR) and protein (flow cytometry) expression was investigated after treatment with MET and mitogen-activated protein kinase (MAPK) targeting drugs .We employed an erlotinib-resistant NSCLC cell line with MET gene amplification. Targeted inhibition of MET significantly decreases both gene and protein expression of PD-L1. Further, we demonstrate that inhibiting MAPK also results in a significant decrease in PD-L1 expression. Taken together these results show that expression of PD-L1 in the erlotinib-resistant cell line is associated with MET activity, and the downstream MAPK pathway.We demonstrate that PD-L1 expression is increased in erlotinib-resistant non-small cell lung cancer (NSCLC) cells with MET gene amplification and that the increase can be averted by targeted inhibition of MET.Our results demonstrate that PD-L1 expression is increased in erlotinib resistant NSCLC cells with  EGFR) of patients with adenocarcinoma histology. Today, treatment with EGFR-targeting tyrosine kinase inhibitors (TKIs), like gefitinib and erlotinib, is part of the routine treatment of patients harboring these mutations. This change in treatment has markedly increased the survival in this subgroup of patients [EGFR T790M mutation, MET proto-oncogene (MET) gene amplification, development of epithelial to mesenchymal transition (EMT), and transition to a small cell lung cancer (SCLC) phenotype [Worldwide, lung cancer is one of the most frequent cancers and mortality is high. The vast majority of lung cancers are non-small cell lung cancers (NSCLC). Over the past decade several oncogenic drivers have been identified , though patients , though patients . Severalhenotype .Treatment of lung cancer with immunotherapy has gained much attention after promising clinical trials. Especially blockade of the pathway activated by the programmed death receptor 1 (PD-1) and its ligand PD-L1 is being massively investigated. PD-1 is expressed on the surface of T-cells, and upon activation by the ligand, PD-1 initiates inactivation of the cell . ExpressEGFR mutations in NSCLC [in vitro through the mitogen-activated protein kinase (MAPK) pathway, and inhibition of EGFR using a TKI correspondingly decreases expression of PD-L1 [in vivo [In vitro data suggest that the T790M resistance mutation is accompanied by increased expression of PD-L1 [Studies of PD-L1 expression have shown an association with activating in NSCLC –14. Thouin NSCLC . Activatof PD-L1 –18. This[in vivo . Despiteof PD-L1 , and a rof PD-L1 . If the PD-L1 in erlotinib-resistant cells. The cells acquired resistance through MET gene amplification, and we further wanted to investigate if targeting the new oncogenic driver and the downstream proliferative pathway would affect the PD-L1 expression.The aim of this study was to investigate expression of The erlotinib-resistant cell line HCC827ER was generated over approximately 4 months where the erlotinib concentration was gradually increased to a maximal concentration of 5 μM. A full description of the resistant cells is presented in Jakobsen et al. .MET and PD-L1 by qPCR at each erlotinib concentration during the establishment of HCC827ER. We observed that MET gene expression initially decreased but started increasing at approximately 200 nM , PD-L1 expression had exceeded the expression level of the parental cells.We investigated the gene expression of y number . We obsePD-L1 gene expression.We used flow cytometry to investigate the expression of PD-L1 protein on the surface of the cells. As can be seen from Figure PD-L1 in erlotinib sensitive cells [MET and PD-L1 gene expression was also present in erlotinib-resistant cells. We therefore wanted to investigate if inhibition of MET, using a MET-targeting TKI (crizotinib), affected the expression of PD-L1 in these cells.Previous studies indicate a general link between receptor tyrosine kinase (RTK) activity and expression of ve cells , 17, 22.The viability of HCC827ER cells following treatment with crizotinib has previously been determined . We foun5 μM erlotinib showed a tendency towards decreasing PD-L1 gene expression (data not shown), but this effect was not significant and neither was the trend seen on the protein expression see . CombineThese results demonstrated that inhibiting MET using crizotinib resulted in decreased PD-L1 expression.MET gene amplification, and we saw that crizotinib inhibited phosphorylation of MAPK, we wanted to investigate if PD-L1 expression in HCC827ER was dependent on the MAPK pathway. Cells were treated with the MAPK inhibitor SCH772984. Cell viability was only mildly affected by the inhibitor at concentrations up to 5 μM  had a pronounced increase in PD-L1 protein expression as compared to the original HCC827ER cell line, while the opposite was the case for the EMT clones (clone 4 and 10).We wanted to investigate how PD-L1 expression differed in these clonal subtypes. We measured R Figure . There wThese results further strengthened the hypothesis of a correlation between MET activity and increased PD-L1 expression, while PD-L1 expression seemed to be lowered in EMT clones.EGFR mutational status and in vitro data have confirmed a regulatory function of EGFR [MET and we further demonstrate that expression of PD-L1 increases as expression of MET increases. Treatment with the MET-directed TKI crizotinib decreases expression of PD-L1. Further, targeting MAPK, downstream in the signaling pathway of MET, also effectively decreases expression of PD-L1. These results suggest an association between MET activity and PD-L1 expression.Immune evasion is one of the hallmarks of cancer . One way of EGFR , 17. Tho of EGFR . We founIn vitro RNAi studies and/or in vivo models should be used to investigate expression of PD-L1 in MET knock-out models to present evidence of MET-regulated PD-L1 expression. Furthermore, the biological effect of the increase in PD-L1 expression should be investigated in the presence of a functional immune system.Our results are in accordance with previous findings of RTK regulated PD-L1 expression and its involvement with MAPK , 22, 25.MET-amplified and EMT clones [MET-amplified clones have a markedly higher expression of PD-L1 than does the EMT clones. Lack of induced PD-L1 expression in EMT clones contradicts previous research on the subject. Generally studies have shown that the mesenchymal phenotype (intrinsic or induced by EMT) is associated with increased PD-L1 expression in different cancers [The erlotinib-resistant cell line investigated in the present study consists of subclones that can be divided into T clones . When in cancers –32. In l cancers , we did  cancers . A possiThe data presented here suggest that erlotinib-resistant cells increase the expression of PD-L1 through a mechanism involving MET and MAPK activation. Treatment with MET inhibitors in relevant cases could possibly prevent PD-L1 expression and thereby immune evasion. Furthermore, the PD-L1 fluctuations observed here, urges clinicians to exercise caution when evaluating the use of immunotherapy after TKI-treatment, based on PD-L1 measurements on biopsies taken at the time of diagnosis.The cell line HCC827 was purchased from ATCC. An erlotinib-resistant HCC827 cell line (HCC827ER) was generated as previously described . FurtherFor all mRNA and flow cytometry experiments cells were plated in 6-well plates and incubated for 24 h . After incubation the cells were treated with drug and incubated for 72 h prior to harvest.The inhibitors erlotinib, crizotinib, and SCH772984 were all purchased from Selleckchem.® AQueous Non-Radioactive Cell Proliferation Assay, Promega) following the manufacturer's instructions. Colorimetric measurement was performed using a Multiscan Ascent plate reader (Thermo Electron Corporation).For cell viability studies cells were plated in 96-well plates (5000 cells/well) and incubated for 24 h. Cells were treated with drug as indicated and incubated for 72 h prior to viability assessment. Cell viability was tested using an MTS assay (CellTiter 96® 2.0 Fluorometer (Thermo Scientific). 30 μg protein was resolved on 4–12% Bis-Tris gels (Life Technologies) and transferred to a PVDF membrane (Thermo Scientific). Membranes were blocked in 1X TBST with 5% non-fat dry milk (protein-specific antibodies) or 5% BSA (phospho-specific antibodies). For detection the following primary antibodies and dilutions were used: anti-EGFR , anti-EGFR phospho-Tyr1173 , anti-Akt , anti-Akt phospho-Ser473 , anti-MAPK , anti-MAPK phospho-Thr202/Tyr204 , and anti-Histone H3 . Antibodies were diluted in either 1X TBST with 5% non-fat dry milk or 5% BSA corresponding to the blocking agent. Goat anti-mouse  and goat anti-rabbit secondary antibody  were diluted in 1X TBST with 5% non-fat dry milk and used for detection of total-MET and the remaining proteins, respectively. The membranes were added SuperSignal Dura West Chemiluminescent Substrate (ECL) (Thermo Scientific) followed by development in an ImageQuant LAS 4000 scanner .Protein was harvested using Lysis Buffer 17 (R&D Systems) supplemented with 10 μg/mL of each inhibitor Pepstatin, Leupeptin, and Aprotinin. Harvest was performed according to manufacturer's protocol. The protein concentration was determined using the QubitFor RNA analysis cells were harvested using Nunc Cell Scrapers (Thermo Scientific), centrifuged, and resuspended in 350 μL RLT buffer (Qiagen). Total RNA was extracted using RNeasy mini kit on a QIACube instrument (Qiagen). RNA concentration was determined using the NanoDrop 2000 (Thermo Fisher Scientific). 200 ng RNA was used for cDNA synthesis. cDNA was synthesised in a 20 μL reaction consisting of 2.5 μM Oligo(dT) (DNA Technology), 1 mM of each dNTP (VWR), 2.5 units/μL MulV Reverse Transcriptase, 1 units/μL RNase inhibitors, 1xPCR buffer, and 6.25 mM MgCL2 . Quantitative PCR (qPCR) was performed using LightCycler 480 SYBR Green I Master on the LightCycler 480 platform (Roche).Five reference genes  were tested and compared using the Normfinder Software . Among tFor the experiments presented the following primer sequences, concentrations and annealing temperatures were applied:ACTB: forward 5′- GGCGCCACCACCATGTA CCCT-3′, reverse 5′- AGGGGCCGGACTCGTCATACT-3′, 0.25 μM, 68°C.PD-L1: forward 5′- GGTGGTGCCGACTACAA GCGA-3′, reverse 5′-TGACTTCGGCCTTGGGGTAGC-3′, 0.25 μM, 64°C.MET: forward 5′- TGGAGACACTGGATGGGAGT-3′, reverse 5′-CAGCGCGTTGACTTATTCAT-3′, 0.25 μM, 60°C.All primers were purchased from Eurofins Genomics. Gene expression was measured and calculated using the Lightcycler 480 instrument (Roche) and the second derivative max method using standard curves.All experiments were performed in biological triplicates, except from the analysis of RNA from the resistance development Figure  and 1B aCells were detached from wells using PBS/0.5% BSA/2 mM EDTA, washed once in staining buffer (PBS/0.5% BSA/0.09% sodium azide), and kept on ice. Non-specific antibody binding was blocked with 10% mouse serum  for 15min at 4°C. Samples were then stained with Live/Dead fixable dye near-IR (Life Technologies) and mouse anti-human PD-L1 PE-Cy7  in stain buffer for 30 min (in the dark at 4°C). Cells were washed in stain buffer, fixed in PBS/0.9% formaldehyde (Sigma-Aldrich), and analysed immediately on an LSR Fortessa flow cytometer (BD Biosciences).Compensation was done using single stained beads, for PD-L1 PE-Cy7 Comp Beads Plus (BD Biosciences) were used and for Live/Dead nIR ArC Amine Reactive Compensation beads (Life Technologies) were used. Data was analysed and figures were made using FlowJo 10.0.7 for Mac . For gating strategy, see All flow cytometry experiments were performed using the LSR Fortessa flow cytometer at the FACS Core Facility, Aarhus University, Denmark.All graphs and statistics are produced in Stata 13 .Gene and protein expression data from TKI experiments is collected from three individual setups. In each experiment data was normalised to an untreated control, and the mean of this normalised data is presented in graphs.t-test was used to assess the difference between two groups, and p-values < 0.05 were considered significant.All data is presented as mean ± standard deviation (SD) . The two-tailed Student's


Broadband on-chip optical frequency combs (OFCs) are important for expanding the functionality of photonic integrated circuits. Here, we demonstrate a huge local optical nonlinearity enhancement using graphene. A waveguide is decorated with graphene by precisely manipulating graphene’s area and position. Our approach simultaneously achieves both an extremely efficient supercontinuum and ultra-short pulse generation. With our graphene-decorated silicon waveguide (G-SWG), we have achieved enhanced spectral broadening of femtosecond pump pulses, along with an eightfold increase in the output optical intensity at a wavelength approximately 200 nm shorter than that of the pump pulses. We also found that this huge nonlinearity works as a compressor that effectively compresses pulse width from 80 to 15.7 fs. Our results clearly show the potential for our G-SWG to greatly boost the speed and capacity of future communications with lower power consumption, and our method will further decrease the required pump laser power because it can be applied to decorate various kinds of waveguides with various two-dimensional materials. The synergetic effect of highly nonlinear materials and photonic crystal structures  have emerged because these materials can be monolithically integrated with electrical circuits by using the nanometers-scale process rule. Indeed, the remarkable advantages are not limited to functionality or scalability, but also provide excellent properties such as a high refractive index and high third-order nonlinearity  is 4.2 × 10−18 m2/W in bulk silicon−13 m2/W for graphene−17 m2/W, which is one order of magnitude higher than that of a SWG. Moreover, both the flat material dispersion in the complex refractive index from the visible to terahertz range and ultra-fast response due to the unique gapless Dirac cone2021n2 as the Fermi level increases in the high-doping regime. The SPM effect with a hybrid structure of graphene on a silicon rib-waveguide has recently been characterizedHere, we focused on graphene, a two-dimensional material, for obtaining a huge enhancement of the nonlinear optical properties. The waveguide is decorated with graphene by precisely manipulating the graphene’s area and position. In this study, we achieved locally enhanced on-chip OFC generation with the G-SWG. The local Kerr coefficient nP) of 100 to 300 μm. Since the SWG itself also has strong dispersion and negligible third-order nonlinearity, this entrance margin contributes as a regulator for controlling the chirping of incident light pulse. Note that the total device length is fixed at 2 mm. Once the ultra-short pulse reaches the G-SWG in the center of the device, a gigantic nonlinear effect is induced on the SWG, which can be observed as spectrum broadening in the frequency domain through the output side the SSC. GrL) × 30 μm graphene pattern was defined by O2-plasma etching . The measurement range in our spectral analyzer is limited to less than 1700 nm. The on-chip OFC intensities of each graphene length in Gr is less than 50 μm, the on-chip OFC bandwidth is little different from that without the graphene. However, the broadband on-chip OFC spectrum is generated when the graphene length LGr is more than 100 μm. We found that with current experimental setup, the widest on-chip OFC spectrum (1050–1700 nm) at −60 dB level is generated when the graphene length GrL is 200 μm and graphene position P is 300 μm from the SWG incident side. Since the band gap of 1.1 eV in silicon translates into an absorption edge of 1100 nm, the on-chip OFC spectrum spans up to the absorption edge. The wavelength dependent loss spectrum can be found in our previous paperPrecise manipulation of the graphene length on the SWG plays an important role in achieving the large nonlinear phase shift. We experimentally investigated the characteristics of the broadband on-chip OFC spectra with and without the graphene on the SWG. By using the nonlinear Schrödinger equation with the split-step Fourier method30Furthermore, we experimentally investigated the input laser power dependence of the on-chip OFC intensity. GrL of 200 μm as shown in P from 100 to 600 μm. The difference in laser pulse widths in front of the graphene on the SWG is small, while the difference in the phases is large  lasers as shown in GrL and position P of the graphene on the SWG, the graphene increases the effective Gr is 200 μm. When it is more than 200 μm, the on-chip OFC bandwidth becomes smaller by TPA. On the other hand, the laser pulse widths before Area 2 for P from 100 to 300 μm differ little, while the difference in phases is large. The phase difference influences the laser pulse width in Area 2. The simulation results are reproduced quite well by the experimental results. Although the on-chip OFC becomes wider for P of 600 μm, the laser pulse splits. From the experimental and simulation results, it is found that the widest and highest on-chip OFC spectrum without splitting of the laser pulse can be generated for P of 300 μm from the SWG incident side and LGr of 200 μm. Our work indicates the feasibility of a new compact and broadband light source using graphene and silicon-photonics technology. Our method has versatility because the silicon and graphene can be replaced with transparent materials  and two-dimensional materials , respectively. By using these attractive materials, the required pump laser power can be further decreased in integrated photonic circuits. Although SWGs show the influence of TPA with a pump pulse light source at the wavelength of 1.5-μm, Si3N4 waveguides32We have experimentally and numerically demonstrated the enhancement of broadband on-chip OFC generation by decorating an SWG with graphene. The results show that, by precisely controlling length A  of the electric field of short pulses along the length z of a nonlinear medium is described by the generalized nonlinear Schrödinger equationThe propagation of the temporal envelope α is the linear propagation loss, βm is the m-th order dispersion coefficient, βTPA is the TPA coefficient, γ0 = ω0n2/cAeff is the nonlinear Kerr coefficient, ω0 is the optical frequency, n2 is the nonlinear index coefficient, and Aeff is the effective area. The effective n2,Gr of 10−11~10−17 m2/W18202122n2 in graphene between theory36n2_Gr of 1 × 10−13 m2/W and two photon absorption coefficient β2_Gr of 1 × 10−7 m/W in graphenen2_Si of 4.2 × 10−18 m2/W and 2_Siβ of 8 × 10−12 m/W in bulk silicon−17 m2/W.where How to cite this article: Ishizawa, A. et al. Optical nonlinearity enhancement with graphene-decorated silicon waveguides. Sci. Rep.7, 45520; doi: 10.1038/srep45520 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Pseudomonas syringae pv. pisi effector AvrRps4. AvrRps4 is processed in planta into AvrRps4N (133 amino acids), homologous to the N-termini of other effectors including the native P. syringae pv. tomato strain DC3000 effector HopK1, and AvrRps4C (88 amino acids). Previous data suggested that AvrRps4C alone is necessary and sufficient for resistance when overexpressed in heterologous systems. We show that delivering AvrRps4C from DC3000, but not from a DC3000 hopK1- strain, triggers resistance in the Arabidopsis accession Col-0. Delivering AvrRps4C in tandem with AvrRps4N, or as a chimera with HopK1N, fully complements AvrRps4-triggered immunity. AvrRps4N in the absence of AvrRps4C enhances virulence in Col-0. In addition, AvrRps4N triggers a hypersensitive response in lettuce that is attenuated by coexpression of AvrRps4C, further supporting the role of AvrRps4N as a bona fide effector domain. Based on these results we propose that evolutionarily, fusion of AvrRps4C to AvrRps4N may have counteracted recognition of AvrRps4N, and that the plant RPS4/RRS1 resistance gene pair was selected as a countermeasure. We conclude that AvrRps4 represents an unusual chimeric effector, with recognition in Arabidopsis by RPS4/RRS1 requiring the presence of both processed effector moieties.Bacterial effector proteins secreted into host plant cells manipulate those cells to the benefit of the pathogen, but effector-triggered immunity (ETI) occurs when effectors are recognized by host resistance proteins. The RPS4/RRS1 pair recognizes the  An important component of the plant immune system relies on the detection of pathogen-derived effectors by immune receptors called resistance proteins. Bacterial pathogens inject effectors into the host cell via a dedicated secretion system to suppress defenses and to manipulate the physiology of the host cell to the pathogen's advantage. Usually, a single resistance protein recognizes a single effector, but an increasing number of exceptions and elaborations on this one-to-one relationship are known. The plant Arabidopsis uses a pair of resistance proteins, RRS1 and RPS4, to detect the effector AvrRps4. After injection into the cell, AvrRps4 is cleaved into two protein parts, and it had been assumed that only the C-terminal part needs to be present to trigger RPS4/RRS1. We show here that both AvrRps4 parts are required for triggering resistance in Arabidopsis, and that the N-terminal part, which previously had been assumed to only function in effector secretion into the host cell, in fact on its own has some functions of an effector. This conclusion is supported by the observation that the N-terminal part of AvrRps4 is sufficient to trigger resistance in lettuce. The fusion of the two AvrRps4 parts may have arisen to counteract plant defenses. Plants deploy a multilayered immune system to defend against invading pathogens –3. The fPseudomonas syringae pv. pisi [N, amino acids 1–133) shares 75% identity with the N-terminus of HopK1, a native effector of the Pseudomonas syringae pv. tomato strain DC3000 (DC3000) that is predominantly used for bacterial delivery of effectors in Arabidopsis studies. HopK1 is a 338 amino acid protein that contributes significantly to the virulence of DC3000. Similarly to AvrRps4, HopK1 is processed in plant cells between amino acids Gly133 and Gly134 [C (amino acids 134–221) is a coiled-coil protein which interacts with WRKY proteins, a plant-specific class of transcription factors of which several have been implicated in defense against pathogens [The effector AvrRps4, originally identified in pv. pisi , has curpv. pisi . The prod Gly134 . AvrRps4athogens –14. The athogens . RRS1 woathogens ,17.Ralstonia solanacearum effector PopP2 and an unknown Colletotrichum higginsianum effector [C was sufficient to trigger a hypersensitive response (HR) in turnip [C interacts, directly or indirectly, with the WRKY domain of RRS1 as well [RRS1 and RPS4 belong to the Toll/Interleukin-1 Receptor—Nucleotide-Binding—Leucine-Rich Repeat (TNL) class of resistance proteins ,18 and, effector ,17. As aeffector  and TNL effector ,13,20. Pn turnip , and thaN. benthamiana, or in Arabidopsis when expressed from its native promoter [Additional components of RPS4/RRS1 activation by AvrRps4 are its interaction in the cytoplasm and nucleus with ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), a positive regulator of both basal resistance and ETI, and the interaction between EDS1 and RPS4 ,22. EDS1promoter ,26. Thispromoter  and a supromoter  to the mpromoter .N directly interacts with EDS1, suggesting that AvrRps4N possesses effector functions outside of it being a signal peptide for secretion and a transit peptide for chloroplast localization. In addition, we endeavored to determine the molecular basis of AvrRps4 triggering RPS4/RRS1 by introducing bacterially delivered AvrRps4 to ascertain function of separate domains of this effector. We provide evidence that HopK1N and AvrRps4N at natural expression levels redundantly contribute to RPS4/RRS1-mediated immunity in the presence of AvrRps4C. Consequently, both AvrRps4C and either HopK1N or AvrRps4N are required for the full ETI-inducing activity of AvrRps4 in Arabidopsis. Additionally, AvrRps4N increases bacterial virulence when overexpressed in Col-0, and triggers a robust HR when expressed in cultivars of Lactuca sativa (lettuce) while AvrRps4C triggers no such response. We propose that separate interactions of AvrRps4N with EDS1 and of AvrRps4C with RRS1 are components of RPS4/RRS1 activation.Here we show that AvrRps4in vivo and in vitro, indicating that the interaction is direct and possibly mediated by AvrRps4N [N. Consistent with previous biochemical fractionations of HA-AvrRps4-expressing cells [N transiently expressed in Nicotiana benthamiana was detected in the cytoplasmic microsomal fraction. In vivo co-immunoprecipitation of AvrRps4N and EDS1 in the microsomal fraction confirmed that AvrRps4N is sufficient for this interaction  and absence (AvrRpm1SP-AvrRps4N and AvrRpm1SP-AvrRps4C) of nuclear signal , which like Col-0 possesses functional RPS4 and RRS1 for AvrRps4 recognition [Pseudomonas fluorescens Effector-to-Host Analyzer system (Pfo EtHAn) [N-AvrRps4C chimera indeed caused a strong HR, whereas an AvrRps4N-HopK1C chimera did not. To confirm these results, in planta bacterial growth assays were performed using chimeras delivered from a DC3000 hopK1- mutant into Ws-0. The HopK1N-AvrRps4C chimera triggered resistance similar to full-length AvrRps4, whereas AvrRps4N-HopK1C supported bacterial growth similar to empty vector and HopK1   the AvrRnd HopK1 . Additio AvrRps4 . Taken tN may substitute for mislocalized AvrRpm1SP-AvrRps4N delivered from DC3000 in bacterial growth assays. To address this question, we introduced all of our AvrRps4 constructs into DC3000 hopK1- [hopK1- failed to reconstitute this strong resistance phenotype , and five times higher in mock- or Dex-treated line 8 (N8) than in mock- or Dex-treated Col-0, whereas the growth of wild-type DC3000 was similar in all plants tested  triggering of resistance in the presence of AvrRps4C in Arabidopsis Col-0, 2) serving as a virulence factor in the absence of AvrRps4C in Col-0, and 3) triggering defense responses in lettuce. These functions of AvrRps4N are consistent with functions of an effector and with its interaction with the positive regulator of immunity EDS1 shown here. Sequence conservation across the entire N-terminal moieties of AvrRps4, HopK1, and the Xanthomonas campestris pv. vesicatoria effector XopO led us to hypothesize that AvrRps4N has functions that extend beyond a T3SS signal and chloroplast transit peptide, which usually are encoded in the first 20–50 amino acids of a peptide [We have demonstrated that AvrRps4 is a bipartite protein, where AvrRps4 peptide . Indeed, peptide ,36. Whil peptide , have beN and is predicted to translocate to chloroplasts, although such predictions may be an artifact of prediction models and the similarity of T3SS and chloroplast targeting sequences [N. benthamiana and Arabidopsis even with N-terminal fusions such as those to AvrRpm1SP. Since such fusions are predicted to block chloroplast import we propose that a subpool of AvrRps4 is processed in the cytoplasm, and that AvrRps4 has multiple virulence targets in multiple compartments. Based on our results it appears that AvrRps4-triggered ETI is initiated in the cytoplasm or nucleus. This does not exclude virulence functions of chloroplast-localized AvrRps4 [Both AvrRps4 and HopK1 are processed in plant cells between amino acids Gly133 and Gly134. It was suggested that this processing occurs exclusively in chloroplasts and that the N-terminal 20 amino acids encode a chloroplast transit peptide . HopAQ1 equences . We obse AvrRps4 .N. benthamiana cells [C triggered measurable resistance in Col-0. To detect bacterial delivery and effector localization in the host cell, effector constructs were tagged with a 13-amino acid split fluorescent protein sequence and were delivered into transgenic plants expressing a split superfolder GFP protein that self-assembles. The strain used for effector delivery, the DC3000 variant Pst CUCPB5500, expresses HopK1 [C by the highly soluble superfolder GFP allowed diffusion of this construct into the nucleus [C in the presence of HopK1N or AvrRps4N triggers resistance.Consistent with results presented here, a recent study also failed to detect chloroplast localization of full-length or processed AvrRps4 in Arabidopsis or na cells . Intereses HopK1 . Interes nucleus . These rC fused to only a 50 amino acid signal peptide, but Xanthomonas XopAK is a homolog of HopK1C fused to a signal peptide lacking the 94 amino acid effector domain of AvrRps4N and HopK1N [N or HopK1N homologs, which presumably targeted hubs important to immunity such as EDS1. The interactions of these proteins with EDS1 may have triggered resistance on resistant plants, and successful bacteria evaded this resistance by terminal reassortment [C or HopK1C to suppress resistance. On the plant side, genes such as the RPS4/RRS1 pair arose to recognize the addition of AvrRps4C. Evidence for this include our data which show that AvrRps4N alone is sufficient to enhance bacterial virulence in Arabidopsis Col-0 when overexpressed whereas addition of AvrRps4C again makes Col-0 fully resistant to bacteria expressing AvrRps4N and AvrRps4C. The virulence function of AvrRps4N was occasionally discernible in in planta bacterial growth experiments using DC3000 hopK1- but was not significant overall, as was reported previously [N delivery and functional redundancy, as observed with several groups of sequence-unrelated DC3000 effectors [N virulence effects at natural protein levels.Searches of NCBI’s protein sequence database using the BLAST algorithm  failed td HopK1N . We thersortment  with eiteviously . Low levffectors , likely N/HopK1N still exists in some plants. Indeed, we identified lettuce as one plant species which retains the ability to recognize AvrRps4N. Future work includes a large-scale screening of Arabidopsis accessions to identify those which can recognize AvrRps4N. In addition, it would be informative to determine whether AvrRps4C also counteracts the virulence function of AvrRps4N in an rps4/rrs1 background, or only attenuates the avirulence function of AvrRps4N. Our finding that AvrRps4N functions as an effector within plant cells suggests a possible contribution of this moiety when assaying candidate non-bacterial effector molecules by delivery with the Effector Detector Vector 3 (pEDV3), which enables bacterial secretion of effectors into host cells using the first 136 amino acids of AvrRps4 [N encoded in pEDV3 should not interfere with assays using Pseudomonas pathogens which contain HopK1 or AvrRps4 natively.A prediction of this model is that the capacity to recognize AvrRps4 AvrRps4 . HoweverN is involved in resistance-triggering in addition to serving as a virulence factor. We previously proposed that EDS1 has roles both upstream and downstream of resistance protein activation [N with EDS1 as a guardee may constitute the first step of RPS4/RRS1 activation but is insufficient to trigger immunity in the absence of concurrent RRS1 activation by AvrRps4C. Alternatively, AvrRps4N through interactions with EDS1 boosts EDS1 immune signaling in the presence of AvrRps4C-activated RPS4/RRS1. Based on the virulence function of AvrRps4N we favor the first model [An outstanding question is how AvrRps4tivation . Interacst model . While ain vitro pull-down assays, GST- EV, AvrRps4N, AvrRps4C, and AvrRps4FL were cloned into pDEST15 via Gateway recombination as previously described [EDS1 was cloned into pET28a by inserting amplified EDS1 into SalI/XhoI using restriction digestion  and HA-AvrRps4 N-terminus or HA-AvrRps4 C-terminus (strain C58C1) were infiltrated into N. benthamiana leaves at 0.05 OD. After 48 hours 1 g of tissue was collected and ground in 2 ml buffer H . Lysate was centrifuged at 1600 x g for 20 minutes. Supernatant was then spun at 100,000 x g for 1 hour to pellet microsomes. Pellets were resuspended in 1 ml buffer H with 1% NP-40. Protein concentration was measured by Bradford assay, and samples were adjusted to 0.25 mg/ml protein. 20 μl of HA-conjugated beads (Sigma) were added to each sample and incubated at 4°C for 1 hour. Beads were washed three times with buffer H with 0.2% NP-40.For in vitro pull-down assays, BL21(DE3) competent cells were transformed with GST- EV, AvrRps4N, AvrRps4C, AvrRps4FL or His-T7-EDS1 individually (GST purification) or with His-T7-EDS1 and individual AvrRps4 constructs together (nickel purification). Overnight cultures from fresh colonies were used to inoculate 200 mL LB, incubating 3–4 hours at 37°C under selection. Expression was induced with 0.2 mM IPTG overnight at 22°C. Cultures were centrifuged 10 min at 3800 x g and resuspended in 20 mL TBS with 0.01% NP-40 and 1x cOmplete EDTA-free protease inhibitor (Roche) for GST purification, or equilibration buffer  for nickel purification. Working lysate was prepared by French press lysis followed by centrifugation. Nickel  3 days post-infiltration. For HR assays in lettuce, N-terminally HA-tagged AvrRps4 full-length, AvrRps4N, AvrRps4C, HopK1 full-length, HopK1N, HopK1C, and HA-pBA empty vector were transiently expressed in Lactuca sativa cv. Kordaat using A. tumefaciens C58C1 infiltrated at an O.D. of 0.3. Cell death phenotypes were visualized three days post-infiltration. Electrolyte leakage experiments were performed as described previously [2O, and vacuum-infiltrated. The six leaf disks were then transferred to a 20 mL GC vial containing 10 mL ddH2O. Conductivity was measured using a conductivity meter.AvrRps4-GFP-containing plasmids were moved from eviously . Lettuce4 cfu/mL densities. Leaf discs were recovered in triplicate samples and thoroughly ground in 10 mM MgCl2 and plated on Difco Pseudomonas Agar  containing appropriate antibiotics. Unless noted otherwise, statistical analyses of differential bacterial growth between treatments were performed using ANOVA with Holm correction for multiple comparisons.Plants were grown in short-day conditions as previously described . Arabidoin vitro secretion assays, bacteria were grown on plates containing appropriate antibiotics overnight. Cells were diluted to an O.D. of 2 x 108 cfu/mL into tubes containing 10 mL minimal media [in planta cleavage assays, Col-0 plants were infiltrated with 109 cfu/mL of wild-type DC3000 or DC3000 hopK1- containing full-length AvrRps4 with or without the AvrRpm1 signal peptide. Tissues were harvested at 0.5 and 7 hpi and ground in 8 M urea for Western blot analysis with HA antibody.For al media  at 19°C avrRps4 and PR1. ACTIN2 was used as an internal control. Oligonucleotide primer sequences used in semi-quantitative RT-PCR are listed in Reverse transcription PCR was conducted as described previously . BrieflyS1 FigN or AvrRps4C-GFP, but not with GFP alone, in N. benthamiana. For the GFP control, a GFP variant with an ER retention signal was used [In vitro interaction of His-T7-EDS1 with GST-AvrRps4N or GST-AvrRps4C, but not with GST alone, in E. coli. These experiments were repeated once with similar results.(A) Co-IP of microsomal Myc-EDS1 with GFP-AvrRps4was used . (B) In (PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig4 cfu/mL suspensions of wild-type DC3000 carrying the indicated constructs, and tissue was harvested in triplicate at the 0 and 3 day time points. Values are averages from three independent experiments with triplicate samples, and error bars denote standard deviation, with letters indicating statistically significant differences (P<0.0001).Col-0 plants were inoculated with 5x10(PDF)Click here for additional data file.S4 FigN, and AvrRps4C with or without the AvrRpm1 signal peptide were C-terminally tagged with GFP and expressed in N. benthamiana together with free RFP to mark the cytoplasm and nucleus. Cells were visualized after two days.AvrRps4 full-length, AvrRps4(PDF)Click here for additional data file.S5 FigIn planta bacterial growth analysis of DC3000 hopK1- secreting wild-type AvrRps4 or chimeric HopK1N-AvrRps4C. Ws-0, eds1-1 or rps4-21 plants were inoculated with 5x104 cfu/mL suspensions of bacteria. Values are averages from two independent experiments with triplicate samples, and error bars denote standard deviation, with letters indicating statistically significant differences (P<0.01).(PDF)Click here for additional data file.S6 FigProtein precipitated from the growth medium (left) and total bacterial pellets (right) were subjected to immunoblot analysis using HA (top) antibodies, and NPTII (bottom) antibodies to probe for a non-secreted protein as a control for cell lysis.(PDF)Click here for additional data file.S7 Fig9 cfu/mL of DC3000 or DC3000 hopK1- containing indicated effectors were harvested at given time points and subjected to immunoblot analysis using HA antibodies. Asterisks indicate full-length or processed C-terminally tagged AvrRps4 proteins, confirming that AvrRpm1SP-AvrRps4 fusion proteins are still processed in planta.Arabidopsis Col-0 leaves infiltrated with 10(PDF)Click here for additional data file.S8 FigavrRps4 and PR1 in transgenic plants expressing avrRps4N (N2 and N8) and wild-type Col-0 by semi-quantitative RT-PCR analysis. Expression of PR1 was inversely related with that of avrRps4N. Total RNA was isolated from 10-day-old seedlings grown on MS media containing 1 μM Dex (+) or mock (-). ACTIN2 was used as an internal control. (B) Protein levels of AvrRps4 and PR1 in transgenic plants expressing AvrRps4N (N2 and N8). Total protein was isolated from 3-week-old plants 24 hours after spraying with 50 μM Dex (+) or mock (-). GAPDH was used as a loading control.(A) Transcript levels of (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Meanwhile, the graphene-based LPFG SPR sensor exhibits excellent response characteristics and repeatability. Such a SPR sensing scheme offers a promising platform to achieve high sensitivity for gas-sensing applications.A graphene-based long-period fiber grating (LPFG) surface plasmon resonance (SPR) sensor is proposed. A monolayer of graphene is coated onto the Ag film surface of the LPFG SPR sensor, which increases the intensity of the evanescent field on the surface of the fiber and thereby enhances the interaction between the SPR wave and molecules. Such features significantly improve the sensitivity of the sensor. The experimental results demonstrate that the sensitivity of the graphene-based LPFG SPR sensor can reach 0.344 nm% The fiber optic surface plasmon resonance (SPR) sensor ,2,3 has To overcome these drawbacks, the fiber grating–assisted SPR sensors are designed by integrating metal overlays on the fiber gratings, which exhibits advantages over the modified fiber-based SPR sensors, such as preservation of the structural integrity and circular symmetry of the fiber . These pCompared to the metals, graphene ,18,19,202 laser. Then the LPFG SPR sensor is fabricated by coating Ag film on the LPFG surface and fixing it on a SiO2 substrate. Finally, the chemical vapor deposition (CVD)-grown graphene monolayer is covered onto the Ag film surface of the LPFG SPR sensor to form the graphene based LPFG SPR sensor. Its longitudinal section is shown in Λ and the length L of the LPFG are 600 μm and 1.5 cm, the diameter of the corroded fiber is 100 μm, the diameter D of the fiber core is 10 μm, and the thickness T of the Ag film is 50 nm.The structure of the proposed graphene-based LPFG SPR sensor is schematically shown in ith cladding mode, Λ is the grating period, ith SPR-FC modes, respectively. As illustrated in S of the sensor is defined as the ratio of the shift in the resonant wavelength to the change in the external refractive index surn expressed as:Once the light is coupled into the SPR-FC hybrid modes, it will decay immediately due to the scattering losses, which provides attenuation bands at the resonant wavelength in the transmission spectrum. Due to coupling with the SPR, the propagating constant of this hybrid mode is highly sensitive to the refractive index changes of the analyte. A change in the external refractive index induced by the variation of the gas concentration leads to the change in the propagating constant of the hybrid mode, thereby resulting in the variation of the resonant wavelength. Then the change of the refractive index can be measured by detecting the variation of the resonant wavelength. The sensitivity ith SPR-FC modes and the grating period of the LPFG. When the LPFG SPR sensor is further coated with a graphene layer, it is expected to increase the intensity of the SPR-FC modes at the fiber/metal interface and consequently improve the sensitivity of the sensor.This equation indicates that the sensitivity of the sensor is proportional to the effective refractive index of the 2 laser  with a computer control at a speed of 2.4 mm/s. The laser beam was focused to a spot with a diameter of ~50 μm. In our work, the length of the processing area is 1.5 cm with grating period Λ of around 600 μm. The LFBG processing area was then corroded to 100 μm in hydrofluoric acid for 12 min, followed by the thermal evaporation of 50 nm Ag film. The thickness of the depositing silver film on the fiber core can be controlled and monitored in real time, which is measured by a quartz crystal monitor in the thermal evaporation machine.The fabrication process of the proposed graphene-based LPFG SPR sensor is schematically demonstrated in 2 (70 mTorr) and 30 sccm CH4 (340 mTorr) gases at 1000 °C. A layer of PMMA thin film was coated on as-grow graphene on Cu foil as a transfer supporting layer by a spin coater. The graphene on the other side was removed by oxygen plasma processing. After completely removing Cu by the solution of FeCl3 and HCl, the PMMA-supported floating graphene film was rinsed with deionized water twice and transferred onto the top of the fabricated sensor probe fixed on the SiO2 substrate. It was then directly covered around the Ag film surface of LPFG SPR sensor to form a graphene-based LPFG SPR sensor. Finally, the supporting PMMA layer were removed by acetone treatment.Then, the large-area graphene monolayer was synthesized by chemical vapor deposition (CVD) method on a high purity copper foil , with flowing mixture of 3 sccm HThe experimental setup for the sensing system is schematically depicted in −1 (D band), 1582 cm−1 (G band), and 2691 cm−1 (2D band) [−1 corresponds to the graphitization of the sp2-bonded carbon atoms in the two-dimensional ordered hexagonal graphene. The peak of the 2D band at 2691 cm−1 is related to the inter-valley double-resonant Raman scattering. The high ratio of the peak intensities of the 2D band to the G band (ratio ≈ 2.5) indicates the Raman feature of the monolayer graphene. Meanwhile, the small peak of the D band near 1350 cm−1, corresponding to the disorderly carbon and defects, suggests the high quality of the graphene. The scanning electron microscopy images of the graphene-based LPFG SPR sensor are shown in 2 laser pulses, three loss peaks can be observed at 1512.8 nm, 1537.7 nm and 1569.1 nm in the considered spectral window, indicating the successful fabrication of the LPFG on the fiber core. This is due to the conversion of the fundamental core mode to the co-propagating cladding modes with different diffraction orders, which leaks out from the fiber and interacts with the sensing medium. Meanwhile, the loss peak located at 1537.7 nm possesses the minimum value of transmission losses of −18 dB, which is deeper than that of the other two peaks. It means that the coupling efficiency of this loss peak is larger compared to the other two peaks, which provides a stronger interaction between the cladding mode and sensing medium, and thereby results in a higher sensitivity. Therefore, we mainly focus on analyzing the sensing performance of the sensors using this loss peak (1537.7 nm) in the following context.To verify the successful inscription of the LPFG on the fiber core, the transmission spectra of the fiber before and after fabricating LPFG are provided in We first investigate the spectral features of the three kinds of fabricated sensors, i.e., the LPFG sensor, the Ag-coated LPFG SPR sensor and the grapheme-based LPFG SPR sensor. It is found that the interaction between the incident light and the sensor can be enhanced by covering the Ag film and monolayer graphene onto the surface of the LPFG. The transmission spectra of these sensors are recorded and shown in y-axis error bars indicate that the signals of the sensors are very stable. The calculated sensitivities of these sensors are 0.116 nm%−1, 0.262 nm%−1, and 0.34 nm%−1, with corresponding detection limits of 0.086%, 0.038%, and 0.029%, respectively, considering that the resolution of the optic spectrometer is 0.01 nm [Then the sensitivities of the LPFG sensor, Ag-coated LPFG SPR sensor and graphene-based LPFG SPR sensor are evaluated by exposing the sensors directly to methane with various concentrations. The transmission spectra of the three kinds of sensors are shown in  0.01 nm ; these vresT  of the sensor was measured to be 50 s, which is comparable to that of the traditional LPFG gas sensor. However, the recovery time recT  was nearly 65 s. In addition, the signal can recover to the initial resonance wavelength with a minimum standard deviation of less than 0.05 nm, which demonstrates that the graphene-based LPFG SPR sensor possesses good recoverability.We further explore the response characteristics of the graphene-based LPFG SPR sensor. Experiments were performed successively exposing the sensor to a 3.5% methane concentration. The signal was collected every 5 s, and the extracted resonance wavelength versus the time is plotted in Finally, the reusability of the graphene-based LPFG SPR sensor is investigated. The sensor was alternatively exposed to methane gas with different concentrations. The signal was collected every 30 s, and the extracted resonance wavelength is plotted in 0 = e2/4ћ in the considered spectral region [e is the elementary charge and ħ is the reduced Planck’s constant. The calculated electric field mode profile of the grapheme-based LPFG SPR sensor is displayed in 4 V/m, which significantly enhances the light-matter interaction. To further quantitatively estimate the penetration characteristics of the mixed SPR-FC hybrid mode, the electric field intensity along the Y-direction (white dashed line) which is perpendicular to the sensing interface is extracted from e. For the Ag/graphene structure, the penetration depth of the electric field into the sensing medium is 1260 nm, which is larger than that of the structures without graphene (1210 nm). Thus, the introduction of graphene can enhance the intensity of the electric field surrounding the sensing layer and enables the sensor to be more sensitive to the change in the gas concentration.To explain the sensitivity enhancement mechanism due to the presence of graphene, a physical model is built using the finite element analysis (FEA) method employing COMSOL Multiphysics. In the model, the dielectric constant of Ag (50 nm) comes from the handbook of Palik , the refl region , where e−1, which is improved 2.96 and 1.31 times with respect to the traditional LPFG sensor and the Ag-coated LPFG SPR sensor, respectively. Furthermore, the graphene-based LPFG SPR sensor exhibits good response characteristics and repeatability. Such a SPR sensing scheme points to a promising future for the graphene-based gas sensor with high sensitivity.In summary, we proposed a graphene-based LPFG SPR sensor combining the advantages of LPFG and graphene. The experimental results show that the sensitivity of the graphene-based LPFG SPR sensor to changes in the concentration of methane gas is 0.344 nm%


The variation in drain current was positively related with increased working temperature and hydrogen concentration. The proposed dual-gate FET gas sensor in this study has potential applications in various fields, such as electronic noses and automobiles, owing to its low-power consumption, easy integration, good thermal stability and enhanced hydrogen sensing properties.A low power, dual-gate field-effect transistor (FET) hydrogen gas sensor with graphene decorated Pd-Ag for hydrogen sensing applications was developed. The FET hydrogen sensor was integrated with a graphene-Pd-Ag-gate FET (GPA-FET) as hydrogen sensor coupled with Pt-gate FET as a reference sensor on a single sensor platform. The sensing gate electrode was modified with graphene by an e-spray technique followed by Pd-Ag DC/MF sputtering. Morphological and structural properties were studied by FESEM and Raman spectroscopy. FEM simulations were performed to confirm the uniform temperature control at the sensing gate electrode. The GPA-FET showed a high sensing response to hydrogen gas at the temperature of 25~254.5 °C. The as-proposed FET H Recently, hydrogen has been extensively used in various applications, such as fuel cells, power generators, automobiles, aerospace engineering, and chemical processing. As a major drawback for using H2 is its low level of explosion limit (4%), therefore, the precise sensing and controlling systems are essential for hydrogen utilization in storage, handling, transportation and any other usages4.Hydrogen  based field effect transistor (FET) sensors have more advantages than other conventional sensors, such as small size, low power consumption, intrinsic safety, corrosion resistance, ease of fabrication, high stability, high sensitivity, fast response and recovery time6. When hydrogen molecules adsorb and dissociate on the catalytic layer, the difference in work function between the catalytic layer and the semiconductor substrate of the FET changes, which causes a shift in the threshold voltage of the FET7. These sensors can detect even a very low concentration of hydrogen gas at a specific position and can be mounted to detect hydrogen gas.It is indeed needed to realize the high-sensitive, high-precision, rapid, robust, real-time, online, and long-distance monitoring of hydrogen concentration. Recently, active and vibrant research activities are involved in the development of various hydrogen sensors9. Tsukada et al. developed dual gate FET with Pt-gate and Ti-gate to detect H2 gas10. The Pt-FET showed a good response to H2 gas while the Ti-FET did not show any response to hydrogen gas. Our research group also reported a dual-gate FET H2 sensor with Pt-gate by DC/MF sputtering technique11. Similarly, the surface of sensing gate was modified with a nano-bumpy shaped Pd film using polystyrene nano-beads as a template to increase the surface area of the catalytic metal film12. The surface morphologies of the gate electrode affect H2 gas sensing performance. Hence, it is necessary to study in detail the performance of different nanostructures towards detection of H2 gas.Several types of hydrogen gas sensors based on FET have been studied2 gas sensor since last two decades. However, a pure Pd metal causes the problem of H2 embrittlement and mechanical instability13. To overcome these obstacles, alloying of Pd with other nanomaterials offer high catalytic activities. Pd is chosen as the one of the gate element due to its higher hydrogen sensitivity. Silver (Ag) is being a perfect material to combine with Pd for H2 gas sensing due to its structural properties and low cost14. Addition of Ag to Pd increases the mechanical stability, hardness, chemical inertness and hydrogen permeability15. In addition to these, graphene is a two-dimensional (2-D) structure with high carrier mobility, high surface area, and outstanding structural properties. It is a good option for surface modification to gain high sensitivity towards H2 detection16. The main reason for using graphene_Pd-Ag nanocomposite instead of only Pd-Ag is that the diffusion of H atoms increases along the graphene layer as compared to only Pd-Ag17. In addition, the highly conductive nature of graphene enhances the conduction path of the as-proposed sensor. Therefore, graphene with Pd-Ag alloy is a suitable candidate for H2 detection due to its high electrical conductivity and large surface area for H2 molecular adsorption.FETs with palladium (Pd) gates have been used for H2 gas sensor. A dual-gate FET sensor with sensitive FET was fabricated having a response towards H2 while reference FET was not affected by the H2 gas. The main aim to use dual-type FETs gas sensor is due to the difference of reference and sensing FETs that affect the threshold voltage offsets because of different gate materials. A multilayer sensor chip was fabricated using a simple MEMS process. To reduce heat loss in the dielectric membrane, an isolated silicon island including the Pt micro-heater was produced using Si bulk micromachining process. To increase the heat efficiency, a Pt microheater was placed on the sensor platform. A micro platform of the multi-layer type structure was fabricated using a MEMS process. The main reason for using graphene decorated Pd-Ag electrode is that decoration of graphene with Pd-Ag nanoparticles would improve the adsorption of hydrogen molecules due to large number of active sites for H2 adsorption from Pd/Ag alloy nanoparticles and high carrier mobility of bottom layer graphene. Surface morphologies of the deposited nanostructures were studied. The electrical and thermal characteristics of the FET sensor chip were also measured, and a hydrogen sensing measurement was performed on the fabricated sensor.In the present work, nanostructures of graphene-Pd-Ag were deposited on the sensing area to develop the H11. The schematic of hydrogen sensor platform with sensing and reference FETs is shown in Fig. 2), reference gate and metal contact line (Pt), a passivation layer (Si3N4), and Si bulk micromachining patterns. The reference and sensing gate electrode pattern (Pt layer), and micro-heater were patterned simultaneously. The back side of the silicon chip was ground by chemical mechanical polishing (CMP) to reduce the thickness up to 100 µm. Finally, sensor platform was diced into the small chip with laser beam cutter. The as proposed H2 sensor was outlined with chip dimensions and sensing gate electrode of 4.0 mm × 4.0 mm and 0.11 mm × 1.2 mm, respectively as shown in Fig. A modified dual-FET sensor platform was designed and fabricated by previously reported method+, VERMES). Before dispensing, the metal mask was applied on the sensor platform to deposit desired sensing area. The graphene nanoplates were deposited at a constant rate of 0.5 L/min for 1 min. The graphene nanoplates deposited sensor platform was then heat treated at 100 °C for 2 hr under vacuum, following by sputtering of palladium (Pd) and silver (Ag) metal. The thickness of sensing gate electrode was around 30 nm that is controlled by dispensing and sputtering parameters.The surface of sensing gate electrode was modified with graphene-Pd-Ag nanostructures Fig. . Initial18. The microheater region was heated homogenously up to high temperature, while the temperature of the outer substrate area was substantially low. The fabrication of microheater near to sensing region can minimize power consumption; provide better temperature uniformity, and low thermal mass. The lowest and highest temperatures of the micro-heater were around 102.4 °C and 192.6 °C respectively. When the heater voltage of 3.0 V was applied, the temperature distribution around the area of the membrane was consistent. The numerical simulation done by FEM explains that the operating temperature of sensing area was controlled well by embedded micro-heater in sensor platform. The main purpose of a microheater is to have high working temperature of the as-formed sensor with low power consumption. The as-proposed H2 sensor shows low power consumption of 45.4 mW at 150 °C, suggesting that the H2 sensor design can attain a thermal isolation with less heat loss by chemical mechanical polishing (CMP). Accurate temperature control of the sensing electrode and the power consumption of proposed H2 sensor was attained by micro-heater operation19.As shown in Fig. 2 bond, although D bands are related to the different types of defects such as sp3 defects that arise from grain boundary edges, electron doping etc.21. In the present study, the D, G, and 2D peaks are compared between graphene and graphene-Pd/Ag nanocomposites as shown in Fig. Raman spectroscopy is a powerful tool for studying the structural properties of the carbon structures. The graphene and graphene-Pd/Ag nanocomposites were investigated to detect the doping effects. In Raman spectroscopy, the G and 2D bands occur due to the in-plane bond stretching of the C–C sp−1 to 1349.5 cm−1, whereas the position of G-peak was shifted from 1577.8 to 1563.48 cm−1; the 2D-peak is also shifted from 2690.3 to 2706.04 cm−1 in the case of the graphene-Pd/Ag to the pristine graphene. The intensity of D peak is increased by 9–10 times in case of the graphene-Pd/Ag as compared to graphene due to more defects and reduces the size of the in-plane sp2 domains and the partially ordered crystal structure. The G peak is shifted due to the interaction between the Pd/Ag and the graphene matrix, as it resembles the graphitic nature of graphene. The deviation in G-peak proposes that charge impurities on surface cause electron donating molecules and heterogeneous charge distribution ratios decrease due to doping effect22. Furthermore, the downshift of G-peak and upshift of 2D Raman shift can be experienced due to the dynamic effect of carrier population and electrical gating.The position of D-peak is changed slightly from 1352.9 cmFigure 23. In this case, substrate nature corresponding to the orientation, presence of defects, strain and interface bonding influences the substrate-adatom24.The morphology and nature of the Pd-Ag films grown on the graphene substrate were shown in Fig. 25. Defects in graphene can be exploited as the active sites for the nucleation and the growth of metal clusters due to its strong tendency to pin the adatoms26. In addition, the nanostructures vary with respect to the mobility of metal adatoms as well as the type of defect on which it is nucleating28. For instance, line defects like step edges act as an active site for metal adsorption and can be used as a nucleating site for one-dimensional nanostructures29. It was reported that palladium atomic diffusion is higher than silver and cesium along the carbon-rich SiC grain boundaries30. Hence palladium adatoms are either directly adsorbed on the step edge or adsorbed on the terrace thereby moving towards the step edges due to its high mobility and low free energy on the step edges, attributing to the nucleation and growth of Pd adatoms along the step edges forming Pd nanowires as shown in Fig. In the present work, the graphene used as the substrate had defect peak as shown in Raman spectrograph . In both cases (presence of hydrogen and absence of hydrogen), the Vpo are 4.5 V and 2.85 V and the corresponding saturation currents are 4.86 mA and 4.06 mA, respectively. In hydrogen atmosphere, the Vpo and IDsat increased as the channel width increased.The I-V curves were measured for the sensing FET as shown in Fig. DS), the drain current (ID) increases linearly and then saturate. At specific, the saturated drain current (IDsat) is 0.48 mA at 4.0 V VGS. By increasing the drain bias beyond the (VDSAT) results in the pinch-off to travel more into the channel, closer to the source end. Electrons in the channel are pulled into pinch-off region and travel at the saturation drift velocity due to the high longitudinal electric field along the channel that results in the saturation of drain current33.The electrical properties (transfer characteristics) of the as-formed FET sensors are studied as shown in Fig. 35, the drain current changes very slightly with temperature. Figure D due to the change in threshold voltage. Variation in drain current (ID) with temperature can be signified as the temperature dependence of charge carrier density, carrier mobility, and threshold voltage36. The drain current increases with increasing temperature since the charge carrier density in channel between source and drain increases. The charge carrier density increases due to formation more electron-hole pairs by increasing temperature. Moreover, the H2 sensing response of as-formed FET sensor is increasing with increase in temperature due to the hydrogen adsorption process of the as-formed FET sensor is an endothermic reaction. At high temperatures, H2 atoms will gain much high energy to facilitate the surface chemical reaction like adsorption, dissociation, and diffusion37. As shown in Fig. 11.As mentioned in several reports2 FET sensor upon introduction and elimination of hydrogen gas and using nitrogen gas as a carrier gas at the temperature of 125 °C at the H2 concentration of 2000 ppm under the applied the drain-source voltage (VDS) = 3.8 V and gate-source voltage = 4.0 V. Firstly, the N2 gas is put in test chamber to set a drain current baseline of 0.528 mA. By putting the H2 gas, the drain current increases rapidly. After few minutes, a quasi-saturation phenomenon becomes deceptive due to dynamic equilibrium between the H2 absorption and desorption39. Under this condition, the maximum variation in drain current was 8 µA. The as-formed FET hydrogen sensors display better stability, even after many testing cycles  is introduced, the equilibrium of H2 concentration between the Pd/Ag alloy nanoparticles and its oxide is abruptly shattered that leads to the sharp drop in the sensing signal (drain current). After some time, the sensing signal enters stage 2 which defined as the intermediate stage. In this stage, more H2 atoms are desorbed due to the fast recombination of H2 atoms that leads to the lessen H2 atoms on Pd/Ag surface. This may result in fast recovery rate. In stage 3, defined as the final stage, the amount of the absorbed H2 atoms on the Pd/Ag surface will increase with time that leads to the increase the binding sites for the H2 atoms40.Figure 2 sensor and enlarges data at low hydrogen concentration up to 500 ppm, respectively. It is clear from Fig. 41. The drain currents change quickly for all the hydrogen concentrations. The proposed H2 FET sensor at a low H2 concentration (<10 ppm) showed a good response to hydrogen, therefore the limit of detection (LOD) of the as-formed FET sensor was around 1 ppm. The dual gate-type FET sensors show high response even at low hydrogen concentration below 1 ppm. The high response is due to the interaction between the graphene-Pd/Ag and H2 gas. Inset Fig. 42. As compared to other reports44, as-proposed H2 sensor shows better sensing properties (fast response and recovery time), the limit of detection (LOD) is around 1 ppm.The relationship of drain current with hydrogen concentration is shown in Fig. 2 sensing mechanism in the graphene-Pd-Ag nanocomposite. The work principle of the GPA-FET will depend on the difference of work function of the graphene_Pd-Ag (gate electrode) during exposure to H2 gas45. Initially, the source needs to be grounded then the conductivity between drain and source can be changed by altering the gate voltage (VGS). During H2 atmosphere, the H2 atoms diffuse at the interface between gate electrode (graphene-Pd-Ag) and insulator (SiO2) to form a dipole layer which leads to change in gate voltage, which finally results in the change in drain current related to the H2 concentration9. As H2 is a reducing gas and donates electrons to the graphene-Pd-Ag during interaction that leads to the increase in drain current (IDS). The increase in the drain current of the graphene- Pd-Ag-gate FET (GPA-FET) can be considered with the process of absorption (physisorption and chemisorption) and desorption taking place on the surface of the graphene-Pd-Ag. With exposure of graphene-Pd-Ag to hydrogen molecules, the formation of metal hydrides takes place that reduces the work function of the Pd-Ag and results in the transfer of an electron from metal hydrides to the graphene46. The fast recovery of the as-formed FET sensor is due to the physisorption as it takes place faster than chemisorption. Practically, it is important that the catalytic component (Pd-Ag) is well dispersed in the electrically conductive path (graphene) to maximize the drain current which simply the charge transfer. Figure 2 absorption the H2 molecules or atoms are absorbed on the surface of Pd/Ag alloy nanoparticles while in case of H2 desorption, all H2 molecules or atoms were desorbed from the graphene _Pd-Ag surface.Figure 2 gas above 1 ppm. The GPA-FET showed a high response to hydrogen gas at temperatures of 25–254.5 °C. The fast response and recovery time were credited to the interaction between the graphene _Pd/Ag nanocomposite and H2 molecules. The as-formed FET sensor shows a linear increase in the drain current with H2 concentration. The proposed dual-gate FET gas sensor in this study has potential applications in various fields, such as electronic noses and automobiles, owing to its low-power consumption, easy integration, good thermal stability and enhanced hydrogen sensing properties.A facile method to design and fabricate the dual-gate FET hydrogen gas sensor was developed with embedded microheater. The dual-gate FET hydrogen sensor was integrated with a graphene-Pd-Ag-gate FET (GPA-FET) as sensing FET to detect hydrogen and a Pt-gate FET as a reference one on a single platform. The FEM numerical analysis showed that the operating temperature of sensing area was well controlled by an embedded micro-heater in the sensor platform. The sensing layer of gate electrode was modified with deposition of graphene by an e-spray technique, followed by Pd-Ag sputtering. FESEM results reveal that the graphene-Pd-Ag nanocomposites are uniformly distributed on the sensing electrode and Raman spectroscopy studies the alteration in D, G, and 2D band after the interaction Pd-Ag nanoparticles in graphene and pristine graphene. The GPA-FET showed a good response to H