A room temperature microfabrication technique using SU8, an epoxy-based highly functional photoresist as a sacrificial layer, is developed to obtain suspended aligned carbon nanotube beams. The humidity-sensing characteristics of aligned suspended single-walled carbon nanotube films are studied. A comparative study between suspended and non-suspended architectures is done by recording the resistance change in the nanotubes under humidity. For the tests, the humidity was varied from 15% to 98% RH. A comparative study between suspended and non-suspended devices shows that the response and recovery times of the suspended devices was found to be almost 3 times shorter than the non-suspended devices. The suspended devices also showed minimal hysteresis even after 10 humidity cycles, and also exhibit enhanced sensitivity. Repeatability tests were performed by subjecting the sensors to continuous humidification cycles. All tests reported here have been performed using pristine non-functionalized nanotubes.  Ever since their discovery in 1991 by Iijima ,3. Owing2 with minimal hysteresis [Humidity Sensors play a major role in environment monitoring in various locations such as homes, automobiles and in medicine . A host steresis . Howeversteresis  using a steresis ,23. Moresteresis . HoweverThe process starts with a Silicon wafer with a 300 nm oxide a. A 3.5 Vacuum filtration enables the formation of a uniform nanotube network and allows a precise control over the thickness of the film depending on the amount of solution used. More importantly, vacuum filtration is a room temperature deposition method and is of low-cost compared to other techniques. The nanotube film is deposited on a nitrocellulose membrane. After vacuum filtration, the film is washed in deionized (DI) water to remove any traces of surfactant that may be present on the surface. Then, the carbon nanotube film is transferred onto the substrate. The film is cut into the desired shape and size and transferred onto the substrate by dipping the nanotube film in chlorobenzene. The substrate is then dipped in acetone for 60 min to dissolve the nitrocellulose membrane. The resulting nanotube film thickness is between 0.5–0.7 µm depending on the amount of solution used. The nanotube film is then patterned by UV lithography. After development, the nanotube film is etched using oxygen plasma e. After The concept of using SU8 as a sacrificial material was adopted from . The reaThe samples were imaged using a MEB 3600 Scanning Electron Microscope. The samples were metallized by sputtering a 20 nm Gold layer to make the nanotubes conductive for the SEM. It was found that the sputtering did not affect the properties of the nanotubes. As shown in The humidity-sensing mechanism of the nanotubes has been extensively studied and described in works such as ,30. The The longer recovery time of the non-suspended devices could be due to the presence of water molecules on the nanotube network during the desorption process and to the reduced surface area available for desorption. One of the most important parameters for gauging sensor performance is long-term stability. The sensor should show consistent values and response even after prolonged duration. To demonstrate long-term stability, the resistance of the suspended devices was measured after certain time intervals with similar testing conditions as the initial tests. The suspended nanotube sensors exhibited stable performance over the time period as shown in HR is the resistance at the measured value of resistance, and R0 is value of baseline resistance. Since the suspended nanotubes beam is not connected to the substrate, the water molecules have both the top and bottom surface to adhere to which increases the sensitivity of the sensor. It has been shown in this work that the sensitivity of the sensor is improved by the suspension of the carbon nanotubes beam. The sensitivity factor, in percent, is given byIt can be gauged from this plot that the sensitivity of the suspended nanotubes is much greater than the non-suspended networks. From a base humidity of 15%, the increase in resistance at 98% humidity was of 246.9% for the suspended device compared to only 46.83% for the non-suspended devices. The results of this study are a clear indication that the use of suspended structures for sensing applications yields significant advantages in comparison to traditional substrate bound materials. It is clear that the response and recovery times presented in this work are not suitable for commercial use, as the nanotubes have not been functionalized. However, the aim of this work is to demonstrate the advantages of suspended nanotube networks, in terms of response time, hysteresis and sensitivity. Many functionalization schemes have been reported in the literature ,31,32 anSuspended carbon nanotubes have been investigated as an alternative to conventional non-suspended devices. We have demonstrated an easy, low-temperature fabrication process to obtain suspended carbon nanotube beams using a common polymer sacrificial layer. Notably, the low-temperature of the process is well suited to the integrated of devices directly above CMOS integrated circuits. Moreover, this process with some modifications can also be used to create devices such as nanotube micromechanical resonators. It was found that suspended carbon nanotubes have better all-round humidity-sensing performance as compared to non-suspended architecture. The suspended nanotube sensor exhibited repeatable performance with good response times and recovery times as compared to the non-suspended sensor. The significantly reduced hysteresis in the suspended nanotube humidity sensor is a major advantage, along with their enhanced sensitivity and response time. The performance of the devices can be enhanced by chemical modification of the nanotube network and future work is to functionalize the nanotubes and study their humidity response in a suspended architecture.


Depletion of amyotrophic lateral sclerosis (ALS)-associated transactivation response (TAR) RNA/DNA-binding protein 43 kDa (TDP-43) alters splicing efficiency of multiple transcripts and results in neuronal cell death. TDP-43 depletion can also disturb expression levels of small nuclear RNAs (snRNAs) as spliceosomal components. Despite this knowledge, the relationship between cell death and alteration of snRNA expression during TDP-43 depletion remains unclear. Here, we knocked down TDP-43 in murine neuroblastoma Neuro2A cells and found a time lag between efficient TDP-43 depletion and appearance of cell death, suggesting that several mechanisms mediate between these two events. The amount of U6 snRNA was significantly decreased during TDP-43 depletion prior to increase of cell death, whereas that of U1, U2, and U4 snRNAs was not. Downregulation of U6 snRNA led to cell death, whereas transient exogenous expression of U6 snRNA counteracted the effect of TDP-43 knockdown on cell death, and slightly decreased the mis-splicing rate of Dnajc5 and Sortilin 1 transcripts, which are assisted by TDP-43. These results suggest that regulation of the U6 snRNA expression level by TDP-43 is a key factor in the increase in cell death upon TDP-43 loss-of-function. Transactivation response (TAR) RNA/DNA-binding protein 43 kDa (TDP-43) has been identified as an amyotrophic lateral sclerosis (ALS)-associated protein. TDP-43 is mainly localized in the nucleus and shuttles between the nucleus and cytoplasm to maintain several RNA-associated functions  . HoweverAlternatively, since TDP-43 knockout in murine motor neurons causes progressive motor neuron degeneration , loss-ofTDP-43 depletion both in murine brain and mammalian cultured cells causes widespread alterations of the RNA-splicing state such as changes in exon inclusion –21. DefeTherefore, to unveil the relationship between cell death and the alteration of snRNA expression during TDP-43 depletion, we investigated whether the expression level of U6 snRNA can be modified using TDP-43-knocked down murine neuroblastoma Neuro2A cells, which show a significant increase of cell death post-TDP-43 depletion. Finally, we investigated whether cell death during TDP-43 depletion can be prevented by restoring the expression levels of U6 snRNA.Plasmid DNAs encoding GFP (pmEGFP-N1) and pmEGFP-N1 to express C-terminally GFP-tagged TDP-43 (TDP-43-GFP) were prepared as established previously . Synthet5) were plated in a 10 cm cell culture dish . A sense and antisense TDP-43-targeting siRNA duplex , or a non-targeting siRNA  as a negative control, were transfected using 24 μL Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) and 160 pmol of each siRNA in Opti-MEM I medium (Thermo Fisher Scientific). A sense and antisense U6 snRNA-targeting siRNA duplex  was modified from a previous study [Murine neuroblastoma Neuro2A cells were kindly provided by Prof. Kazuhiro Nagata at Kyoto Sangyo University. The strain of the cell was as same as that in the previous studies, and culture conditions were reported previously ,27. For us study .5) were plated in a 10 cm culture dish. After a 20 h incubation, cells were transfected using 12.8 μL Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) and a mixture of 0.5 μg pmEGFP-N1 and either 4.5 μg pU6 or pEV. The transfection mix was incubated with the cells for 4 h. After the incubation, the medium was replaced and siRNA was transfected following the protocol described above  and purified using PureLink RNA Mini Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Extracts were treated with DNase I  before performing additional assays.To detect non-coding RNAs, total RNA (750 ng) was used for synthesis of first-strand cDNA using Mir-X miRNA First Strand Synthesis Kit (TaKaRa) according to the manufacturer’s instructions. Real-time quantitative PCR (qPCR) was performed using SYBR Premix Ex Taq , ROX Reference Dye II (TaKaRa), 0.2 μM forward primer, 0.2 μM reverse primer, and 2 μL template cDNA . Amplification and detection were performed using a real-time PCR system . The PCR was performed in a two-step protocol under the following conditions: initial denaturing at 95°C for 30 s, followed by 35 cycles of denaturing at 95°C for 10 s, and annealing/extension at 60°C for 20 s.To detect the splicing state of transcripts, first-strand complementary DNA synthesis was performed using a transcriptase  according to the manufacturer’s instructions. PCR was performed using a thermal cycler . The PCR was performed in a three-step protocol under the following conditions: initial denaturing at 98°C for 30 s, followed by 21 cycles of denaturing at 98°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 60 s. PCR products were separated in 5% polyacrylamide gel and detected using LAS 4000 mini . PCR primers are described in Samples including total extract RNA (2.75 μg) were mixed with loading buffer consisting of 1.5× Tris-acetate EDTA (TAE), 10 M urea, 10% (w/v) sucrose, 0.05% (w/v) bromophenol blue, and 0.05% (w/v) xylene cyanol, incubated at 65°C for 15 min, and kept on ice until electrophoretic separation. Samples were separated in a 12% polyacrylamide gel containing 7% urea in 1× TAE buffer. RNAs were stained using SYBR Gold (Thermo Fisher Scientific). Fluorescent intensities were detected using LAS 4000 mini (Fujifilm) using a 460 nm excitation light and a long-pass filter . Quantification was performed using ImageJ 1.47 software .Dead cells were stained by 1.0 μg/mL propidium iodide  and observed using an LSM 510  and a Plan-Neofluar 10×/0.3 NA objective as reported previously ,27.5) in 100 μL cell culture medium were re-plated into a 96-well plate  and cultivated for 20 h. Thereafter, 10 μL WST-1 mixture  was added to the cell culture medium, samples were incubated for an additional 4 h, and then the medium was collected. Absorbance at 490 and 690 nm was measured using a DU 800 spectrophotometer . Absorbance at 490 nm was subtracted from that at 690 nm.For alternative cell viability assays based on metabolism, at 96 h after siRNA transfection, cells (0.5 × 10Neuro2A cells were passaged into four 10 cm culture dishes at a ratio of 1/5 and transfected using 16.0 μL Lipofectamine 2000 transfection reagent and 4.0 μg pmEGFP-N1 or TDP-43-GFP expression plasmid (TDP-43-GFP). After incubation for 30 h, the CLIP assay was performed as previously reported . Cells wt test was performed to evaluate statistical significance.Student’s We first sought to evaluate the timeline of TDP-43 depletion following knockdown. To knock down TDP-43, siRNA targeting TDP-43 mRNA (T43-siRNA) or non-coding siRNA (NC-siRNA) were transiently transfected into Neuro2A cells. TDP-43 protein levels were assessed by Western blot at 72, 96, and 120 h after transfection. T43-siRNA-transfected cells showed a significant decrease in TDP-43 levels compared with NC-siRNA-transfected cells. In turn, NC-siRNA transfection did not have any significant effect on the levels of TDP-43 compared with mock-transfected cells. These results indicate that T43-siRNA-mediated knockdown can achieve an efficient reduction in the protein levels of TDP-43 . MoreoveNext, we analyzed the timeline of cell death after TDP-43 depletion. Quantification of cell death showed that the proportion of live cells remained stable at 72 and 96 h after transfection with siRNA and significantly decreased at 120 h after siRNA transfection . Given tNext, we checked the expression level of U6 snRNA in TDP-43-knocked down Neuro2A cells at 72 h after the transfection of siRNAs using qPCR. The amount of U6 snRNA in T43-siRNA-transfected cells was significantly decreased compared with that in NC-siRNA-transfected cells . The amoWe next elucidated the expression levels of other U snRNAs using urea-polyacrylamide gel electrophoresis (Urea-PAGE). The migration pattern of U1, U2, U4, and U6 snRNA was unequivocally assigned using their predicted molecular sizes . Unfortunately, U5 snRNA was not distinguished in this migration pattern . The decMoreover, to investigate whether TDP-43 can associate with U6 snRNA, we quantified the amount of U6 snRNA co-immunoprecipitated with TDP-43 using qPCR. The amount of U6 snRNA did not differ between lysates of TDP-43-GFP-expressing and GFP-expressing cells ; INPUT. To investigate whether U6 snRNA is involved in cell death, U6 snRNA was knocked down using siRNA. At 72 h after transfection of U6-siRNA, U6 snRNA expression in Neuro2A cells was decreased to 72% compared with that in NC-siRNA-transfected cells . The proTo determine whether restoration of U6 snRNA expression rescues cell death during TDP-43 depletion, we examined cell viability when exogenous U6 snRNA was transiently expressed using a plasmid DNA encoding U6 snRNA (pU6) in T43-siRNA-transfected Neuro2A cells. Transfection with an empty vector (pEV) as a negative control decreased the proportion of live cells during TDP-43 knockdown at 120 h .Next, to confirm whether transfection of pU6 effectively increased the amount of U6 snRNA, we performed a qPCR at 76 h after transfection with pU6. The amount of U6 snRNA in NC-siRNA-transfected cells showed a slight increase by transfection with pU6, but the difference was not significant (We confirmed that the proportion of live cells among T43-siRNA-transfected cells was restored by exogenous expression of a TDP-43 that is resistant to knockdown by T43-siRNA. Expression of exogenous TDP-43 tagged with GFP (T43-GFP) in NC-siRNA-transfected cells did not alter the proportion of live cells . QuantifIn addition, exogenous expression of U6 snRNA in TDP-43-knocked down cells did not alter the amount of endogenous TDP-43 until 120 h after transfection of T43-siRNA . TherefoTo investigate a plausible mechanism by which an increase in the U6 snRNA level ameliorates the viability of TDP-43-knocked down cells, we examined the aberrant mRNA-splicing state during TDP-43 depletion. Based on previous reports –20, 30, Next, we analyzed whether expression of exogenous U6 snRNA changes the amount of the spliced form of these transcripts in TDP-43-knocked down cells. The amount of the exon-excluded form of Dnajc5 and Sort1 transcripts was slightly increased by exogenous expression of U6 snRNA in TDP-43-knocked down cells . These rvia RNA polymerase III and/or (ii) stability of U6 snRNA. In the first case, U6 snRNA is transcribed by RNA polymerase III, while U1, U2, and U4 snRNAs are transcribed by RNA polymerase II [via RNA polymerase III may not contribute to the decrease of U6 snRNA levels during TDP-43 depletion. In the second case, the U6 snRNA sequence does not contain typical UG repeats, which TDP-43 preferably recognizes; however, CLIP showed that a low amount of U6 snRNA binds to TDP-43 [via an intermediate process . However, it is unknown whether TDP-43 binds to U6 snRNA directly or via other proteins, and this should be clarified in a further study.We show here that TDP-43 knockdown downregulates the amount of U6 snRNA but not of U1, U2, and U4 snRNAs in Neuro2A cells. Two possible mechanisms could explain this downregulation: TDP-43 could be involved in (i) transcriptional promotion erase II . Howevererase II , our reso TDP-43 . We confo TDP-43 . These fAlthough a previous study using human SH-SY5Y cells reported that the amount of U6 snRNA was not decreased by TDP-43 knockdown ,26, we sIn addition, our results show that expression of U6 snRNA in TDP-43-knocked down cells slightly improved the mis-splicing efficiency of Dnajc5 and Sort1 transcripts. Although the direct effect of U6 snRNA on the alternative splicing of transcripts in the TDP-43-knockdown condition has not been clarified yet, we expect that restoration of U6 snRNA levels during TDP-43 depletion might maintain the alternative splicing efficiency of these transcripts. On the other hand, TDP-43 loss-of-function inhibits vesicular trafficking, including endosomal trafficking and autophagosome-lysosome fusion ,34. ThesAlthough our results are confined to the cellular level, a report showed a decrease in the U6 snRNA levels in spinal cord from ALS patients . TherefoS1 Table(A) Synthetic oligonucleotides used to construct the U6 snRNA expression plasmid. (B) PCR primers used to detect transcripts and small RNAs. † denotes a commercially available primer included in the Mir-X First Strand Synthesis Kit (TaKaRa).(PDF)Click here for additional data file.S1 Fig(A) Transfection of T43-siRNA, total RNA extraction, protein extraction, and staining of dead cells. Green indicates the period of siRNA transfection. (B) Transfection of U6 snRNA plasmid DNA prior to transfection of T43-siRNA, total RNA extraction, protein extraction, and staining of dead cells. Blue and green indicate the period of plasmid DNA and siRNA transfection, respectively.(TIF)Click here for additional data file.S2 Figt test: *p < 0.05.Relative PCR-amplified amounts of U6 snRNA, 18S rRNA, 7SL RNA, and snoRNA 202 in T43-siRNA-transfected cells compared with those in NC-siRNA-transfected cells at 72 h after siRNA transfection . Significance was tested by Student’s (TIF)Click here for additional data file.S3 Figt test: *p < 0.05 and ***p < 0.001 .Absorbance at 450 nm was subtracted from that at 690 nm. Cells were cultured for 120 h after transfection of TDP-43-siRNA or NC-siRNA, with or without exogenous U6 snRNA expression. Significance indicated in the graph was tested by Student’s (TIF)Click here for additional data file.S4 Figt test: *p < 0.05. N.S. denotes no statistical significance.(A) Western blot analysis of TDP-43 and α-tubulin in T43-siRNA- or NC-siRNA-transfected cells transiently expressing U6 snRNA. Time corresponds to the amount of time after siRNA transfection. (B) Quantification of TDP-43 expression by Western blotting using anti-TDP-43 and anti-α-tubulin antibodies . Significance indicated in the graph was tested by Student’s (TIF)Click here for additional data file.S5 Figt test: *p < 0.05 and ***p < 0.001. N.S. denotes no statistical significance. Numbers in each bar show mean values.(A) Images of migrated bands of spliced forms of Madd transcripts. RPS18 was used as an internal loading control. Spliced forms of transcripts were distinguished using splicing-dependent pairs of PCR primers. (B) Relative amount of the exon-excluded form of Madd transcripts . Significance was tested by Student’s (TIF)Click here for additional data file.


Relative humidity (RH) was measured at the left mid-thigh, right mid-thigh and coccyx. Sensors were shown to be unaffected by loading and showed highly reliable responses to measured changes in humidity, varying little from the traceable standard (<5%). Field-test data, smoothed through a moving average filter, revealed significant differences between the three chosen locations and between the gel and foam cushions. Maximum RH was attained in less than five minutes regardless of cushion material (foam or gel). Importantly, RH does not appear to distribute uniformly over the body–seat interface; suggesting multiple sensor positions would appear essential for effectively monitoring moisture in this interface. Material properties of the cushions appear to have a significant effect on RH characteristics (profile) at the body–seat interface, but not necessarily the time to peak moisture.Little is known about the changes in moisture that occur at the body–seat interface during sitting. However, as increased moisture can add to the risk of skin damage, we have developed an array of MEMS  humidity sensors to measure at this interface. Sensors were first evaluated against traceable standards, followed by use in a cross-over field test ( Among the sedentary life-style related chronic diseases, epidermal ulceration is one of the most painful, hardest to treat successfully and has the potential to create life-threatening complications . This siTreatment of skin ulcers is costly ,4 and raInitial studies of wheelchair or office seat surface (or cushion) materials were based upon humidity probes ,7, whichIn order to achieve the task of continuously acquiring RH information in real time, a parallel analogue-to-digital converting (ADC) module was considered the most appropriate choice. Based on factors such as reliability and stability, the Pico ADC-11/12  was selected as the data sampling unit. The Pico ADC-11/12 has 11 ADC channels with 12-bit resolution and up to 1 kHz sampling frequency per channel. To implement device initialisation, real-time data storage and off-line analysis, a user-friendly graphic user interface was developed with the help of the Visual Basic Integrated Development Environment . In addition, the whole measurement system was powered through the computer universal serial bus (USB) port, which increased portability and prevented possible errors when connecting the power supply to the humidity sensors.The humidity sensor , which is able to convert the moisture information into a corresponding analog voltage signal, was chosen to detect the RH level at the body–seat interface. The advantages of HIH4000 include: (1) simple connection to a microcontroller; (2) easily solderable pins; (3) integrated signal conditioning circuit and (4) multilayer protection construction suitable for hazardous applications. To evaluate the output accuracy of humidity sensors, they were verified over a range of values (10% to 90% RH) using a traceably calibrated humidity chamber  with 10% RH increment each time. Averaged outcomes of the three sensors for the humidity increasing assessment  showed tR2 = 0.998) in relation to the preset values of the standardised humidity chamber. In addition, strong correlations were seen between the increasing and decreasing trail results for each sensor .To evaluate the presence and degree of hysteresis for the humidity sensors, a trial using stepped decreases in humidity was also conducted with 10% RH decrement each time: the averaged output of the three sensors also exhibited an approximately linear relationship  and one on either side of the mid line of the cushion approximately where the middle parts of each thigh would be. An hour-long trial was conducted in a vacant research room with ambient temperature 24.7 °C ± 0.2 °C and 38.6% ± 0.4% RH. To avoid any possible disturbance coming from the change of environmental conditions, the door of the research room was closed during the whole period of the experiment.p > 0.1) between the three humidity sensors under the 50 kg (25 kg × 2 = 50 kg) mass over the period of 1 h. The experiment further attested the measurement consistency of humidity sensors in the same environmental RH. It could be concluded that any difference among the humidity sensors in prolonged sitting experiments could quite confidently be considered as not deriving from the pressure of the mass being applied.Experimental results  indicateTwo medium density foam wheelchair cushions were used for these experiments with one containing a high-density gel pack placed in a well formed on the uppermost surface . Dimensions of the two cushions were 430 × 484 × 77 mm, except that the gel cushion requires an additional contoured region to accommodate the gel pack. For the purpose of being unobtrusive and imperceptible to subjects, humidity sensors were placed in small recesses (30 mm × 40 mm × 20 mm) cut into the cushion foam . There was only one exception for this protocol and that was for the coccyx region of gel pack, where the sensor was placed on the top of it. According to previous studies [Prior to the sitting experiment, both sensor-embedded cushions were covered with the standard black upholstery . After that, they were mounted on identical wheelchairs  having a backrest, armrests and adjustable footrests. These additional functions to the wheelchairs created conditions that appeared to generate more consistent sitting postures between subjects . Since uAs RH values between the body and the seat would not be expected to vary abruptly over short periods (<1 s), the sampling frequency of the measurement system was set at 1 Hz/sensor. Matlab  and Excel  were used to process and analyse recorded humidity information.−2. The experimental protocol comprised each subject sitting in their natural  posture for 20 min on either of the wheelchair cushions. Duration of sitting was based on our previous studies [Eleven university students  took part in the sitting experiment which had been approved by the Faculty of Health, Sport and Science ethics sub-committee, University of Glamorgan . All volunteers who participated in the trial gave written informed consent form prior to the experiment. Age range for the subjects was from 21 to 40 years old, while their body mass index (BMI) was in the range 19.31–26.44 kg·m studies ,10 whichith data point. For our practical application, It was necessary to suppress unwanted noisy disturbance combined in the raw data before exploring moisture characteristics of different cushions for prolonged sitting. A moving average filter  was usedTm) extracted from the 20-min sample data of each participant. Beyond those parameters, data from 30-s epochs at 5th min, 10th min, 15th min and 20th min were used to analyse RH characteristics at different measurement locations.To study the effect of cushion properties on RH, several parameters were employed including average, maximum and median RH values as well as the time to reach the maximum value  and p < 0.01) regardless of time epochs or underlying cushion materials.For each participant, data from 30-s epochs prior to the end of the time stamps  were employed to investigate RH features of the three measurement locations. The recorded RH values from each of the three positions were significantly different from each other  might exist between the three measurement positions for this small, relatively homogeneous population under these controlled conditions  at the regions of right mid-thigh and coccyx. However, the difference is not significant between the foam and the gel at the area of the left mid-thigh .The average, maximum and median of RH values  show thaIn the calibration test, all the maximum deviations were within the range given by the datasheet, however, there was no predictable deviation from the preset values of the humidity chamber. For the increasing test, the maximum deviation (5.0% RH) occurred when the preset value of the chamber was 90% RH, whereas, for the decreasing test, the maximum deviation was 6.9% RH when the preset value of the chamber was 80% RH. Though the hysteresis phenomenon between increasing and decreasing tests was not obvious, we would suggest that sufficient time (≥1 h) should be provided to allow humidity sensors to go back to its environmental moisture when undertaking sitting trials where the same seat sees repeated use (as reported here).p > 0.1) with the maximum deviation between the three sensors being 1.5% RH  is considered, coccyx and right mid-thigh exhibited higher averaged RH values than the left mid-thigh . This cop < 0.05). Furthermore, significant differences existed between foam and gel (p < 0.05) based on the averaged data over the entire surface. To further investigate the effect of cushion material properties on this result, we conducted an extreme experiment by shaping a chipboard kitchen worktop into a wooden “cushion” equivalent with the same dimensions as the other two cushions (foam and gel). The humidity sensors were embedded in the same positions as the foam and gel cushions, with the unlaminated side facing upward and the rounded edge facing forward and the same upholstery used. In accordance with the experimental protocol, the same subjects  were invited back to sit on the upholstery-covered wooden ‘cushion’, which was mounted on the same wheelchair for 20 min. An interesting finding was that the averaged RH output for the wood ‘cushion’ over the 20 min lay between foam and gel cushions, while the trend in RH was similar to foam: decreasing . This finding may be beneficial for cushion manufacturers as less time will be needed to examine those properties of different materials that affect moisture at the user–seat interface [In addition, all cushions show that the maximal RH can be reached within five minutes  and female (n = 5). However, no significant difference (p > 0.05) was seen between the two groups of data, at any of the three locations for both cushions. This finding further indicated that moisture changes at the contact surface were resulting from aspects of the cushions’ property.To study the possible impact of different sexes on RH outcomes, recorded data were divided into two groups: male . Therefore, there is a push-pull effect between interface pressure, sweat (sensible moisture build up) and humidity. Another factor linked to surface moisture is associated with thermophysiological responses to changes in relative humidity under different thermal environments . Having However, since no material currently offers excellent performance in all aspects of seating comfort , a multi-layer theoretical model has been proposed for clinical applications which divides a cushion into different layers and employs a specific material to that region ,19. Our We report the development and testing of a sensor-based measurement system capable of allowing investigation of RH at the user–seat surface interface. The results indicate that the system can reliably monitor RH changes at this important interface and appears unaffected when simulating pressures imposed on sensors during normal sitting.Compared with previous studies, the system introduced here closely examined the stability and reliability of humidity sensors using traceable calibration methods. These results confirm what we have previously presented with a greater degree of confidence, especially in the stability of the sensors over a period of use, and show if they were affected by the pressure of sitting rather than the humidity alone. Additionally, new data processing methods were employed including that of 30 s epochs statistical analysis and the moving average filter. Furthermore, this paper reports data from a reduced number of sensors and experimental time; reductions being based on our pilot research and confidence in the sensors .RH properties at different measurement locations vary . Thus, it is of importance to deploy multiple sensors at the user–seat interface if greater resolution of the RH is needed to determine the highest ‘at risk’ regions. In future research, the optimal number of humidity sensors and their locations will be investigated.p < 0.05). As cushion composition may have significant impact on moisture at the user–seat interface, it is vital to consider the effects on RH of different materials when selecting cushions for prolonged sitting applications with those in need, such as wheelchair-dependant patients. In addition, this system might help in the future design of materials which can reduce the build-up of moisture at this critical interface zone.RH and changes in RH at the three locations are different for the gel and foam cushion (For both foam and gel cushions, the time to reach a maximum humidity value during uninterrupted sitting is less than five minutes. This means a shorter testing duration may be possible when evaluating transient humidity properties related to different cushion materials. However, it is important to note that these subjects were not impaired and as a result might have created movements capable of increasing ventilation and reducing interface RH. Therefore, this study should be extended into those populations which are dependent on wheelchairs and may not have the capability of sensing changes at the seat interface.The system also appears capable of discriminating between two commonly used, commercially available cushions and can supply information such as:


Ninety standardised benthic communities were grown on PVC tiles deployed at two shallow-water volcanic CO2 seeps and two adjacent control sites in Papua New Guinea. Along the CO2 gradient, both the upward facing phototrophic and the downward facing cryptic communities changed in their composition. Under ambient CO2, both communities were dominated by calcifying algae, but with increasing CO2 they were gradually replaced by non-calcifying algae . Responses were weaker in the invertebrate communities, however ascidians and tube-forming polychaetes declined with increasing CO2. Differences in the carbonate chemistry explained a far greater amount of change in communities than differences between the two reefs and successional changes from five to 13 months, suggesting community successions are established early and are under strong chemical control. As pH declined from 8.0 to 7.8, rates of gross photosynthesis and dark respiration of the 13-month old reef communities (upper and cryptic surfaces combined) significantly increased by 10% and 20%, respectively, in response to altered community composition. As a consequence, net production remained constant. Light and dark calcification rates both gradually declined by 20%, and low or negative daily net calcification rates were observed at an aragonite saturation state of <2.3. The study demonstrates that ocean acidification as predicted for the end of this century will strongly alter reef communities, and will significantly change rates of community metabolism.Ocean acidification is expected to alter community composition on coral reefs, but its effects on reef community metabolism are poorly understood. Here we document how early successional benthic coral reef communities change  Predicting how marine communities will respond to OA is complicated, as many of these chemical alterations can act as drivers of change [T) [2, while the response of communities remains largely uninvestigated. To better predict how communities will be shaped under OA, there is thus a need for studies which investigate the response curves of communities to increasing CO2.The oceanic uptake of anthropogenic carbon dioxide (COion (OA) . OA not f change ,2. For eange [T) ,5. As OAange [T) ,7, as weange [T)  along gr2 analogues, such as volcanic CO2 seep sites [Coral reefs are likely to be among the ecosystems most affected by OA . Predictep sites –13 or otep sites –16, as wep sites –19. Whilep sites .in situ [3 accumulation will decline. However, these investigations have mainly examined effects due to changes in seawater carbonate chemistry, without fully accounting for changes due to the longer-term shifts in benthic community composition that may occur under OA. For example, Ω declines may directly reduce calcification rates in numerous taxa [Coupled with the predicted changes in community composition under OA will likely be changes in community metabolism. However, scaling up OA effects on metabolic processes from individuals and species to the community level has proven difficult, and our current understanding is poor . To datein situ ,26 and iin situ ,27, as win situ ,19,28,29ous taxa , but if ous taxa ,31, or iThe frequency and severity of disturbances affecting coral reefs is increasing , and sclin situ, under natural levels of light and shade, temperature and water flow, along CO2 gradients at two volcanic CO2 seep and two control sites in Papua New Guinea. Benthic communities developing on the upper light exposed tile sides, as well as the shaded crevice-dwelling taxa on the lower sides were investigated after five and 13 months, and their successional changes, and taxa-specific responses along the CO2 gradients were explored. Response curves in community photosynthesis, respiration and light and dark calcification were then determined after 13 months.In this study we investigate how OA will shape the composition of early-successional benthic communities that live on the carbonate substrata of coral reefs, and the metabolic rates of the non-scleractinian components of reef communities that have developed under altered carbonate chemistries. To do so, non-carbonate settlement tiles were deployed 2 seeps and adjacent control sites in Milne Bay Province, Papua New Guinea. Seep and control sites at Upa-Upasina and Dobu are located adjacent to Normanby and Dobu Islands, respectively, and are described in detail by Fabricius et al. [2 gas emerges from the seafloor, locally altering the carbonate chemistry of the seawater without altering its temperature [This study was conducted at two tropical shallow water (<5 m depth) COs et al. . At bothperature . The seeperature ,11. Thiset al. [T) [T).Data on the seawater carbonate chemistry for the sites has been published by Fabricius et al. ,11, as het al. ,20,36. S al. [T) . Given set al. [In December 2011, 90 labelled settlement tiles were evenly distributed across seep and control sites at both Dobu and Upa-Upasina reefs (n = 15 control and 30 seep site tiles per reef). Tiles were 11.5 * 11.5 cm, made of 3 mm thick polyvinyl chloride (PVC) and roughened on both sides with sandpaper. Kennedy et al.  have shoThe benthic community composition of the upper and lower sides of the tiles, from the two census periods, was assessed on the tile photographs using the evenly spaced point analysis method . To do sin situ temperatures from a 2m deep intake (30°C). To minimise boundary layers, a 35 mm magnetic stirrer bar was placed into each chamber, activated with a custom made system of rotating magnets and pullies placed under the water baths. Communities from control sites were incubated in seawater at pHT 8.08, while those originating from the seep sites were placed in seawater with pHT 7.70. Four chambers per seep, and two per control site, were incubated with a blank settlement tile as a control. Water obtained from the seep sites was mixed with control seawater immediately prior to incubations to make a large batch at the target pH level, which was then used to fill the chambers. pH was determined with a portable pH meter  calibrated on the NBS scale. Measurements of pHNBS and AT from the incubation water were used to calculate carbonate chemistry parameters using the macro CO2SYS with the constraints set by Dickson and Millero [et al. [To determine rates of community gross photosynthesis, dark respiration, and light and dark calcification, all settlement tiles were incubated in the light and dark on the day of their collection in January 2013. For the incubations, tiles were transferred from their holding containers in running seawater into clear rectangular-prism chambers (920 mL volume), atop spacers which left ~1.5 cm gap between the tile and the walls, bottom and lid of the chamber. Chambers were placed in black flow-through bins that served as water baths and were maintained at ambient  Millero , followi [et al.   and a sample of water was retained and fixed with saturated HgCl2 (>7 g L-1) for calcification assays. Tile communities were allowed >30 min dark adaptation in the water baths under black lids, before the chambers were closed again and the process was repeated in the dark. Light and dark calcification rates were determined with the alkalinity anomaly technique [et al. [T is only affected by the removal  or addition (dissolution) of CaCO3, however some organisms in the tile communities (e.g. molluscs) would have altered AT through the release or uptake of nutrients [T from blank-tile control chamber values that ranged from -219 to -6 μmol kg-1 SW in the light, and -76 to +92 μmol kg-1 SW in the dark. Primary AT standards  were titrated to calculate titrant concentration, and replicate secondary seawater standards (n = 15) were interspaced and titrated throughout incubation samples and were highly consistent (SE = 2.19 μmol kg-1 SW). Rates of gross photosynthesis and dark respiration (μg O2 cm-2 min-1), and light and dark calcification (μM CaCO3 cm-2 min-1), were calculated by subtracting the values of blank tile O2 or AT values, at the end of the incubation runs, from values of chambers with tile communities, and then standardised to incubation time and planar surface area of each tile. Changes in the surface area of the tiles due to differences in settling benthos were not accounted for. Estimates of daily net photosynthesis and calcification were calculated by combining light and dark rates, assuming a square profile of 11.5 hrs light and 12.5 hrs dark. This daily estimate equated to a cumulative daily light integral of 7.45 mol photon m-2 d-1, which is similar to those at the study site and depth [Light incubations were conducted in black bins under four white fluorescent tubes (10 000K) at their maximum output of 180 μmol photons mechnique , using o [et al. . The alkutrients . This woutrients ,44, and utrients . The incnd depth .T, and across Reef (Dobu and Upa-Upasina) and Time (five vs 13 month censuses). Median pHT was used in models as it was directly measured many times at each tile , and correlated better with the other carbonate chemistry parameters than AT (which is not directly affected by CO2 addition). Percent cover data were fit using a quasibinomial distribution accounting for the larger proportion of values at the upper and lower bounds of the distribution. Non-significant (p > 0.05) main effects and interaction terms were removed in the final models. For comparison to control values , percent coverages of taxa were also predicted for seawater pHT 7.80 or ΩAr = 2.50, using the predict function in R, as this value may be expected by the year 2100 in tropical oceans [Generalised linear models (GLM) were used to examine how percent cover of the different benthos OTUs on the tiles differed with median pHl oceans .T at each tile as a continuous quantitative variable.Redundancy analysis (RDA) was also conducted to visually examine how the community data separated out in two-dimensional space, and to further test the significance of each explanatory variable via permutation. The explanatory variables in the analysis included reef and census period as categorical factors, as well as the median pHT of the tile origin as predictors for measurements of gross photosynthesis and respiration, and Reef and ΩAr for calcification. For the calcification assay, two chambers opened, and five outliers  were removed from final models (n = 81). A second series of GLMs was conducted on the metabolism measurements which incorporated the carbonate chemistry of the tiles, as well as the taxonomic benthic cover data (n = 66 due to the loss of 15 photos). An identity link function was used when response parameters approximated a Gaussian distribution. Quasipoisson distributions and log link functions were used for over-dispersed data. The appropriate model distribution was selected by comparing dispersion factors, preferring those which approached a value of 1. All statistical procedures were conducted with the statistical software R version 3.2.5 [GLMs were fitted to the metabolic measurements from all tiles, using Reef and median pHon 3.2.5  using th2 gradients at both the Dobu and Upa-Upasina seeps (T over the tiles ranged from 8.0 ± 0.001 (SE) at the control sites of both reefs, to 7.7 ± 0.10 as lowest values at the Upa-Upasina seep, and to 7.4 ± 0.10 at Dobu seep (Ar) was 3.84 ± 0.05 at the control sites, declining to 2.10 ± 0.25 and 1.16 ± 0.43 at the Upa-Upasina and Dobu seeps, respectively (Ca) values decreased from 5.75 ± 0.10 at the control sites along the carbonate chemistry gradient to 3.14 ± 0.38 and 1.73 ± 0.64 at the Upa-Upasina and Dobu seep, respectively. While none of the communities were exposed to median ΩAr values <1.0, 55% had median ΩAr <3.0, a value which has been suggested to be the limit for reef development [The settlement tile communities were developed along clear COna seeps . Both coobu seep . Median ectively . Similarelopment .2, CT and AT were also spread along the carbonate chemistry gradient. The daytime pCO2 at the control sites averaged 392.15 ± 9.32 μatm, increasing to 1008 ± 291.99 and 2253 ± 759.63 μatm at Upa-Upasina and Dobu seeps respectively. Control CT values averaged 1926 ± 0.70 μmol kg-1, and increased to >2100 at the seeps. Median AT values were slightly elevated within the seep sites (perhaps due to CaCO3 dissolution), and differed from control values by no more than 6%. AT values increased from an average of 2252.68 ± 3.33 μmol equivalents kg-1 at the controls to 2339.60 ± 16.29 and 2329.68 ± 18.89 μmol equivalents kg-1 within the Upa-Upasina and Dobu seeps, respectively , and macroalgae (on the lower sides of high CO2 tiles).Results of the RDA agreed with the GLM analyses: all explanatory variables accounted for significant variation in benthic communities , and tile pH explained more of this variation than differences between census periods or reef . In the ordination, the first RDA axis separated communities between seep and control sites. This was primarily driven by high cover of cyanobacteria, macroalgae, and green and brown filamentous algae in the high COol sites . Communius algae . At 13 m2 cm-2 hr-1, increasing by 10% to 11.54 ± 0.3 μg O2 cm-2 hr-1 at pHT 7.8. Increased gross photosynthetic rates were also positively related to the increasing cover of non-calcifying algae in the upper communities, but declined with the increasing cover of non-calcifying invertebrates along the pH gradient  to those at pHT 7.8 (6.21 ± 0.2), and continued to increase along the pH gradient . Given rates of light calcification were greater than those in the dark, 90% of the tiles recorded positive daily net calcification, with negative rates all observed at <2.3 ΩAr .Rates of community calcification in the light and dark, and net 24-h community calcification, all declined along the Ωgradient . Mean ligradient , Table 2<2.3 ΩAr . Interesgradient , where Ω2 and community composition also affected community metabolism; rates of gross photosynthesis and respiration increased with increasing CO2, and with the cover of certain taxonomic groups, while 24-h net calcification decreased to low or even negative values.Ocean acidification is predicted to fundamentally alter benthic marine communities. Here we report a drastic shift in the composition and metabolism of early successional benthic coral reef communities along seawater carbonate chemistry gradients. The carbonate chemistry explained a far greater amount of change in communities than successional changes from five to 13 months, and differences between the two reefs. Shifts were more pronounced in the algae compared to the invertebrate communities, where a suite of non-calcifying algal groups largely replaced CCA on seep site tiles. Changes in CO2 seeps in the temperate Mediterranean [The present study adds to the mounting body of evidence predicting ecosystem-wide changes in benthic communities under OA. Here we found rapid increases in non-calcifying algal cover as pH declined along the carbonate chemistry gradients, and little evidence of threshold responses for these taxa. This pattern was largely consistent between light exposed and cryptic communities, with green filaments establishing dominance on the upper sides, and cyanobacteria and macroalgae on the lower sides. While results are not universal , patternerranean ,49–51, aerranean ,12, concerranean  and anemerranean , may alserranean , howevererranean , which m2 gradient. The cover of the lightly calcifying algae Peyssonnelia spp. increased at lower pH, albeit only in high-light environments. Some Peyssonnelia species have increased in abundance at other seep sites [T for photosynthesis. The steep decline in CCA cover is consistent with data from multiple seep sites [Peyssonnelia spp. on the upper tile surfaces, indicates light intensity is playing a role in the response of these taxa to OA.Calcifying algae on the settlement tiles displayed dissimilar results between taxa along the COep sites ,51, suggep sites ,53, perhep sites ,12 and iep sites  with potep sites . The ste2, however a previous study found the abundances of ascidians on natural reef substrata also declined with CO2 exposure at the Milne Bay seeps [3 skeletons are somewhat protected from the surrounding seawater through external tissue layers [2, with some species negatively responding, while species with phototrophic symbionts or siliceous spicules may respond positively [Invertebrate responses to carbonate chemistry changes varied between taxa, and overall their cover and abundances did not respond as strongly as the algae did. It is important to note that the invertebrate communities in the present study were in relatively early successional stages, and consisted of shade-adapted communities without scleractinian corals (no invertebrates were found on the upper tile surfaces). The number of tube-dwelling polychaetes per tile declined with pH, as did the cover of ascidians. Similar declines were seen in tube-dwelling polychaete species at a Mediterranean seep , possiblay seeps . Our stue layers ,57. Sponsitively .Peyssonnelia spp., other macroalgae and the invertebrate groups) changed between census periods, this did not significantly alter the patterns in the rest of the tile communities. For example, patterns along the CO2 gradients in the cover of non-calcifying algae and CCA, which accounted for the majority of the tile communities, were largely consistent between census periods. This consistency between census periods contrasts a similar study at Mediterranean seeps, where Kroeker et al. [et al. [Patterns in the 13 month old tile communities were largely established in the first five months, and successional changes between census periods were considerably weaker than the influence of the carbonate chemistry gradient for the majority of taxa. While the cover of some ephemeral  and slower growing taxa . Previous work at the Milne Bay seeps has similarly shown higher turf and macroalgae cover, and lower CCA cover, on natural substrate within the seep reefs compared to control reefs . Regardlin situ under high CO2. Here we documented a 10% increase in gross photosynthesis and a 20% increase in respiration at pHT 7.8 compared to control sites with a pHT 8.0, but no change in net community production. Gross photosynthesis may increase under OA by directly stimulating photosynthesis [per se. Respiration may increase as a consequence of increasing biomass or increased metabolism. Biomass estimates are unavailable for the tiles, however our models indicated that declining seawater pH, and increasing invertebrate cover, both significantly contributed to the observed increase in respiration at lower pH.The present study presents the first investigation of metabolic changes for combined surface and cryptic subsurface reef communities that have developed entirely ynthesis ,31, and/ynthesis ,20,27,28Ar gradient, and our models indicated that it was these changes in ΩAr, rather than shifts in tile communities, that were responsible. While results are not universal, OA is widely reported to reduce calcification in individual coral reef organisms [et al. [3 substrata decreased linearly along CO2 gradients, and became negative by pHT 7.8. The stronger response found by Enochs et al. [3 blocks as settlement substrata, and attracted many macro-boring organisms, yet did not include cryptofauna on the lower surfaces.OA is likely reducing reef calcification rates on coral reefs ,26. Lighrganisms  and at trganisms –20,29. F3 [et al. [2, with half of this being attributed to sediment decalcification. Some estimates suggest that even if coral calcification rates are maintained under OA, the dissolution of carbonate sediments alone would result in reef loss [3 substrata, are thus likely to considerably under-estimate reef-wide dissolution rates expected under OA. Instead, they provide insight into how the calcification dynamics of a part of the biological components of coral reef communities may respond.When predicting OA effects on coral reef calcification, one must also take the permeable carbonate matrix and sediments into consideration. These are the largest sources of reef CaCO3 , and the3 ,23,59,60 [et al.  documenteef loss . Results2 seep sites from perfectly representing the future of the world’s oceans. Firstly, they are relatively small, and scaling up predictions to the world’s coral reefs will undoubtedly introduce some uncertainty. Secondly, the altered carbonate chemistry at the seeps is occurring in isolation from the warming that is also predicted for a high CO2 world [T is slightly elevated , which may increase calcification rates at the seep sites [T, however sediment dissolution- and dilution-rates from the surrounding seawater are largely unknown [There are several factors that preclude COO2 world , and theO2 world . Thirdlyep sites . Increas unknown . And fin unknown  with unc unknown . On the 2 analogues [in situ seasonal comparisons [3 accumulation rates will likely decline, owing to the carbonate chemistry changes and the dissolution of carbonate sediments and increased bio-erosion [2 emitted into the atmosphere, the less deviation we will see from the reefs of today.Results from the two seep sites investigated here, as well as other naturally occurring high COnalogues –14, genenalogues ,10,65 tonalogues ,25,29,66parisons ,44 and qparisons –69. All -erosion . Unfortu-erosion ,26 have S1 FigPercent cover of the non-calcifying algal groups responsible for the increases seen along the pH gradient at Upa-Upasina (white points) and Dobu (grey points): green filamentous algae on the upper tile sides (a) and cyanobacteria (b) and macroalgae (c) on the lower tile sides. Left and right hand panels represent the five and 13 month census periods, respectively. The black lines represent the modelled means, while the grey lines are confidence intervals.(EPS)Click here for additional data file.S2 FigPercent cover of the sum of non-calcifying (a) and calcifying (b) invertebrates on the lower tile sides at Upa-Upasina (white points) and Dobu (grey points) in relation to median pHT. Left and right hand panels represent the five and 13 month census periods, respectively. The black lines represent the modelled means, while the grey lines are confidence intervals.(EPS)Click here for additional data file.S1 TableSome OTUs were further classified into more general grouping for analysis (Group).(DOCX)Click here for additional data file.S2 TableStandard errors are shown in brackets. N = 2 and 4 per control and seep site.(DOCX)Click here for additional data file.S3 TableUp and low refer to the upper and lower sides of the tiles, respectively. Non-significant terms removed from final models. Caption as in (DOCX)Click here for additional data file.S4 TableAr was used in calcification models. For calcification, the inclusion of the biotic OTUs did not improve on the GLM fit to ΩAr. Generalised linear model results, with G and Q denoting Gaussian and Quasi Poisson distributions used respectively, and ^0.25 indicates square root transformation. Caption as in Median pH was used as predictor in oxygen production and consumption models , while Ω(DOCX)Click here for additional data file.


The concordance rate of PD-L1 between primaries and metastases and correlation with clinicopathological factors were analyzed. PD-L1 expression was higher in squamous cell carcinoma, wild-type EGFR, and smokers than in non-squamous carcinoma, mutant EGFR, and never smokers, respectively. PD-L1 positivity was observed in 18.9% of primaries and 21.6% of metastases. IHC demonstrated 78.4% concordance of PD-L1 positivity between primary and metastatic cancers. In 10.8% of cases, PD-L1 positivity was higher in primaries than in metastases, and vice versa in the remaining 10.8%. By PD-L1 RISH, 35.1% of primaries and 27.0% of metastases demonstrated PD-L1 positivity. There was 62.2% concordance in PD-L1 by RISH between the primaries and metastases. Our results thus highlight the clinical importance of replacing metastases with primary archival tissue, particularly when re-biopsy is difficult at recurrence.We investigated the concordance of programmed death-ligand 1 (PD-L1) expression between primary cancer at initial diagnosis and metastasis at recurrence in resected non-small cell lung cancer (NSCLC). PD-L1 expression was evaluated using the SP142 assay in 37 NSCLC patients with paired primary lung cancer and surgically resected metastases at recurrence. PD-L1 positivity was defined as immunohistochemistry (IHC) and also evaluated by RNA  Non-small cell lung cancers (NSCLCs) comprise nearly 80% of lung cancers and have a dismal prognosis, with an overall 5-year survival rate of only 15% –7. PD-1 Currently, PD-L1 expression evaluated by immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissue samples is used as a companion diagnostic test to predict the response to PD-1 blockade –5, 10. Tin situ hybridization (RISH) might circumvent these limitations [et al. recently demonstrated that PD-L1 mRNA expression above the detection threshold showed statistically better outcomes by RISH than by IHC in two cohorts of NSCLC patients [PD-L1 positivity has been found to be a predictive factor for non-squamous histology but not for squamous cell histology , 3. Althitations , 19, 20.patients . The vispatients , 19. SucHere, we aimed to investigate the concordance of PD-L1 level between archival primary tumors at initial diagnosis and metastasis at recurrence in surgically resected NSCLC by IHC and RISH.EGFR mutations, including L858R at Exon21 (n = 3), Exon19 deletion (n = 4), and Exon 20 insertion (n = 1). The remaining 29 patients (78.3%) harbored wild-type EGFR. The sites of resected metastatic tumor were the lung , brain , pleura , and others . The median time from initial diagnosis to recurrence was 17.8 months .The baseline demographics of the 37 patients analyzed are summarized in Table EGFR than in adenocarcinoma and mutant EGFR, respectively  of primaries and 21.6% (8/37) of metastases to be PD-L1 positive. PD-L1 expression was found to be correlated with clinicopathological features. In primary tumors, higher PD-L1 positivity was observed in squamous cell carcinoma (SCC) and wild-type  of primaP = 0.02); in 10.8% cases, the PD-L1 score of primaries was higher than that of metastases, and in the remaining 10.8%, it was the other way around ; in 24.3% of cases, the PD-L1 score of primaries was higher than that of metastases, and vice versa in 13.5% cases  of primaries and 27.0% (10/37) of metastases were PD-L1 positive. PD-L1 RISH positivity tended to be high in SCC but low in never/light smokers or individuals with s Figure . We alsovs. 12.5 months, P = 0.06) and those without PD-L1 RISH of metastasis showed numerical improvement of RFS  surgical resection of primary NSCLC with a curative aim at initial diagnosis, (2) surgically resected metastatic lesions at recurrence, (3) availability of paired primary and metastatic tumor tissue, and (4) availability of clinical data on smoking status and survival. Paired tumor samples from 37 patients were used for examining PD-L1 expression. Tumors were classified according to the seventh American Joint Committee on Cancer (AJCC) TNM cancer classification system and the World Health Organization system. A predesigned data collection format was used to review the patients’ medical records for evaluating clinicopathological characteristics and survival outcomes. The study was approved by the Institutional Review Board of Severance Hospital.Sections of FFPE tissues were prepared and stained with hematoxylin and eosin. IHC was performed on 4-μm tissue sections using the Ventana Bench Mark XT Autostainer  and the SP142 antibody . PD-L1 positivity was defined as a membranous staining intensity of ≥5%. IHC scoring was done on a 0–2 scale  . The sem® eZ-L assay  on the Leica Bond III Immunohistochemistry and ISH Staining System (Leica Biosystems). Each case was assayed using probes for PD-L1. The ViewRNA eZ Check Human probe  was used as the positive control to determine RNA integrity. The Bacillus subtilis probe DapB was used as the negative control to determine assay background. FFPE sections on slides were processed automatically from deparaffinization to ISH staining and hematoxylin counterstaining. Briefly, 4-μm-thick FFPE sections were baked for 1 h at 60°C and placed on the Bond III for processing. Next, slides were rinsed with water, air-dried for 30 min at room temperature, mounted using Dako Ultramount , and visualized using a standard bright-field microscope. Punctate dot-like red hybridization signals in the cytoplasm of tumor cells indicated positive staining. RISH scoring was done on a scale of 0–3 as follows: 0, <1 dot/cell at 400× magnification; 1, 1–5 dots/cell at 400× magnification; 2, 6–20 dots/cell at 400× magnification; and 3, >20 dots/cell at 400× magnification. At least three high-power fields were analyzed before assigning an RISH score to each case.RISH was performed on FFPE sections using the ViewRNA2 correction or Fisher's exact test for categorical variables. To compare dichotomized values, we calculated the proportion of discordance between both procedures together with 95% confidence intervals, as well as Cohen's κ coefficient of agreement. Survival variables were estimated using the Kaplan–Meier method. Categorical variables were compared via the log-rank test, and quantitative variables by a Cox regression model and the associated Wald chi-square statistic. Overall survival (OS) was defined as the time from the initial diagnosis until death or the most recent follow-up. Relapse-free survival (RFS) was measured from the time of surgery to initial tumor relapse  or death from any cause. Patients with no signs of relapse were censored at the most recent follow-up or death. The median follow-up duration for the overall population was 46.3 months. A two-sided P-value of <0.05 was considered statistically significant. All the statistical analyses were performed using SPSS 20.0 for Windows.Correlations between immune markers and patient characteristics were analyzed using the chi-squared test with χ


The prepared nano-composite was further characterized via XRD, SEM, TEM, Raman and XPS. The photocurrent analysis was further tested for its photocatalytic reduction of CO2 using gas chromatography (GCMS-QP2010 SE). To further improve the the photo-catalytic efficiency, a sacrificial agent (Na2S/Na2SO3) was added to the WSe2-graphene nanocomposite and was found to improve the photo-catalytic efficiency, with the methanol yield reaching 5.0278 µmol g−1h−1. Our present work provides a convenient way to prepare nanomaterials various morphologies that have future applications for environmental remediation.A nanowire-like WSe One such technology is photocatalytic CO2 reduction, which is a most valuable approach to overcome both global environmental and energy problems due its low cost, clean energy production, and environmental friendliness3. In particular, the photocatalytic reduction of CO2 can convert noxious gasses (carbon dioxide) into profitable solar fuels, i.e., CO, HCHO, CH3OH, and CH4 by using solar energy5. To date, many photocatalysts such as TiO2, CdS, g-C3N4, ZnO and Bi2WO6 have been shown to achieve the photo reduction of CO210. Unfortunately, there are limits to the practical applicability of these semiconductors because the efficiency of the CO2 conversion into useful products is very low due to many factors of the CO2 reduction process, e.g., band gap tuning, charge carrier recombination, and light utilization11. Due to the wide band gap energy, most materials are unable to absorb visible light, and on the other hand, small band gap semiconductors exhibit a fast recombination phenomenon. To overcome this and to find an achievable process, highly efficient, low cost transition metal dichalcogenides (TMDC) have been investigated15. The generalized formula for TMDs is MX2, where M = transition metal element of group 4, 5 or 6 and X represents a chalcogen. Tungsten diselenide (WSe2) is a semiconductor that belongs to the TMDC family. Its structure consists of Selenium and tungsten held together by a weak van der Waals force that allow the material to be exfoliated into monolayers17. Moreover WSe2 is a layered semiconductor with a small band gap of approximately 1.6 eV18. It has unique electrical transport performance, and its use was recently reported in many advanced energy storage applications, such as in superconductors19, lithium ion batteries20, photodetectors21, and photocatalytic hydrogen production22 in solar cells. Furthermore, Li, Yanguang et al. and Merki, Daniel et al. indicated that both the theoretical calculation and experimental results for the hydrogen evolution reaction of transition metal dichalcogenide (WSe2) materials appears to be from active edge sites that play an important role in hydrogen evolution reaction catalysis24. However, due to the intrinsic interlayer Van der Waals attraction, the layered TMDC materials can be easily re-stacked as a result of reducing the active edge sites. Therefore, to improve photocatalyitc activity, the TMDC (WSe2) materials are combined with 2D carbon material such as graphene.The increase in the amount of the CO26. Therefore, coupling with a photocatalyst may improve the photocatalytic reduction efficiency of CO2 to fulfill the practical requirements. Liange et al. first described that coupling with a semiconductor is one of the best ways to boost the photocatalytic reduction efficiency of CO227. Recently, graphene coupled with TMDCs (WSe2) was reported for the hydrogen evolution reaction (HER)28, high-performance oxygen reduction reaction (ORR)29 and in a superconductor30. However, the WSe2 chalcogenide family has not yet been analyzed for the photocatalytic reduction activity of CO2. In the present study, we report on WSe2-graphene nano composites prepared via ultra-sonication. The prepared samples (WSe2-graphene) were used for CO2 reduction, and the results exhibit a high efficiency for photo catalytic CO2 conversion under UV and visible light irradiation.Graphene has unique properties, including a large surface area, good conductivity, and high flexibility, and it is used in high-performance energy storage devices2-graphene nanocomposites is shown in Fig. 29. However, no other diffraction peaks form any other chemical species were observed in the prepared sample. For the GO analysis, a sharp peak (002) occurs at 13.44°, revealing that the graphite power was converted into graphene oxide by expanding the d-spacing from 3.5 to 6.78 Å32. Moreover, the results indicate that the XRD signals of GO are very weak and are overlapped with those of WSe2-graphene at 13.54°. Therefore, the measurements were unable to detect the weaker diffraction peaks of GO33. Further EDX spectra confirm the presence of the main elements in the catalyst composites. The Fig. The XRD patterns of the WSe2-graphene at different magnifications. Figure 2 morphology composed of interlaced and ultrathin nanosheets uniformly dispersed on the graphene nanosheet. Figure 2 nanowires extensively grow on the surface of the graphene nanosheet. Figure 2 shapes that are properly coated on the graphene surface, indicating that graphene nanosheets provide a good platform for the nucleation and successive growth of WSe2 layers, and growth on WSe2 in graphene is possible due to the precursors that are attached to the graphene oxide through functional groups 36. Figure 2-graphene nanocomposites. Figure 2-graphene nanocomposites at different magnifications. The WSe2 is clearly seen to have dark imaged compounds that are almost very small in a spherical form, layer form, and a highly agglomerated structure attached to the surface of the graphene sheets. Moreover, several layers of the WSe2 are covered with the graphene nano sheets, and the average size of the WSe2 is measured to be approximately 6 to 10 um using the ImageJ software.SEM observations were carried out to investigate the surface and structural morphology. Figure 2 nanosheets. Different WSe2 flakes indicating that the particles sizes fluctuate between 30 nm to 190 nm. When compared to the bulk material, the size of the WSe2 layers decreases, which is may be due to the discontinuity of the particles induced by sonication and particle selection by centrifugation37. The precise structural properties of the WSe2-graphene were analyzed via Raman spectroscopy. Figure 2 nannocomposites , Fig. 2 located at 200 to 400 cm−1. The typical Raman spectra for WSe2 shows a band at the A1g (out-of-plane) (255 cm−1) and E12g (inplane) mode (250 cm−1)38.Figure −1, which shows the presence of disorder in the atomic arrangements or edge effect of graphene, while the G band appears at 1590 cm−1. Both the G and D band show the vibration of the carbon atoms in disorder or defect sites and the in-plane vibration of sp2 and sp3 bonded carbon atoms39. These two bands  indicate an interaction between rGO and selenide nanosheets, which improves the stability of the catalyst interface and provides a high stability for the nanocomposites. A 2D band appears at 2690 cm−1 to express the degree of graphitization. Moreover, the 2D band is smaller than the G band in our spectrum, which indicates the presence of a few layers of graphene sheets43.Figure 2-graphene nanocomposite. Figure 2-graphene nanocomposite. Figure 7/2, Wf5/2 and W5p3/2 at positions of 31.90, 34.80 and 37.90 eV, respectively. The W4f7/2 and Wf5/2 binding energy peaks express the elemental chemical binding state of W, while the peak positioned at 37.70 eV is attributed to the core level of W5p3/2 from WO3 due to the partial oxidation of the WSe2 layers45. The binding energy for the Se 3d3/2 core level peak of 54.90 eV confirms a lattice Se−2 of the WSe2-graphene hybrid  and visible (λ > 400) light irradiation, and every 12 h interval sample withdraw manually from the reactor using a gas-tight syringe, then the collected sample was centrifuged and characterized via gas chromatography. Figure 3OH yield for the pure WSe2 and WSe2-g nanocomposites. The control experiment determined that no methanol yield was produced in the absence of photocatalysts or light irradiations or both. The gas chromatographic study shows that only CH3OH was successfully achieved as a reduction product. Furthermore, graphene is an electron donor for the reduction of CO2 as a substitute carbon source to produce CH3OH, and H2O was a reactant of the CO2 reduction. Figure 2 with pure WSe2 and WSe2-graphene under UV light was higher than that for the same composite under visible light.We assessed the photocatalytic activity of the pure WSe2-graphene nanocomposite with different time intervals and different conditions under UV/Visible light irradiation are shown in Tables 2-graphene (12 h), WSe2-graphene (24 h), WSe2-graphene (36 h) and WSe2-graphene (28 h) under visible light were 2.2434 (0.3285), 2.7021 (0.4435), 3.3243 (0.5346) and 3.5509 (0.5779) µmol g−1h−1, and under UV light were 2.8854 (0.4596), 3.1835 (0.3165), 3.7778 (0.6040) and 4.3257 (0.7053) µmol g−1h−1, respectively. Na2SO3 was used as a sacrificial reagent to further enhance the catalytic activity of the binary graphene-based nanocomposites in the photoreduction of CO2, as shown in Fig. 2 and WSe2-graphene nanocomposite under UV/visible light is almost two times greater than that of the nanocomposite without using scavenger (Na2SO3), and Fig. 3OH, the products were recovered after 18 h and 24 h of irradiation and analyzed by 13 C NMR (Proton decoupled)  was reused for six consecutive runs, and only a minor change in the CH3OH yield rate was found, indicating that the prepared nanocomposite highly stable and can be used for a continuous photocatalytic reduction system of CO2.The methanol yields and quantum yields for the WSeraphene 2 h under ed) Fig. . A singl2 nanomaterial absorbs light of the solar spectrum and creates photo-generated charge carriers (holes and electrons). However, due to the narrow bandgap of the WSe2 nanocomposite, these electron-holes recombine very quickly, and their photocatalytic efficiency is therefore limited. To improve the photocatalytic efficiency, the WSe2 nanocomposite was attached to a graphene nanosheet since graphene is an electron accepter/transporter that plays an important role in the separation of the transport electron-hole pairs in the binary system55. The excited electron and hole in the conduction band of WSe2 can be conveniently shifted to the graphene nanosheet, which decelerates the recombination of the electron-hole pairs and thus promotes the electron transport to the catalytic sites for the photo reduction of CO2. The graphene with a large surface area and many defective sites absorbs the CO2, and the photo-generated electrons on the WSe2 are transmitted to the catalytic sites of the graphene and then reduce the absorbed CO2 into CH3OH56. However, the photocatalytic reduction mechanism contains a series of water oxidation and the reduction processes, as shown in Fig. 2-g VB could absorb water molecules to form hydroxyl radicals (OH·), and then the hydroxyl radicals further oxidize the protons (H+) and oxygen. In the meantime, electrons in the conductor band transfer and absorb CO2 to form ·CO−2. The ·CO−2 reacts with ·H radical, which then leads to the formation of a series of radicals, finally producing CH3OH.A further explanation of the proposed photocatalytic mechanism is given in Fig. Reaction Mechanism.2 reaction.CO3), selenium powder , ammonium hydroxide , Sodium sulfite , Nitric acid (HNO3) and ethyl alcohol (94%) were purchased from Duksan Pure Chemical Co. Ltd., Korea. All chemicals were used without further purification, and all dilutions were carried out using distilled water.Tungsten (VI) oxide  are put in de-ionized (DI) water and are stirred continuously for one hour at 0 C. After that, 45 g of KMnO4 are slowly mixed with the solution (graphite + H2SO4) and are constantly stirred at a temperature of 35 °C until it becomes a dim brownish color. Then, the container is sealed and kept at 100 °C with vigorous stirring for 30 min. Meanwhile 20% H2O2 is added drop wise within 5 min. After that, the solution is washed with acetone and 10% HCl several times to remove the residual metal ions. The solution was then heat-treated in a dry oven at 90 °C for 12 h to obtain the graphite oxide power, then 250 mg graphite oxide power were added to 200 ml DI water, were vigorously stirred for 30 min, and were then ultrasonicated  for 2 h. Finally, the resulting solution was refined and washed several times with hot water and kept in a dry oven for 6 h to obtain the graphene oxide powder.Graphene oxide was prepared in the laboratory following Hummer-Offeman’s method, as previously reported in the literature3) are dispersed in deionized water, 0.5 M nitric acid are then added drop wise in a three-necked flask (100 mL), and the mixture is heated to 120 °C to eliminate H2O and O2. In a separate flask, 1.5 g of anhydrous sodium sulfite (Na2SO3) and 0.3 g crude selenium (Se) powder were dispersed in 200 ml of ethylene glycol with continuous magnetic stirring at 80 °C until a selenium salt was obtained. In the next step, both solutions are transferred to a stainless steel autoclave with a Teflon liner with 20 mL capacity for 24 h at 250 °C in an electric furnace. Finally, the WSe2 precipitates are cooled to room temperature, the prepared solution is filtered using 47-mm Whatman filter paper, and the remaining material is heated to a temperature of 350 K for 12 h to obtain a WSe2 power.In a typical synthesis process, 0.675 g tungsten (vi) oxide . The reaction solution was allowed to cool and settle at room temperature after filtering with 47-mm Whatman filter paper with a pore size of 0.7 mm. The resulting powder was washed with distilled water multiple times and was dried in a vacuum oven at 80 °C for 12 h before heat treatment at 500 °C for 1 h with (Ar) inert atmosphere. The prepared sample was then labeled as WSe2-graphene.Graphene oxide (200 mg) was added in 150 ml ethylene glycol and was then exfoliated to generate a graphene oxide nanosheet (GONS) dispersion solution via ultrasonication for 30 min. The WSe3OH, a Standard Gas Chromatograph-Mass Spectrometer (GCMS-QP2010 SE) was used with a long column. Photoelectrochemical measurements were performed using a self-made photoelectrochemical system installed a 250-W halogen lamp as the irradiation source. The photocurrent measurement was performed by a computer-controlled Versa-STAT-3 electrochemical analyzer. A WSe2-graphene modified photoelectrode with an active area of 1 cm2 was used as the working electrode, and a Pt wire and saturated Ag/AgCl were used as the counter and reference electrodes, respectively. All the photocurrent measurements were conducted by dipping the WSe2-graphene modified photoelectrode into a mixture of 0.1 M KCl and 0.5 M TEA at a constant potential of 0 V vs. Ag/AgCl.The crystal structure and morphology of the prepared samples were measured using monochromatic high intensity Cu Kα radiation (λ = 1.5406 Å) in XRD (Shimadzu XD-D1), Energy dispersive X-ray spectrometer (EDX) was used to measure the atomic percentage of W, Se, and C elements, SEM  and TEM  observations. X-ray photoelectron spectroscopy (XPS) was performed using a VG Scientific VISACA Lab 2000 device with a monochromatic Mg X-ray radiation source, and the Raman spectra of the prepared samples were observed using a spectrometer (Jasco Model Name NRS-3100) with an excitation laser wavelength of 532.06 nm. For quantitative analysis of the CH2 with H2O in the photocatalytic experiments was carried in a reactor designed in our laboratory, as shown in Fig. 2 + N2 gas (N2 gas was used to remove gasses from the reactor). 100 mg of photocatalyst (WSe2-graphene) and Na2SO3 as hole scavenger59 were added in 20 ml distilled water containing sodium bicarbonate  and were constantly stirred for one hour. Ultra-high-purity grade CO2 gas was purged through the reactor for 30 min, and then the suspension solution was magnetically stirred and irradiated with visible light using a metal halide lamp . The distance between the light source and the photocatalyst remained at 10 cm, and a heat sink was equipped in the left side of the chamber to remove the lamp heat. Furthermore, the temperatures inside the reactor were kept at 283.15 K. The reaction continued up to 48 h, and in every 12 h interval, the reactor was allowed to cool down naturally for CH3OH desorption from the catalyst. Then, the reaction product was taken from the reactor for GC analysis (GCMS-QP2010 SE).The reduction of CO3OH yield, noting that six electrons are required to reduce CO2 to CH3OH. The equation is as follow.−2)The reaction quantum yield (QE) is estimated using the CHS is the irradiated area of the reactor (30 cm × 15 cm)−19 J at 400 nm)E is the photon energy (4.97 × 10−1)NA is Avogadro’s constant (6.02 × 1023 mol2) materials are attached to the graphene nanosheet via ultra-sonication. The SEM and TEM images of the prepared samples show that the WSe2 have nanowire morphology that is uniformly distributed on the graphene sheets, and the average size of the nanowires is verified to be from 30 to 130 nm. The graphene sheet-supported WSe2 nanowire resulting from the binary structure present excited charge carriers with the combined effect of WSe2 and graphene, improving the recombination time. Raman spectroscopy and XPS measurements show an intimate contact and chemical binding interaction between the WS2 and GO. Graphene plays a role as an electron mediator to support a binary system and to help provide stable photocatalyst materials. The CO2 photo reduction experiment was carried out to investigate the photo catalytic reduction of CO2 with the WSe2-G nanocomposite, achieving a maximum photocatalytic efficiency after 48 h. The WSe2-graphene with added Na2SO3 (48 h) showed the highest photocatalytic efficacy and obtained a total CH3OH yield of 5.0278 µmol g−1h−1. Our present work indicates that the attachment of WSe2 on the graphene sheet can further increase the photocatalytic performance, opening new ways to deploy novel, next-generation heterojunction photocatalysts for environmentally-related applications.In summary, the TMDC (WSe


Surface enhanced Raman spectroscopy (SERS) is a novel method to sense molecular and lattice vibrations at a high sensitivity. Although nanostructured silver surface provides intense SERS signals, the silver surface is unstable under acidic environment and heated environment. Graphene, a single atomic carbon layer, has a prominent stability for chemical agents, and its honeycomb lattice completely prevents the penetration of small molecules. Here, we fabricated a SERS substrate by combining nanostructured silver surface and single-crystal monolayer graphene (G-SERS), and focused on its chemical stability. The G-SERS substrate showed SERS even in concentrated hydrochloric acid (35–37%) and heated air up to 400 °C, which is hardly obtainable by normal silver SERS substrates. The chemically stable G-SERS substrate posesses a practical and feasible application, and its high chemical stability provides a new type of SERS technique such as molecular detections at high temperatures or in extreme acidic conditions. The origin of the intense Raman signals in SERS is enhancement of local electromagnetic fields via the localized surface plasmon resonance effect4 in nanostructured metal surface or metal colloids. The main contribution of SERS enhancement is electromagnetic (EM) enhancement up to 1010 times7, and another minor contribution is chemical enhancement including the resonance Raman effect, the charge-transfer effect, and the adsorption effect8. Coinage metals such as silver, gold, and copper, are the most common elements for SERS due to its large SERS enhancement and well-known fabrication methods. Gold and copper have interband absorption in the visible wavelength range, which decreases the maximum SERS intensity9. On the other hand, interband absorption of silver is located in the ultraviolet wavelength range, which leads the largest SERS intensity for visible light among these metals10.Surface-enhanced Raman spectroscopy (SERS) is a novel method to sense molecular and lattice vibrations11. Therefore, graphene can be useful for a prominent protecting layer of nanostructured silver surfaces for SERS. Besides, graphene has only 2.3% absorption from near infrared to visible range12, minimizing the loss of SERS signals from silver.Despite the superior enhancement, silver is difficult to use in acid or heated environment with oxidative gas because of dissolution or oxidation of silver. Graphene, a single atomic carbon layer, has a prominent stability for chemical agents, and its honeycomb lattice completely prevents the penetration of small molecules like hydrogen and water14, reduced graphene oxide17, mechanically exfoliated graphene from bulk graphite19, and graphene grown by chemical vapor deposition (CVD)22. Although there are a lot of works on SERS using graphene, there are no reports to focus on the effectiveness of graphene as a protective layer for the plasmonic metal surfaces. Here we studied the chemical stability of graphene in the SERS substrate, consisting of silver nanostructures and single-crystal monolayer graphene24. Since the SERS for physisorbed molecules on graphene/plasmonic metal has been referred to as graphene-mediated SERS (G-SERS)19, we call our developed SERS substrate G-SERS substrate. It is found that the G-SERS substrate showed SERS even in concentrated hydrochloric acid (35–37%) and in heated air up to 400 °C, which is unattainable by the bared silver SERS substrates. The enhancements of Raman signals were observed not only for the protective graphene but also rhodamine 6G (R6G) molecules adsorbed on the graphene, indicating G-SERS can be used for the molecular sensing. Unstable peaks due to nonuniform molecular adsorption, structural change of molecules induced by laser, and metal-molecular interaction are usually observed on the bared silver SERS substrate but G-SERS substrate does not show such behavior which enables us to measure stable and reliable Raman spectra. The high chemical stability of G-SERS can be utilized for new types of SERS techniques such as molecular detections at high temperatures or in extreme acidic conditions.Composites of nanostructured silver and graphene for SERS have been widely reported using various types of graphene materials such as graphene oxide2/Si, where the graphene as a protective layer was prepared by CVD and transfer process, and the silver as plasmonic metal was deposited by sputtering. Figure 2/Si. The sputtering resulted in the formation of nanoparticles with the average height of 6.4 ± 0.7 nm which is considered as the average diameter of the nanoparticles. Figure 26, and the size of the domains was approximately 1 mm in diameter. The present CVD graphene is monolayer which is confirmed by Raman spectrum shown in Fig. 2 , graphene/SiO2/Si (middle), and G-SERS substrate (lower). To compare peak intensities, all spectra were taken at the same conditions . The number located above each spectrum shows the magnification for the intensity scale. The spectrum of Ag/SiO2 shows various intense peaks indicating SERS, although these peaks are not reproducible in peak position, width, and intensity ). The structure of the adsorbed carbonaceous species can be changed by laser heating30, which is called photocarbonization29. The photocarbonization can be seen in Fig. Figure ity Fig. . These n−1, respectively. The larger intensities of G’ than G, and small D peak  on each substrate. Prominent Raman peaks of R6G at 1180, 1361, 1507, and 1647 cm−1 for R6G/Ag/SiO2 and 1195, 1361, 1506, and 1652 cm−1 for R6G/G-SERS are observed, in agreement with previous reports36. Slight peak shifts in the R6G peaks for the G-SERS substrate would be due to the different charge transfer effect between R6G and silver or graphene. The peaks located at 1584 and 2673 cm−1 are G and G’ peak, respectively. The other peaks in R6G/Ag/SiO2 cannot be assigned, but would be from carbonaceous impurities as mentioned above. In contrast, there are no additional peaks in G-SERS, indicating that G-SERS can be suitable for molecular sensing.Figure 19. Xu et al. revealed that monolayer graphene shell on Au nanospherical dimer enhances the localized electromagnetic fields rather than decrease19. The enhancement mechanism would be same also in our G-SERS since the structure of G-SERS is basically same. The relatively small enhancement for R6G on the G-SERS would be due to structural change of Ag nanoparticles during the process of graphene transfer  with continuously taking Raman spectra. Figure ger Fig. . The sizopy Fig. . The cheopy Fig. .Figure 3−1 in Ag/SiO2/Si and of G’ peak in G-SERS with respect to time after immersing in HCl. The intensity of G’ peak initially decreases by 40%, and then keep constant, while the peak intensity of 1600 cm−1 in Ag/SiO2/Si is completely disappeared. Since the average enhancement factor is ~10 , indicating its high chemical stability.Figure ~10 Fig. , the G’ 39. As a demonstration for the G-SERS, we measured tertiary butyl chloride (TBC) which was synthesized by tertiary butyl alcohol (TBA) and HCl.To show a potential of G-SERS for practical applications, we focused on the chemical reaction which requires HCl. Normal Ag SERS substrate cannot be used in such chemical reactions, although monitoring chemical reaction is one of the SERS application2/Si substrate. For TBA, a Raman peak at 749.7 cm−1 was observed, corresponding to C-C-O (C3C-O) symmetric stretching mode40 in the molecule. The peak intensity of C-C-O stretching was 2.4 times higher on G-SERS than that on SiO2/Si. For TBC, the peak position of C-C-O was shifted to 745.8 cm−1, which corresponds to C-Cl stretching mode41 in the molecule. The peak intensity of C-Cl stretching was 1.8 times higher on G-SERS than that on SiO2/Si. The observed enhancement factors are relatively small, which would be due to weak adsorption of TBA and TBC onto the graphene.Figure 39.Since the peak shift from C-C-O to C-Cl stretching was clearly observed by G-SERS, G-SERS has enough potential to monitor such chemical reaction. For the practical application for monitoring chemical reactions by G-SERS, its enhancement ability has to be improved, for example, by designing the structure of nanoparticlesin situ Raman spectra of G-SERS and Ag/SiO2/Si at elevated temperatures in air, respectively. These spectra are taken under the same conditions , and are shown on the same intensity scale. The G-SERS sample shows several large peaks around the G peak at lower than 250 °C  before and after heating at 200 °C for 15 min  as shown in Fig. Subsequently, thermal durability of G-SERS was examined. Figure  °C Fig. . The peain Table , and fou2/Si and G-SERS. To give clear difference for the long time stability in SERS, Ag/SiO2/Si and G-SERS were heated at 150 °C in air up to 63 hours (h).To validate the protective function of graphene for long heating duration time, we measured Raman spectroscopy of benzoic acid (BA) dissolved in isopropyl alcohol (IPA) on Ag/SiO−1 on heating duration time. The intensity of BA on Ag/SiO2/Si was drastically decreased in the first 1 h, and kept nearly constant until 30 h, and completely disappeared after 63 h. The intensity of BA on G-SERS was fluctuated for the first 2 h, and reached maximum at 30 h, and decreased to be nearly half of the initial count (~1000) but existing. To examine the reason of the intensity changes in Fig. 2/Si. AFM analysis revealed that the aggregation of Ag particles proceeds gradually by belonging the heating duration time. The speed of aggregation of Ag particles is slower in G-SERS than Ag/SiO2/Si  and graphene/SiO2/Si at each temperature in air, respectively. In the G-SERS substrate  at room temperature with a silver target of 99.99% purity in argon atmosphere. The deposition pressure and rate were 1 Pa and ~1.3 Å/s. The deposited thickness (assuming uniform film structure) was measured by a quartz oscillator in the sputtering chamber to control the particle size of silver. The deposited thickness was used to control the particles size. The deposited thickness of silver ranging from 2 to 6 nm shows similar order enhancements in Raman signals for 532 nm wavelength.24. The purities of H2 and Ar gasses are 99.99999% (7N), and 99.9999% (6N), respectively. The chamber pressure has been kept to be near atmospheric pressure (0.123~0.128 MPa) during the CVD process. 100 μm-thick commercial Cu foils  with the size of ~2 cm × 10 cm were used as catalytic substrates for the growth. The grown graphene was transferred onto arbitral target substrate with a conventional method, which uses a spin-coated polymethyl methacrylate (PMMA) support layer, ammonium peroxodisulfate for etchant of Cu, and the mixture of acetone, isopropyl alcohol, and methyl isobutyl ketone for removal of PMMA47.Graphene was grown by a custom-made atmospheric pressure chemical vapor deposition (CVD) system−8 Pa and a monochromatized AlKα (1486.6 eV) X-ray source. Raman spectra were recorded with a Raman microscope  at a wavelength of 532 nm in the backscattering geometry. In situ heating experiments were carried out in the temperature controllable stage .Surface morphologies of samples were microscopically observed using an atomic force microscope  in the peak force tapping mode and an optical microscope . Surface elemental analyses were performed by XPS  under a base pressure of ~6 × 10Supplementary Information