PD-L1 or PD-L2, driving elevated expression in the corresponding gene. In addition to the previously described upregulation of the PD-1 immunosuppressive pathway in Epstein-Barr virus (EBV)-positive stomach tumors, we also observed upregulation of the PD-1 pathway in cytomegalovirus (CMV)-positive tumors. Furthermore, we found signatures of T-cell and B-cell response in HPV-positive HNSC and EBV-positive stomach tumors and HPV-positive HNSC patients were associated with better survival when T-cell signals were detected. Our work reveals that viral infection may recruit immune effector cells, and upregulate PD-1 and CTLA-4 immunosuppressive pathways.Viruses drive carcinogenesis in human cancers through diverse mechanisms that have not been fully elucidated but include promoting immune escape. Here we investigated associations between virus-positivity and immune pathway alteration for 2009 tumors across six virus-related cancer types. Analysis revealed that for 3 of 72 human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSC) the HPV genome integrated in immune checkpoint genes  Cao et al. show that human papillomavirus-positive, head and neck squamous cell carcinoma patients are associated with better survival when T-cells are activated. This study suggests that viral infection may recruit immune effector cells and that it may activate PD-1 and CTLA-4 immunosuppressive pathways. These genomic biomarkers may aid in therapy selection6. Although large-scale sequencing projects such as The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) continue to catalog variants across cancer types, only a minority of patients harbor tumors with genomic aberrations associated with sensitivity to targeted therapy.Since the sequencing of the first genomic DNA from a leukemia patient8. PD-L1 and PD-L2 on tumor cells or antigen-presenting cells suppress T-cell immune response by binding to PD-1 on T-cells10. To escape attack by immune cells, tumor cells overexpress PD-L1 by gene amplification, utilization of an ectopic promoter, and disruption of 3′ untranslated regions (3′ UTRs)11, in addition to PTEN loss-of function12 and EGFR mutations13. Other studies indicate that EGFR mutations are not associated with an increased PD-L1 expression and a better clinical response of PD-L1 immune checkpoint inhibitors15. Elevated PD-L1 expression creates an immunosuppressive microenvironment that facilitates tumor progression16. Anti-PD-1 and anti-PD-L1 immune checkpoint blockades show favorable clinical outcome for treating patients with high PD-1 and PD-L1 expression20. Another important immune checkpoint pathway involves CTLA-4 and its ligands CD80 and CD86. CTLA-4 serves as a negative regulator of T-Cell activity. The anti-CTLA-4 blockade is also an effective therapeutic strategy to kill tumor cells22.Complementary to targeted therapy, cancer immunotherapy utilizes the host immune response to kill tumor cells24. Despite the importance of immune infiltrates and their theoretical associations with viral-positivity25, there is no systematic study of associations between virus-positive samples and the immune response except for some limited studies on human papillomavirus (HPV) in head and neck squamous cell carcinoma (HNSC)29. Our previous large-scale study demonstrated viral positivity in multiple cancer types30. Here, using TCGA RNA-Seq data for six virus-associated tumors, we systematically study the associations between virus-positivity and the tumor microenvironment, as measured by expression of PD-L1, PD-L2, PD-1, CD80, CD86, CTLA-4, Tim-3, LAG3, and 4-1BB and the prevalence of infiltrating immune cells across multiple types of human cancers. Specifically, we found the enrichment of a T-cell immune signature in HPV-positive and EBV-positive tumors compared to non-viral tumors. The increase of T-cell immune response is associated with a better prognosis in HPV-positive patients. In addition, we found HPV integrations at PD-L1 and PD-L2 are associated with high expression of these genes. Higher levels of PD-L1, PD-L2, PD-1, CD80, CD86, CTLA-4, Tim-3, LAG3, and 4-1BB expression were found in Epstein-Barr virus (EBV)-positive stomach adenocarcinoma (STAD) and cytomegalovirus (CMV)-positive colon and rectum adenocarcinoma (COADREAD) tumors compared to virus-negative tumors, providing the rationale for treating virus-positive tumors by anti-PD-1, and anti-CTLA-4 immune therapy. Besides PD-L1, PD-L2, PD-1, CD80, CD86, CTLA-4, Tim-3, LAG3, and 4-1BB, we found the increase of an inducible co-stimulator (ICOS) expression in both HPV-positive head and neck squamous cell carcinoma and EBV-positive stomach adenocarcinoma tumors. ICOS is the immune checkpoint protein, functionally and structurally related to CD2831. A positive ICOS signature may indicate a better clinical outcome of anti-CTLA-4 immune therapy in HPV-positive head and neck squamous cell carcinoma and EBV-positive stomach adenocarcinoma patients32.Immune infiltration of the tumor microenvironment correlates with improved survival in cancer patients30. The 413 HNSC tumors with clear HPV status were most common in two ethnicity groups: 364 Caucasians and 30 African Americans. There was no significant difference in HPV status between the two ethnicity groups. We additionally found that HPV-positive HNSC tumors were mostly from males (92%). See Supplementary Table PD-L1 or PD-L2 by using discordant read pair analysis . Although different integration patterns and anatomic sites were observed in the three tumors, HPV integrations at PD-L1 or PD-L2 were all accompanied by higher expression levels of these genes compared with those in virus-negative tumors .We analyzed 498 TCGA HNSC tumors using VirusScan and identified 72 HPV-positive tumors (numbers of virus-supporting reads per hundred million reads mapped (RPKM) > 100) and 341 virus-negative tumors (RPKM < 5)ors Fig. , and PD-PD-L1 or PD-L2 in other tumor types, we analyzed 229 TCGA HPV-positive cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) tumors and found no putative HPV integrations at either gene. We also checked for integrations of viruses, including HPV, hepatitis B virus (HBV), EBV, and cytomegalovirus, in other cancer types and found no integration events at PD-L1 or PD-L2. This result indicates that high PD-L1 or PD-L2 expression induced by HPV integration is a phenomenon that is selectively associated with HPV in HNSC tumors, with PD-L1 or PD-L2 integrations occurring in 4.2% of HNSC HPV-positive tumors.To examine the prevalence of HPV integrations at NR4A2, TBC1D1, BTNL9, DTX1, FOXP1, INPP4B, PDE4D, and STAT4 with HPV integration events are expression outliers (see Methods) and all these events are associated with the increase of expression.We further looked the relationship between HPV integration and expression in T-cell and B-cell genes. Supplementary Fig. PD-L1 and PD-L2 expression in HPV-positive HNSC, we next correlated any viral infection with PD-L1, PD-L2, PD-1, CD80, CD86, CTLA-4, Tim-3, LAG3, and 4-1BB expressions. In Fig. PD-L1 or PD-L2, i.e., TCGA-CV-5443 and TCGA-T2-A6X0 with 11.8 and 9.2 for PD-L1 and TCGA-HL-7533 with 10 for PD-L2 . RSEM stands for RNA-Seq reads by expectation maximization, which is widely used for quantifying gene expression34. Overall, no significant difference in PD-L1, PD-L2, CD80, or CD86 expression between HPV-positive and virus-negative HNSC tumors was found. A similar observation was also made for HBV , CD80 (p = 0.01), and CD86 (0.04) associated with EBV or cytomegalovirus positivity  between HPV-positive and virus-negative HNSC tumors  genes such as HLA-DQB1, HLA-DQA1, HLA-DMA, HLA-DMB, HLA-DRB5, and HLA-DOA. These genes play an important role for the formation of major histocompatibility complex (MHC) class II/peptide complex, which recognizes microbial antigens and cancer neoantigens45.Next, we identified 78 T-cell genes with significant differential expression  between EBV-positive and virus-negative STAD tumors Fig. . In the tus Fig. . B-cell tus Fig. , 4d. ThiICOS increases about two-fold in EBV-positive STAD and HPV-positive HNSC compared to virus-negative samples. ICOS is an immune checkpoint gene in CD28 and CTLA-4 family, which plays important role in regulating the immune response and enhances the antitumor immune response in anti-CTLA-4 blockade32.From our analyses, 78% of T-cell signature genes in EBV-positive tumors overlapped with those from HPV-positive HNSC, indicating that similar T-cell immune responses are associated with EBV and HPV. For instance, an ICOS signature was identified in EBV-positive STAD  passed the cut-off. We note that for PLAC8, a gene that is associated with pancreatic cancer progression46, was highly elevated in HPV-positive samples with about 16-fold increase compared to the virus-negative HNSC samples. However, in EBV-positive STAD sample, the expression of PLAC8 was downregulated by about four-fold compared to virus-negative STAD sample  and CD45 compared with virus-negative HNSC and colon and rectum adenocarcinoma tumors. Also, HPV-positive HNSC tumors and EBV-positive tumors had a higher expression of CD103, a marker for resident T-cells, compared to virus-negative samples, but not in cytomegalovirus-positive colon and rectum adenocarcinoma and HBV-positive liver hepatocellular carcinoma tumors.We further compared the T-cell differentiation phenotype in virus-positive and negative tumors for different viruses by using markers such as CD28, CD27, CD45, CD103, perforin, GMP-17, and granzymeAp < 0.01, Fisher’s exact test, two-tailed)  with frequency greater than 10% in HNSC tumors with elevated immune response. The HPV-positive cohort contained no variants in either TP53 or CDKN2A and only one in FAT1; in the HPV-negative cohort, these genes were frequently mutated. HNSC tumors with wild-type TP53 are more sensitive to radiation therapy than tumors with TP53 mutations51. In addition, we examined differentially expressed genes in the p53 signaling pathway between the two cohorts and found 4 of 16 p53 pathway genes showing substantial expression alteration  compared with virus-negative tumors  (ed) Fig. . We thened) Fig. ; in totaed) Fig. . Notablysee Fig. . Levels p = 0.01, Fisher’s exact test) between HPV integration status at PD-L1 or PD-L2 in HPV-positive HNSC (N = 72) and CESC (N = 229) tumors. Specifically, we found three integrations among the HNSC samples with high expression of PD-L1 or PD-L2 and no integrations among the CESC samples. It is likely that HPV has co-evolved to target PD-L1 or PD-L2 to create an immunosuppressive tumor microenvironment in some head and neck cancers. Further investigation with a large sample set is important for leveraging the observed relationship between virus integration at PD-L1 or PD-L2 in HNSC and the increased expression. We also found samples with HPV integrations in other immune-related genes  have an increased expression of these genes. Previous studies show that high expression of NR4A2, BTNL9, FOXP1, or PDE4D can antagonize immune response or is associated with tumor progression57. In EBV-positive STAD, cytomegalovirus-positive colon and rectum adenocarcinoma and EBV or cytomegalovirus positive stomach and esophageal carcinoma, we found that viral infection associates with high expression of PD-L1 or PD-L2, CD80, CD86, PD-1, CTLA-4, Tim-3, LAG3, and 4-1BB without integrating into the human genome. Moreover, our study indicates that EBV and cytomegalovirus elevate PD-L1 or PD-L2, CD80, CD86, PD-1, CTLA-4, Tim-3, LAG3, and 4-1BB expression in multiple cancers along the gastrointestinal tract including stomach and esophageal carcinoma and colon and rectum adenocarcinoma.In this study, we systematically investigated associations between virus infection or integration and alteration of the tumor microenvironment. We found a significant difference (58 demonstrated elevated PD-L1 expression in both tumor and immune cells across a large number of tumor samples by using immunohistochemistry (IHC) assays. For example, in 101 head-neck tumors, they found 28 and 19% of immune cell and tumor cell, respectively, were positive for PD-L1 in their samples. The anti-PD-L1 antibody works well on PD-L1 positive tumors to neutralize PD-L1 and make the tumor susceptible to attack by the immune system58. Our studies show an elevated PD-L1 expression in EBV and cytomegalovirus-positive samples, suggesting clinical trials of PD-L1 immunotherapy in these patients may be beneficial. Also, in a subset of HPV-positive HNSC, PD-L1 is highly expressed when HPV integrates into the PD-L1, suggesting these patients may have responded to anti-PD-L1 therapies. The previous clinical trial on HNSC has shown longer overall survival in both HPV-positive and PD-L1-positive tumors when treating with ant-PD-1 monoclonal antibody than the standard single-agent therapy59. Furthermore, we found that the expression of PD-L2, CD80, CD86, and CTLA-4 are also elevated in cytomegalovirus and EBV-positive tumor patients, suggesting anti-PD-L2 and anti-CTLA4 immunotherapy may be effective in patients with these types of tumors.A previous study62. In our previous large-scale study, we found a higher abundance of cytomegalovirus in tumors than in adjacent normal samples30. In the current study, we discovered a high level of PD-L1/PD-L2 in cytomegalovirus-positive tumors across the gastrointestinal tract suggesting that cytomegalovirus mediates the tumor microenvironment, which helps tumor cells to avoid the attack of immune cells.In contrast to the clear association between EBV and stomach adenocarcinoma, some controversy exists about the association between cytomegalovirus and colorectal cancerICOS and CTLA-4 in both HPV-positive HNSC and EBV-positive STAD, suggesting these tumors may have had an effective clinical response to anti-CTLA-4 immune therapy. Survival analysis shows high immune response is associated with favorable survival in HPV-positive but not HPV-negative HNSC samples. The different mutational status and expression patterns may lead to different clinical outcomes of the immune response. For instance, we found different expression alteration in key genes involved p53 signaling pathway in two cohorts. The complete retention of wild-type TP53 in HPV-positive HNSC tumors is another key factor driving the difference, as previous studies show better radiotherapy sensitivity in HNSC patients with wild-type TP5351. For cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), we separated tumors into low and elevated immune infiltration cohorts according to HPV-positive T-cell signatures  will enable us to investigate the virus-mediated tumor microenvironment at the protein level30, which is available from Github64. For the identification of virus integration sites in human genome, we first used BWA65 to align RNA-Seq data to the human plus viral reference. From the re-aligned bam file, we extracted the discordant read pairs, where one read of a read pair maps to human, the other to virus. Pindel66 was used to identify exact breakpoints for all samples with ten or more human-virus discordant reads. The breakpoints in Fig. 67.We discovered viruses in the tumor samples by using the VirusScan pipelinet-test to extract differentially expressed genes in virus-positive and negative samples, using FDR = 0.05 as the cut-off. The FDR value was obtained by p.adjust with Benjamini and Hochberg correction from R package. The heatmap figure was generated by using Heatmap.3R package with default parameters.Survival analysis was implemented by using R package survival. We used the Student’s To investigate if genes with virus integrations are expression outliers, we used the Tukey’s standard formula to quantify the outlier score:x − Q3)/IQR for upper tail and (x − Q1)/IQR for low tail,Outlier score =  are constructed from HPV16 protein sequences. We use NetMHC3panFurther information on experimental design is available in the Supplementary InformationReporting SummarySupplementary Data 1Description of Additional Supplementary Files


Highly sensitive electrostatic doping of ReS2 enables fabrication of gate-tunable NAND logic gates, which cannot be achieved in bulk semiconductor materials because of the absence of gate tunability. The vdW heterostructure NAND gate comprising a single transistor paves a novel way to realize “all-2D” circuitry for flexible and transparent electronic applications.Two-dimensional (2D) semiconductors, such as transition metal dichalcogenides (TMDs) and black phosphorus, are the most promising channel materials for future electronics because of their unique electrical properties. Even though a number of 2D-materials-based logic devices have been demonstrated to date, most of them are a combination of more than two unit devices. If logic devices can be realized in a single channel, it would be advantageous for higher integration and functionality. In this study we report high-performance van der Waals heterostructure (vdW) ReS Among them, transition metal dichalcogenides (TMDs) have been actively studied as channel materials because of absence of short-channel effect7. It has been anticipated that a scale-down limit can be overcome by 2D semiconductors for higher integration of electronic devices. In this regard, “all-2D” devices comprising only 2D materials, i.e., van der Waals heterostructure (vdW) devices, have been demonstrated11. As emerging 2D materials, rhenium-based TMDs, such as ReS2 or ReSe2, have exhibited promising properties. Typically, few-layer TMDs outperform monolayer in terms of carrier mobility, while the band structure of TMDs transforms from direct to indirect band structure as the thickness changes from monolayer to few-layers, which limits their use for optoelectronic applications12. However, ReS2 and ReSe2 exhibit direct band structure at all thickness because of the distorted 1 T’ structure and weak interlayer coupling13. Despite the unique properties of ReS2 and ReSe2, there have been only limited studies on them, which reported preliminary results of transistors and photodetectors based on ReS2 and ReSe2 with conventional device geometry19.Ultrathin two-dimensional (2D) semiconducting materials are useful for a number of electronic applications because of their unique properties originating from their atomically thin nature20, which makes it difficult to utilize the 2D semiconductors for practical applications required by the industry. In addition, because the band gap of TMDs increases with decreasing thickness, the Schottky barrier height for monolayer TMDs is significantly higher than that for few-layer TMDs, leading to a lower field-effect mobility22. The use of graphene electrodes has been considered as a solution because of the de-pinning of the graphene Fermi level and absence of chemical bonds at a stacked heterointerface of 2D layers23. In addition, it has been shown that hexagonal boron nitride (hBN), used as an ultraflat dielectric, suppresses charged impurity scattering, resulting in enhanced carrier mobility of hBN-encapsulated 2D materials24.To improve the device performance of 2D semiconductors, contact resistance and carrier scattering should be reduced. Significant contact resistance between deposited metal and 2D semiconducting channels severely deteriorates device performance due to the Fermi level pinning at the interface28. Even though the NMOS inverters using ReS2 have been demonstrated27, all of the logic devices reported so far are made of double devices (two connected devices). Therefore, for higher integration and functionality, a novel device geometry is required for logic devices. Because atomically thin 2D materials are highly sensitive to electric fields with no screening effect, it is possible to locally tune the densities and types of carriers, leading to electrically controlled p-n junction devices comprising graphene or WSe233. Therefore, by locally tuning the Fermi level of a 2D channel, a multi-functional logic device can be realized in one 2D channel.A number of logic devices based on 2D materials have been demonstrated by combinations of two different 2D transistors: n-type and p-type2 channel with a graphene split gate. We first demonstrate ReS2 field-effect transistors (FETs) on an hBN substrate with graphene electrodes. The field-effect mobility (μFE) of an n-type ReS2 FET is 35 cm2/V·s and the on/off ratio is as high as 106 at room temperature. When the graphene split gate is used to locally modulate the channel, two regions, separately tuned by the split gates, act as independent transistors, which can be used as a gate-tunable NAND logic gate.Here we report a NAND gate based on a single ReS2, graphene, and hBN was fabricated by stacking the exfoliated nanosheets, as shown in Fig. 2 substrate22. The ReS2 channel was placed on the hBN to reduce scattering from charged impurities of the substrate and interfacial impurities. As ReS2 has a smaller effective mass along the a-axis, it has anisotropy in conductance, leading to higher carrier mobility along the a-axis27. Therefore, the ReS2 flake, which has an elongated shape along the a-axis, was chosen as a channel. Graphene was used as an electrode to lower the Schottky barrier. Two pieces of graphene were placed on the ends of the ReS2 channel to form source and drain electrodes. After stacking, e-beam lithography and metal deposition (1 nm Cr/50 nm Au) were used to define the metal electrodes. Figure For “all-2D” devices, a vdW heterostructure of ReS2 FET on the hBN substrate with graphene electrodes is shown in Fig. IDS − VDS) at different back gate voltages (VBG) clearly exhibit a gate-dependent linearity, as shown in Fig. 2-graphene interface forms Ohmic contact even at low carrier concentrations. As shown in the transfer curves (IDS − VBG) in Fig. 2 exhibits n-type transport as typically observed in MoS2 or WS234. Because of the low contact resistance and reduced carrier scattering in the vdW heterostructure device, higher mobility (μFE = 35 cm2/V·s) and a high on-off ratio (~106) were extracted, compared with the same ReS2 FET with metal electrodes on a SiO2/Si substrate  of ReS2 devices with metal or graphene electrodes and SiO2 dielectric19. We also fabricated ReSe2 devices using the same procedure and device structure. As ReSe2 exhibits a smaller mobility of 6.6 cm2/V·s than ReS2, ReS2 was used for the logic device.  etching for cutting. The patterned graphene split gates are annealed at 350 °C for 4 hrs in forming gas (5% H2 in Ar) to remove residue. After cleaning, other 2D flakes of hBN dielectric, ReS2 channel, and graphene electrodes were sequentially transferred by the PDMS stamping technique as described in Fig. 2 device with graphene split gates.We modified the previous device structure to demonstrate the logic devices comprising a single 2D channel. To locally tune the ReSVGG1 and VGG2), the corresponding band alignments are shown in Fig. VGG1 and VGG2 are higher than the threshold voltage (Vth), the whole channel region is conductive and the Schottky barrier is lowered because of the simultaneous up-shift of the graphene Fermi level, which results in n-type conductance of the device. When the VGG1 is higher than the threshold voltage and VGG2 is smaller, carrier transport through the ReS2 channel is blocked because of the depletion of the negatively gated ReS2 region, while the Schottky barrier is still small, resulting in carrier injection of the positively gated channel  of the ReS2 device with graphene split gates when VGG1 is varying while VGG2 is fixed. The transfer curves are plotted on a semi-log scale in Fig. VBG < 3 V) and low subthreshold swing (SS) of <300 mV/dec were obtained, which were an order of magnitude smaller in the ReS2 device on the 300 nm-thick SiO2 substrate which is calculated from the device described in Figs VGG1 > −1.5 V), conductance of the whole channel increases with increasing VGG1. However, in the off-state (VGG1 < −1.5 V), the off-current remains at a low level of 10−12 A at different VGG1 because the region where VGG1 is applied is completely off. Therefore, the on-current can be strongly modulated by VGG1 and VGG2. The on-off ratio modulation ranges from 102 to 106, which is beneficial for multifunctional applications  used in this study is 1 V and the dielectric constant (εr = 3–4) of hBN is relatively small compared to previous studies38. Therefore, the gain can be further enhanced by increasing VDD as shown in the inset in Fig. VDD was set as 1 V because the VTC exhibits a mirror reflection and a gain larger than 1 (unity gain), as shown in Fig. VDD.The logic device characteristics of the ReS2 devices as NAND logic devices, output voltages were measured under different combinations of input states, as shown in Fig. VDD, a logic state of 1 can be realized when either Vin1 or Vin2 is −3 V (defined as an input logic state of 0). For Vout of state 0, positive input voltage of 3 V was applied to both graphene split gates of GG1 and GG2. If a gate voltage smaller than −1.5 V is applied to one of the two gates, the whole device is non-conducting. Therefore, ReS2 devices with graphene split gates can be used as a NAND gate, one of the universal logic gates. It should be noted that a NAND gate was fabricated on a single 2D channel material, which is distinct from other reports using two transistors regardless of the carrier type. Output voltages measured at different combinations of the two input signals are shown in Fig. 2 device with graphene split gates operates well as a NAND gate. Unlike bulk materials, gate tunability of 2D semiconducting materials enables independent control of the separated channel regions by split gates. Therefore, NAND logic device can be fabricated within single 2D channel, which is clearly different from conventional logic gates that require more than two separate transistors.To utilize ReS2 transistors by using graphene and hBN as electrode and dielectric, respectively. The high field effect mobility of 35 cm2/V·s and high on-off ratio of 106 were obtained by suppression of Fermi level pinning at the graphene-ReS2 junction and reduction of charged impurity scattering. For the “all-2D” integration, we also fabricated ReS2 devices with graphene split gates for logic devices. The graphene split gates and bottom hBN enables us to locally and effectively modulate channel regions. By independently changing gate voltages from the two split gates, the device conductance can be controlled with a higher degree of freedom: the whole device is turned off by applying a negative voltage to only one gate, while a positive gate voltage is applied to both split gates to turn-on the device. By utilizing local tunability of the channel by split gates, it was shown that the ReS2 device with graphene split gates can be used as an efficient NAND gate, which is one of the most essential elements in integrated circuits. The vdW heterostructure NAND gate comprising a single transistor fabricated in this work provides a further step toward realization of “all-2D” circuitry for flexible and transparent electronic applications.In conclusion, we fabricated high performance ReS2/Si chips, and exfoliated other 2D materials, such as graphene and ReS2, separately onto each cured PDMS flake on glass slides. After careful alignment with a micromanipulator in the transfer stage, the PDMS with 2D materials were attached and detached, as carefully and slowly as possible, at room temperature.PDMS transfer First, the PDMS base (Sylgard 184) was cured with 10 wt% of curing agent at 120 °C for 2 h. We then mechanically exfoliated hBN, which is the bottom of the heterostructure on SiOTo pattern metal electrodes on the as-stacked heterostructure or gap on the graphene for the split gate, we performed e-beam lithography with a Raith Pioneer 2 nanolithography system.−8 Torr, to create the interconnection to the pads for the electrical measurement from the graphene electrodes, where 1 nm of Cr is an adhesive layer. For the lift-off process, the samples were soaked in acetone overnight.We deposited metal electrodes (Cr 1 nm/Au 50 nm or Cr 1 nm/ Pd 20 nm/Au 30 nm) with an e-beam evaporator (Korea Vacuum Tech.) in a high vacuum, of approximately 102 RIE (Femto Science) for only 5 s to prevent etching of e-beam resist.  We then put the samples in acetone to remove the e-beam resist.We etched the developed region after the e-beam lithography using OAll electrical measurements were carried out by semiconductor parameter analyzers at room temperature under ambient condition. (Keithely 4200, Keithely for FET characterization in Figs Supplementary information for “All-2D ReS<Subscript>2</Subscript> transistors with split gates for logic circuitry”


Graphene nanobubbles (GNBs) have attracted much attention due to the ability to generate large pseudo-magnetic fields unattainable by ordinary laboratory magnets. However, GNBs are always randomly produced by the reported protocols, therefore, their size and location are difficult to manipulate, which restricts their potential applications. Here, using the functional atomic force microscopy (AFM), we demonstrate the ability to form programmable GNBs. The precision of AFM facilitates the location definition of GNBs, and their size and shape are tuned by the stimulus bias of AFM tip. With tuning the tip voltage, the bubble contour can gradually transit from parabolic to Gaussian profile. Moreover, the unique three-fold symmetric pseudo-magnetic field pattern with monotonous regularity, which is only theoretically predicted previously, is directly observed in the GNB with an approximately parabolic profile. Our study may provide an opportunity to study high magnetic field regimes with the designed periodicity in two dimensional materials. Presence of nano-bubbles within an atomically thin material generates potentially large pseudo-magnetic fields. Here, the authors report an innovative technique to induce nano-bubbles in graphene with desirable features and high precision through energized AFM tips, and experimentally measure three-fold symmetric pseudo-magnetic fields up to 120 T. The introduced strain can induce local changes of Dirac point position at low-energy, and always together with lattice distortions that will change the electron hopping in graphene. That phenomenon can be equivalence of gauge field imitating the electron behavior in real magnetic field, unveiling the signatures of Landau quantization3. The strain-induced PMFs conserve the time-reversal symmetry unlike real magnetic field and have opposite signs in the K and K’ valleys of graphene forming the low-energy electronic band structure4. In consequence, the gauge field can be used as a building block for valleytronic device5 or the realization of the Aharonov–Bohm effect by building STM interferometer in strained graphene6. The creation of graphene nanobubbles (GNBs) has been proven as an efficient strategy for strain engineering of graphene, thus generating the enormous PMFs8. For instance, the unconsciously introduced GNBs with the triangular shape on a Pt(111) surface exhibit large uniform PMFs up to 300 T at room temperature7, which was not ever obtained previously. Diverse methods had been proposed to create graphene nanobubbles including gas ion irradiation11, electric field stimulus14, water splitting15, or intense laser irradiation16. However, all proposed methods have the limited capability in the manipulation of GNBs including position definition, size control, and shape design, thus greatly impeding the exploration of the relevant physics phenomenon and the practical application of GNBs, though the enormous PMFs exist.Strain engineering in graphene has been proven as an effective method for modifying its electronic structure due to the remarkable feature of Dirac fermions, such as bandgap opening and pseudo-magnetic fields (PMFs) generationHere, rendered by the accuracy of AFM, the programmable GNBs with the tunable size and shape have been successfully obtained on germanium substrate at the pre-defined locations. With controlling the voltage applied on AFM tip, the contour of bubbles can gradually transform from parabolic profile to Gaussian profile, and the diameter of bubbles can be tuned from tens to hundreds of nanometers. Moreover, higher-resolution scanning tunneling microscopy/spectroscopy (STM/S) reveal that the strain-induced three-fold symmetric pseudo-magnetic field with monotonous regularity exists in GNB with approximately parabolic shape, which is only theoretically predicted previously.17, as characterized by phase-sensitive sum-frequency vibrational spectroscopy  surface (see Methods), as shown in Fig. 10 surfacell Fig. . Besides28. Figure R < 50 nm) can be well fitted by a parabolic curve written ashmax is the maximum height of bubble and R is the radius of bubble base. Our fitting results agree well with the membrane model proposed by Yue et al.29 and the unified power form proposed by Lu et al.31. However, the large GNBs (R > 50 nm) exhibit a more complex profile: the bottom part of the bubble can be still fitted by a parabolic curve, but the top part of the bubble changes into a Gaussian contour. The Gaussian contour part of GNBs can be depicted as29. The bending stiffness can be described by the bending rigidity, κ, and the in-plane stiffness is always described by Young’s modulus Y. Beyond the characteristic parameter 10, the stiffness is dominated by in-plane stiffness. For the GNBs in our study, the dimension is much larger than 4 Å, so the in-plane stresses contribute the majority of the elastic energies, while the contribution of the out-of-plane bending stiffness can be neglected.In addition to the tuning of height and radius of GNBs, their contour can also be customized by the applied voltage, thus further regulating the strain distribution of GNBs10 concludes a universal law of hmax/R considering the stress γ is the adhesion energies between graphene and the germanium, the coefficient c ≈ 0.7. Figure hmax/R behaves approximately as a constant, 0.08, on a wide range of radius or volume. This value is comparable to the aspect ratio hmax/R ≈ 0.11 for circular GNBs (R > 50 nm), hmax/L ≈ 0.07 for triangular GNBs (1000 nm > L > 500 nm) obtained on hBN substrate experimentally10, or 0.18 for small GNBs filled with helium gas in the graphene interlayer11. The similar aspect ratios hmax/R suggest the adhesion energy between graphene and hydrogen terminated Ge surface is quite small17. Besides, the transition of GNB from the parabolic profile to the combination of parabolic and Gaussian profile may attribute to the change of vdW interactions, which strongly depend on the distance, h, between the graphene and substrate, and could be trivial in experimental scales for large GNB. For small GNB with the height hmax < 5 nm, the vdW force on the center deflection of GNB may exist though is not comparable to that at the edge of round base of GNB31. Due to the clamping effect at the bubble edge and the internal hydrogen gas pressure, the GNB with parabolic profile is formed. However, when the height of GNB hmax > 5 nm, the vdW force on the center deflection of GNB is negligible33 and the corresponding constriction effect disappears. However, the clamping effect at the bubble edge still exists. Therefore, the profile of central part of large bubble transits from the parabolic profile to Gaussian profile, but the parabolic profile near the edge of round base of GNB preserves, as observed in Fig. In addition, the shape of bubbles is determined by the adhesion energies, which is related to the vdW force between graphene and the substrate. ref. 1. Figure I/dV spectra collected at different rotational angles  caused by strain-induced pseudo-magnetic fields14. In strained graphene, lattice deformation can create PMFs, which affect the behavior of massless Dirac fermions, thus yielding zero-field pLLs quantization. Similar to the influence of a real magnetic field, the PMF gives rise to a sequence of quantized pLLs:The lattice distortion in GNB creates non-uniform strain distribution, which will modify the low-energy electronic band structure of graphene and change the electron hopping amplitude between carbon atoms, in an equivalent way to the effect of real magnetic fields applied perpendicular to the graphene plane, as known as PMFsEDirac is the energy of Dirac point, e is the electron charge, ℏ is the reduced Planck’s constant, n is the level index, 40. The positions of the pLLs peaks indexed as n = 0, 1, 2, 3, and 4 are marked on Fig. Here, BS| data corresponding to each tunneling spectrum can be extracted by linear fitting of the PMFs using Equation  surface7. Moreover, intriguingly, the maxima of the field occur near the angles of which the interval is nearly 60° at both the edge and the center of GNB, such as 30°, 90°, 150°, 210°, and 270°, and the values diminish and even vanish to zero at the angles between two adjacent ones, such as 60° and 180°. Such the evolution has a six-fold (every 60° in circle lines) symmetry, which can be described by the function |BS| = |121sin(3θ)|. Similar |Bs| distribution have also been observed in different GNBs with distinct heights and radii  with alternating signs42. Here it should be noted that, the distribution of PMF in our experiment only change as the rotational angle changes, but keep constant for the certain rotational angle even when the distance between the measured position and the bubble edge varies, which is different from the predicted three-fold symmetric PMF occurring only at the bubble edge27. The discrepancy may be due to the fact that, unlike the theory prediction, the GNBs with the strictly parabolic shapes could not been obtained in the real experiment, and the configuration complexity of the approximately parabolic GNB including the loss of axisymmetry and the anisotropy of strain may play a crucial role in the formation and modulation of the local PMF distribution. Moreover, GNBs can also be created by AFM tip on other substrates pre-hydrogenated, e.g., SiO2 or Si substrate . During the graphene bubbles fabrication, Pt/Ir coated silicon AFM tips with radius of curvature ~30 nm  were chosen.Intrinsic Ge(110) wafers  were used in the experiments. The graphene film was synthesized on Ge(110) substrate by chemical vapour deposition in a horizontal tube furnace with H−11 Torr) with constant-current scanning mode at liquid-nitrogen temperature of ∼77 K. The STM tips were obtained by chemical etching from a wire of Pt0.80Ir0.20 alloys. Lateral dimensions of the STM images were calibrated using a standard graphene lattice as well as a Si(111)-(7 × 7) lattice, and, the STS spectra were calibrated using Ag(111) surface. The STS spectra, i.e., the dI/dV curves, were collected with a standard lock-in technique by turning off the feedback circuit and using a 793-Hz 5 mV a.c. modulation of the sample voltage.An ultrahigh vacuum scanning probe microscope (USM-1400S) from UNISOKU was utilized for STM and STS measurements. Both STM and STS measurements were performed in the ultrahigh vacuum chamber (∼10Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Source Data


The relation between tinea pedis and the internal environment of footwear has not been scientifically proven. This study aimed to determine whether the internal environment of footwear affects the incidence of tinea pedis and tinea unguium. This cross‐sectional, observatory study involved 420 outpatients who were categorized into non‐tinea, tinea pedis or tinea unguium groups based on mycological analysis. External climatic conditions, and temperature, humidity and dew points inside the patients’ footwear were recorded. Univariate and multivariate analyses were used to determine independent risk factors for tinea pedis and tinea unguium. A significant correlation was found between high temperature/high humidity and dew point of the internal environment of the footwear and the season. Furthermore, those who wore footwear with internal environments characterized by high temperature, high humidity, high‐temperature/high‐humidity and high dew point values had a significantly higher incidence of tinea pedis. The internal dew point correlated with the incidence of tinea pedis, whereas the external temperature correlated with the incidence of tinea unguium. The internal humidity and dew point of footwear as well as the frequency with which footwear with a high‐temperature/high‐humidity internal environment were worn was significantly higher in men than in women. In conclusion, the internal environment of footwear is a risk factor for tinea pedis, and this environment is affected by the season. Moreover, the frequency of tinea pedis among men is related to the internal environment of footwear. The dew point is an appropriate index for evaluating temperature and humidity in relation to tinea pedis. For many years, epidemiological studies and clinical experience have indicated that a relationship exists between footwear and tinea pedis (TP).Several risk factors have been reported for TP and tinea unguium (TU).The internal environment of footwear is determined by physical factors of the footwear , human physical factors , socks and environmental factors outside the footwear .In this observational study, 420 outpatients who wore footwear and who presented for examination at the Sasagawa Dermatological Clinic  between 16 May 2011 and 28 November 2014 were enrolled.Based on clinical assessment, subjects with fungal foot disease were classified into TP only, TU only and both (TP only and TU only) (three‐group classification). Subjects were also classified into TP (TP only and both) and TU (TU only and both) in a two‐group classification. For this study, fungal foot disease was considered as including TP only, TU only, or both TP and TU. In addition, the study cohort was divided into three age groups: group I, less than 30 years; group II, 30 years or more and less than 60 years; and group III, 60 years or more. Patient footwear was categorized as follows: (i) natural leather; (ii) synthetic leather; (iii) sneakers; (iv) cloth footwear; (v) sandals; (vi) slippers; and (vii) boots.Clinical assessments included follow‐up examinations, completion of a medical questionnaire and mycological analysis of samples collected from the infected sites. The toenails and feet were examined, and information about the symptoms or characteristics of TU or TP were recorded. Demographic data such as sex, age and employment were also recorded.Skin and toenail scraping smears were collected from patients diagnosed with TP and TU and submitted to mycological testing. Samples were placed on slides and examined with direct microscopy after addition of 15% potassium hydroxide in 40% dimethylsulfoxide solution, using an optical binocular microscope .Temperature, humidity and dew point inside the footwear of all patients were measured using an automatic temperature and humidity data logger monitor . The loggers were positioned on the dorsal side of the feet between the third and fourth toes. Humidity in the fourth interdigital space was also measured in 10 male subjects.Data were recorded while subjects were wearing their footwear, once each minute for a total of 15 min. All measurements were taken while the patients were wearing socks to avoid direct contact with the skin. Patients remained at rest in a seated position for 15 min before measurements were made. Subjects remained at rest in a seated position for at least an additional 15 min after commencement of measurements. Dew points were calculated automatically using software in the logger and were based on temperature and humidity values. A high‐temperature/high‐humidity environment inside the footwear was defined as a temperature of 32°C or more and humidity of 80% or more.External temperature and relative humidity measurements were obtained from climate data records for the Osaka region compiled by the Ministry of Land, Infrastructure, Transport and Tourism, and the Japan Meteorological Agency. In accordance with the designations used by the Japan Meteorological Agency, spring was defined as March to May, summer as June to August, fall as September to November, and winter as December to February. Fall and summer were collectively referred to as the warm season and winter and spring as the cold season.All statistical analyses were performed using IBM SPSS Statistics 22.0 software . Data are presented as mean ± standard deviation. Different tests were used for each analysis. The optimum cut‐off point was determined by the receiver–operator curve (ROC) analysis that separates TP and non‐TP. Details of the tests used are described in This study was conducted in accordance with the ethical guidelines for epidemiological studies designated by the Ministry of Health, Labor and Welfare, and the Ministry of Education, Culture, Sports, Science and Technology after receiving approval from the Institutional Review Board of The Japanese Association for the Promotion of State‐of‐the‐Art in Medicine. All patients  provided written informed consent.Overall, 420 participants  were enrolled in this study. In terms of age demographics, 44.5% of subjects were in the working age group . Among tP < 0.01). The percentage of men with TP only was significantly higher than that of women with TP only (P < 0.05). The prevalence of fungal foot disease was significantly higher in men than in women (P < 0.05). There was no sex‐based difference in the incidence of TU. The subjects were mainly office workers in the working age group, and the incidence of TP was 39.1%. For TU, there was no difference in the incidence between the two sexes.The incidence of TP was significantly higher in men than in women . Furthermore, the mean temperature inside the footwear changed with the season, which was 29.9°C ± 3.4°C in spring, 32.6°C ± 1.5°C in summer, 31.5°C ± 2.6°C in fall and 26.5°C ± 4.1°C in winter. When the four seasons were grouped into two temperature extremes, the mean temperature inside the footwear was significantly higher (P < 0.001) in fall and summer  than in spring and winter . The humidity inside the footwear was also significantly higher (P < 0.001) in the warm season (76.7% ± 9.8%) than in the cold season (69.6% ± 10.2%). A similar trend was found for the dew point   than during the cold season . These results show that the local climate affects the internal environment of the footwear.A significant correlation was found between the internal environment of the footwear and the external environment  . The numThere were significant differences in temperature, humidity and dew point values among the seven types of footwear. Cloth footwear had the highest temperature, humidity and dew point values  and was therefore prone to mustiness . SandalsP < 0.01; Table The temperature, humidity and dew point values of closed footwear  were significantly higher than those of open footwear . The dew point was also higher among the footwear worn by men (26.5°C ± 4.3°C) than by women . The types of footwear most often worn by office workers were synthetic leather (32.7%) and natural leather (12.9%). The types of footwear often worn by men were sneakers (16.8%), synthetic leather (14.2%) and natural leather (10.0%), whereas those worn by women were synthetic leather (20.3%), sneakers (13.5%) and natural leather (9%).Overall, the footwear with a high‐temperature/high‐humidity environment were more commonly worn by men (29.5%) than by women .Average humidity in footwear is 73.84% ± 10.19% . The humidity was significantly higher in men than in women (P < 0.001), humidity  and dew point .The footwear of those with TP registered significantly higher temperature  and internal dew point , humidity , dew point  and high‐temperature/high‐humidity . For TU, temperature was a significant factor  and dew point of 24.56°C  for TP only, and humidity of 74.94%  and dew point of 23.14°C  for fungal foot disease.Previous epidemiological and clinical studies have indicated a relationship between TP and footwear. However, it has not been scientifically proven that the internal environment of footwear and primary climatological factors have an effect on TP risk. To the best of my knowledge, this study is the first to measure the internal environment of footwear and the external environment, as well as assess how they relate to TP and TU incidences. In the present study, measurements were made of the internal footwear environment in the patients’ everyday life setting. These measurements were made during patient hospital visits and associations with TP and TU infections were determined. The results indicate that TP incidence is strongly affected by the internal environment of the footwear, that a significant correlation was found between footwear's internal environment and the season. Moreover, high‐temperature/high‐humidity footwear environments with a high dew point showed a significantly higher prevalence of TP.The footwear's internal environment was more conducive for TP in men than in women.The findings showed that the dew point is a useful indicator of a footwear's internal environment that easily becomes stuffy and is favorable for TP.Although many epidemiological surveys and clinical experiences show a correlation of TP incidence with seasonal changes,Typically, a region's climate has an influence on the footwear's internal temperature.The results presented herein demonstrate a significant correlation between high‐temperature/high‐humidity and high dew point of the footwear's internal environment and the season .Interestingly, 42.3% of TP patients wore footwear that likely have a high‐temperature/high‐humidity internal environment during summer.Many studies have reported the relationship between the footwear's internal environment and footwear type.The type of shoes  is related to TP;Many studies have reported the relationship between sex and TP. These include studies reporting that TP occurs more frequently in men,Trichophyton penetration into the stratum corneum of a host. In vitro studies on invasion and proliferation of Trichophyton rubrum and Trichophyton mentagrophytes, which are major pathogens causing tinea in the stratum corneum, show that both fungi enter the stratum corneum within a day at two different humidity conditions (in an incubator at 35°C: 90% humidity and 100% humidity). The invasion rate of the dermatophytes was slower at 80% humidity,T. rubrum and T. mentagrophytes infiltration was absent at 85% humidity or less, and humidity needed to be at least 90% for dermatophytes to infiltrate and proliferate in the stratum corneum.T. mentagrophytes were able to penetrate the stratum corneum in cases of skin injury even at 70% humidity.Whether temperature or humidity is a critical requirement for dermatophyte infection is currently under debate. However, no studies have elucidated the role of the footwear's internal environment in Further, my results show that the optimal cut‐off point for discriminating between the risk of tinea pedis and non‐tinea pedis is 75% humidity for both patients with TP only and fungal foot disease; humidity and dew point can be assumed to be even higher in the interdigital area and areas close to the skin surface of the foot. Indeed, the average humidity of the fourth interdigital area in the summer was 80%, average maximum humidity was 84%, maximum humidity was 91% and average dew point was 31°C. A humid environment is important for the development of tinea infections;This study has several limitations. First, the study was conducted in a single institution. Second, the research was performed with arbitrary footwear (footwear worn at the visit) and the effect of changing the footwear by the same patients on different occasions was not examined. Finally, the conditions including sex, age, shoe type and experimental environment in this study are also not uniform, as they would be in a controlled experimental study, which is important to completely understand the relationship between footwear type and TP and TU incidences. Thus, a multi‐institutional study with a large sample size should be conducted to verify the results from this study. Verification of the results in countries with climate and lifestyle habits different from those in Japan is also necessary.In conclusion, the internal environment of footwear is a risk factor for TP and TU. The findings confirmed that the footwear's internal environment is affected by the season. Moreover, frequency of TP among men is related to the interior environment of footwear. The dew point is an appropriate index for evaluating temperature and humidity environments in relation to TP. Although this study does not contribute directly to clinical practise, it may enable dermatologists to advise patients on how to wear and maintain footwear, rather than limiting consultations to simply prescribing topical and internal agents. Consequently, dermatologists may play an important role in TP treatment and prevention.None declared.Methods S1. Submission methods.Figure S1. Internal temperature, humidity and dew point of the footwear during cold and warm seasons.Table S1. Demographic and clinical characteristics.Table S2. Tinea unguium (TU) risk factor analysis.Table S3. Fungal foot disease risk factor analysis.Click here for additional data file.


R2 = 0.080–0.921), TICs (Cohen’s  κ = 0.175–0.396) and NTICs (κ = 0.004–0.505). Such diversity may impact on the reliability and reproducibility of PD-L1 IHC assays as a predictor of response to immune checkpoint inhibitors.Programmed cell death ligand-1 immunohistochemical detection (PD-L1 IHC) is a putative predictor of response to PD-1/PD-L1-targeted checkpoint inhibitors. However, there is no gold standard assay in hepatocellular carcinoma (HCC). We evaluated 5 PD-L1 IHC assay platforms  in 100 HCCs reporting PD-L1 expression in malignant (M) and tumour-infiltrating immune cells (TICs) and non-tumorous cirrhotic tissues (NTICs). We found substantial inter-assay heterogeneity in detecting PD-L1 expression in M ( Therapeutic reversal of immune exhaustion with anti-PD-1/programmed cell death ligand-1 (PD-L1)-targeted therapies induces responses in only 20% of patients with HCC.2 However, its utility in HCC remains controversial. Albeit its expanding clinical use, the predictive role of PD-L1 IHC status is limited by analytical variability, a factor that can be potentially controlled by standardisation of PD-L1 protein detection techniques in clinical samples. Compelling evidence in non-small-cell lung cancer (NSCLC) and melanoma suggests significant inter-assay heterogeneity in comparative studies of PD-L1 IHC tests due to geographical heterogeneity in PD-L1 expression, antibody used and interpretation.4 While considerable research efforts are underway to harmonise PD-L1 IHC assays in other solid tumours,5 the performance of the various PD-L1 IHC assays available is unknown in HCC. As a result, no recommendation can be made for an optimal PD-L1 IHC test in HCC, a tumour where PD-L1 expression predicts for adverse prognosis, but whose predictive role in defining an increased likelihood of response to ICPI remains unclear.6 To address this issue, in Blueprint-HCC we performed a quantitative comparison of five antibody clones used for PD-L1 IHC testing in landmark trials of ICPI.PD-L1 expression by immunohistochemistry (IHC) enriches for response to immune checkpoint inhibitors (ICPIs) in selected tumours.n = 41), the academic Liver Unit in Birmingham  and Novara  following ethical approval (Ref. 17/YH/0015). Patients were treated between 2001 and 2016 and clinicopathologic features are presented in Table We constructed tissue microarrays (TMAs) using a multi-centre repository of 100 archival HCC specimens from three tertiary referral centres including Imperial College London . Analysis was performed using PRISM . Pearson’s correlation coefficient and Cohen’s κ were utilised to evaluate the inter-assay agreement in defining the heterogeneity of PD-L1 expression.3PD-L1 expression was scored in M samples as the percentage of immune-positive cells (1% cut-off) multiplied by chromogenic intensity (ranked from 0 to 3) to derive a semi-quantitative H-score.Tumoral PD-L1 expression was lowest for E1L3N , followed by SP263 , 22c3 , 28-8  and SP142  Fig. . In totaR2 = 0.921) and lowest between SP263/28-8 (R2 = 0.080)  Fig. . Low lev80) Fig. . We comp80) Fig. . Cohen’sCC Table .Table 1I8 Compared to other tumours, however, the level of inter-assay heterogeneity observed in HCC samples appears even more substantial. Unlike melanoma and NSCLC, HCC is unique for the presence of an immune cell-rich cirrhotic microenvironment, which adds a further layer of complexity to the classification of PD-L1 status. To address spatial and cellular heterogeneity in PD-L1 immunolabelling, we ensured representation of multiple cores from the centre, periphery of HCC and the cirrhotic microenvironment. With this approach, we were able to discover substantial heterogeneity in PD-L1 immunolabelling of immune cell infiltrates, which was maximal for NTIC (Cohen’s κ range 0.004–0.505) compared to TIC (0.175–0.396). This is not the first report to suggest the relevance of spatial variance as a determinant of PD-L1 IHC status in malignancy, a finding that might explain the suboptimal linkage between PD-L1 expression and response to PD-1/PD-L1-targeting ICPI. Despite the potential for underestimation in determining PD-L1 status due to the focal nature of protein expression in malignant and immune cells, the use of serial TMA sections might have facilitated a more standardised and less subjective evaluation of cancer specimens as shown in previous studies.9 Moreover, in the clinic, the issue of sampling bias is unavoidable as PD-L1 status requires evaluation in biopsy samples, where the quantity of tissue is limited and not dissimilar to that available in a standard TMA section.In Blueprint-HCC we document significant inter-assay variability in tumoral and stromal immunolabelling across the principal antibody clones utilised for PD-L1 IHC testing in clinical trials and routine practice. Our observation mirrors the results generated in melanoma and NSCLC, where a number of companion diagnostic assays have evolved in parallel with the clinical development of PD-1/PD-L1-targeting ICPI.Methodologically, the PD-L1 IHC results reported here have been generated using standardised antigen retrieval, immunostaining and scoring techniques, in an attempt to mitigate potential sources of bias. Importantly, there were no qualitative differences in the staining pattern produced by any of the tested antibodies, all of which reproduced the specific staining patterns in the tested samples comparable with those of appropriate positive control reactions.10Based on our findings, the heterogeneity of PD-L1 IHC assays does not appear dependent on the portion of the PD-L1 protein recognised by the tested antibody. When considering immunostaining in M cores, we did not observe significant differences between the antibodies targeting the extracellular  versus the intracellular domain , a finding of clinical importance due to the existence of diverse splicing variants of PD-L1 with uncertain biologic significance, which might have been differentially captured by each antibody.To conclude, in the Blueprint-HCC study we have shown significant heterogeneity in the performance of PD-L1 IHC assays in HCC. Whether depending on target epitope diversity, differential assay specificity or antibody affinity, our study highlights inter-assay variation to be a critical component of the reliability and reproducibility of PD-L1 expression as a predictive correlate of response to immunotherapy. None of the patients identified in this study have received ICPI treatment for HCC; therefore, we cannot infer a relationship between PD-L1 IHC heterogeneity and response to immunotherapy. Prospective studies evaluating the diverse PD-L1 IHC assays in patient cohorts with adequate linkage with outcomes from PD-1/PD-L1 ICPI treatment are urgently warranted to clarify the clinical meaning of such variation and facilitate patient selection for immunotherapy.Supplementary ResultsSupplementary Materials and Methods


A comparison of the structure and sensitivity of humidity sensors prepared from graphene (G)-PEDOT: PSS ) composite material on flexible and solid substrates is performed. Upon an increase in humidity, the G: PEDOT: PSS composite films ensure a response (a linear increase in resistance versus humidity) up to 220% without restrictions typical of sensors fabricated from PEDOT: PSS. It was found that the response of the examined sensors depends not only on the composition of the layer and on its thickness but, also, on the substrate used. The capability of flexible substrates to absorb the liquid component of the ink used to print the sensors markedly alters the structure of the film, making it more porous; as a result, the response to moisture increases. However, in the case of using paper, a hysteresis of resistance occurs during an increase or decrease of humidity; that hysteresis is associated with the capability of such substrates to absorb moisture and transfer it to the sensing layer of the sensor. A study of the properties of G: PEDOT: PSS films and test device structures under deformation showed that when the G: PEDOT: PSS films or structures are bent to a bending radius of 3 mm (1.5% strain), the properties of those films and structures remain unchanged. This result makes the composite humidity sensors based on G: PEDOT: PSS films promising devices for use in flexible and printed electronics. In recent years, graphene-based gas or vapor sensors have attracted much attention due to their unique sensing performance, room-temperature operating conditions, a variety of feasible designs, and tremendous application perspectives. Over the past few decades, the rapid development of graphene-based sensors for various applications has been observed. Humidity measurement is one of the most significant issues in the fields of medical diagnostics, environmental protection, industrial manufacture, agriculture, etc. Graphene demonstrates great potential for application in resistive humidity sensors . The sen3 or Fe2O3 nanoparticles [One of the organic polymers, poly  doped with poly (styrene sulfonate) (PEDOT: PSS), is a material most often used in humidity-sensor applications . PEDOT: articles ,12, graparticles . The useParticular interest in humidity sensors based on graphene and PEDOT: PSS is due to the possibility of using those materials in flexible, stretchable, and wearable electronics as well as in the printing technology for creating device structures based on such sensors. For an instant, printed PEDOT: PSS electrodes showed good flexibility in several types of flexibility tests, including outer/inner bending, twisting, and stretching . The higIn the present study, we examined the properties of films prepared from the G: PEDOT: PSS composite versus the composition of the material and as dependent on the solid and flexible substrates used. A composition ensuring the maximum sensitivity of the film to water adsorption was identified. It was found that the use of flexible substrates, due to their capability to quickly absorb the liquid components of the solution, leads to a more porous structure of the films, thus making the response of such a humidity sensor increase substantially. An analysis of the properties of such films under deformation proved the films show promise for use in flexible electronics.2/Si wafers, Kapton polyimide films, polyethylene terephthalate (PET) with additional hydrophilic adhesion layer on the surface, and ordinary paper were used as the substrates.A graphene suspension with a graphene content of ~1 mg/ml was used as the starting material in the present study. The suspension was obtained by electrochemical exfoliation of highly oriented pyrolytic graphite, followed by the sonication, centrifugation, and filtration of obtained particles on a track membrane with a pore diameter of 1.2 μm . The sec2/Si substrates. In the case of PEDOT: PSS films, the removal of part of the substrate has allowed us to obtain an image of a free film. Raman spectra of the films are shown in AFM images of the surface of the films obtained using a suspension of graphene and PEDOT: PSS, and AFM images of the surface of the C1a1 and C1c structures with one and the same film composition yet printed on different substrates are shown in The response of the structures with different compositions observed on the humidity change to 90% is shown in 2/Si substrates, the C1a2 structure with a minimum film thickness of 300 nm exhibited a maximum response of 27%–60%, and at a film thickness of 3.5 μm, and that response dropped to 15%–35%. In the case of relatively thick G: PEDOT: PSS composite films formed on paper (6.5 to 7.5 μm thick), the film thickness was no longer an obstacle for reaching a pronounced response due to the porosity of the film formed on paper. Moreover, a decrease in film thickness to 3 μm led to a decrease in response to 72%.No less important parameters for the response to moisture is the film thickness. Among the structures fabricated on SiOIn general, it should be noted that the rate of change of the resistance is determined, first of all, by the inertia of the measurement chamber. The inset in r at the substrate thickness t = 100 μm and film thickness d amounts to =t+d)/2r or compressive (negative) strains could be generated. The strain estimated from the bending radius (t+d)/2r . On a ch(t+d)/2r ,20. In t(t+d)/2r ,22,23,24The obtained experimental results can be summarized as follows. (a) The content of the composite was identified at which the response of the sensor to humidity was maximum. This is a composite in which a suspension of graphene and PEDOT: PSS is in a proportion of 1:(1–2). It is shown that the response of the system is unambiguous, the resistance increases by increasing the humidity up to 95%. Film recovery occurs on the same time scales in the entire range of humidity. Estimations of the 1:1 G: PEDOT: PSS film weight content after drying of printed layer gives the value of 10% addition of PEDOT: PSS to graphene and 5% in the case of 1:2. In our paper  we have (b) The use of flexible substrates capable of absorbing the liquid ink component permits the formation of sensor elements with high porosity. As a result, the relatively large thickness of such films does not lead to a loss of layer sensitivity.2/Si substrate a maximum sensor response was observed for the thinnest film.(c) As it could be expected, in the case of using a rigid SiOThe used G: PEDOT: PSS composites and test structures possess properties promising for the use of such humidity sensors in flexible and printed electronics: When the structures are bent to a bending radius of 3 mm (1.5% deformation), their properties remain almost unchanged. A pronounced reduction of the scatter of resistance values was obtained for zigzag-shaped test structures.The bending-induced effects in flexible electronics have emerged as a relatively new experimental and theoretical research area. The optimum device design for flexible electronics requires a comprehensive understanding and accurate modeling of strain-induced effects in sensor devices . The shaThe properties of graphene: PEDOT: PSS composite films are compared as a material for resistive humidity sensors for moisture range 30%–95% fabricated on flexible and rigid substrates. It is shown that the resistance increases almost linearly depending on the environmental humidity. The composite films G: PEDOT: PSS provide the maximum response (increase in resistance) up to 220% for film content 1:(1–2) (10–5 weight % of PEDOT: PSS in graphene film) when exposed to a humid environment without the restrictions typical of humidity sensors prepared from PEDOT: PSS (80% RH). It is found that the response of the sensor depends not only on the composition of the layer but, also, on its thickness, film structure, and substrate used. Formation of the porous film, for instance, due to the capability of flexible substrates to absorb the liquid component of the ink, used to print the sensing layer, leads to increase the response to humidity. However, in the case of using paper as a substrate, a hysteresis occurs in the resistance value under a two-sided variation of humidity. This hysteresis is associated with the capability of such substrates to absorb water. The measurements of the resistivity of G: PEDOT: PSS films under bending deformation showed that down to a bending radius of 3 mm (1.5% deformation), the resistance of the structures remained almost unchanged. Moreover, the transition from strips to zigzag structures reduces the scatter of resistance values observed during multiple measurements. The response of the test device structures also remained almost unchanged for a tensile bending radius from 3 cm to 3 mm. Thus, used composite material and humidity sensors prepared from it are highly promising for use in flexible and printed electronics.


To evaluate the effectiveness and safety of Qingpeng ointment on eczema-associated pruritus. This single center randomized double-blinded placebo-controlled trial enrolled 60 patients with nonexudative eczema, who were randomized at 1:1 ratio to the Qingpeng ointment and placebo control groups (n=30 in each group). The investigational and control ointment were applied on lesions twice daily for two weeks. Visual analogue scale (VAS) and pruritus symptom scores were used to assess pruritus severity, frequency, and duration. Eczema lesions were evaluated by eczema area and severity index (EASI) and lesion morphology scores. Subjects were evaluated after the first treatment and at the end of the first and second week. P < 0.05). The scores of pruritus severity, frequency, and duration were significantly lower in the Qingpeng ointment group than in the control group at the end of week 1 and week 2 of the study . The scores of lesion morphology at the end of week 1 of the study were not significantly different between the two groups, but the Qingpeng ointment group showed significantly lower score than the placebo control group at the end of week 2 of the study. No adverse event was observed in the study. The average age and disease duration were 50.1±13.5 years and 30.9±16.0 weeks, respectively. Baseline EASI and VAS scores were similar between the two groups. VAS scores of the Qingpeng ointment group were significantly lower than those of the placebo control group at 10 minutes and 30 minutes after the first treatment and the first and second week follow-up (all Qingpeng ointment can effectively alleviate pruritus and reduce skin lesions in patients with nonexudative eczema. The antipruritic effects occurred early and at a greater magnitude than the effects on lesion attenuation. Eczema, as a common skin disease, is characterized by a complex etiology and apparent pruritus. The clinical manifestation of eczema is diverse, and eczema lesions can develop in the whole body and are usually symmetric and exudative . PrurituThe neurophysiological mechanism of pruritus remains unclear. Recent studies have confirmed the contributions of various factors to pruritus, such as factors that induce pruritus, specific receptors to the factors, afferent nerve fibers, and the specific regions of the central nervous system where scratch reflex occurs , 6. KeraKeratinocytes express multiple receptors that are specifically associated with pruritus, such as histamine receptor, neuropeptide receptor, neurological factor receptor, cannabinoid receptor, protease activated receptor-2, and transient receptor potential vanilloid subtype 1 , 10. His Oxytropis falcata Bunge, Rheum lhasaense, Aconitum pendulum Busch, Chebulae Fructus (without the kernel), Fructus Terminaliae Billericae, Phyllanthus emblica Linn, Benzoin, Tinospora sinensis (Lour.) Merr., and artificial musk. The main chemical components of Oxytropis falcata Bunge are flavonoids, alkaloids, and steroids. The roots, rhizomes, and the whole plant of Oxytropis falcata Bunge can be used as herb medicine. The herb has anti-inflammatory, analgesic, wound-healing, hemostatic, and anti-itching effects. The 3-acetate aconitine from Aconitum pendulum Busch can inhibit some early symptoms of inflammation, such as capillary hyperpermeability, exudation, edema, and leukocytosis. The main chemical component of Chebulae Fructus, Fructus Terminaliae Billericae, and Phyllanthus emblica Linn is tannin. Galloyl-containing tannin can block or reduce the release of inflammatory factors and thus attenuate inflammation. The main chemical components of Chebulae Fructus are tannin and polyphenols. The water extracts of Chebulae Fructus can terminate the free radical cascade and have strong antiallergic and antioxidant effects. Extracts from the leave of Phyllanthus emblica Linn can inhibit the bacteria, Staphylococcus aureus, Escherichia coli, and Candida albicans in vitro, and thus can block the allergic reactions caused by those bacteria [Multiple recent articles have shown that the traditional Tibetan herb medicine, Qingpeng ointment, can treat dermatitis and eczema effectively –16. Howebacteria , 16. Polbacteria –17. Studhttp://www.chictr.org.cn/enIndex.aspx and the trial identification number is ChiCTR1800016190.This was a single center, randomized, double-blind, placebo-controlled study. The study protocol was approved by the Institutional Review Board of Beijing Friendship Hospital . All participants signed the informed consent form. The trial has been registered at May 17th 2018. The trial was registered at All patients, who were diagnosed with nonexudative eczema in the Department of Dermatology of Beijing Friendship Hospital from May 2018 to September 2018, were screened. Inclusion criteria were age was 18-75 years; clinical symptoms met the diagnostic criteria for nonexudative eczema [Computer-generated random number was used to allocate participants into the test and control groups at 1:1 ratio. A study nurse, who was blinded to the group allocation, assigned the participants to the testing or placebo control group using the random numbers. Both participants and investigators were blinded to the group allocation.The investigational drug was Qingpeng ointment provided by Tibet Qizheng Tibetan Medicine Co., Ltd. (Batch number 170901). The placebo control was sham ointment, which is the nonmedicine component of Qingpeng ointment. The sham ointment was also provided by Tibet Qizheng Tibetan Medicine Co., Ltd. According to the fingertip unit , QingpenThe effectiveness of the drug on pruritus and skin lesions was evaluated. The patients were followed up at the end of the first and second weeks of the study. VAS score for pruritus and pruritus symptom scores were evaluated before the treatment and at the first and second week follow-up visits . VAS scoEfficacy index = (EASI before treatment - EASI after treatment)/EASI before treatment × 100%. Complete cure was defined as efficacy index ≥ 90%. Significant improvement was defined as 60% ≤ efficacy index < 89%. Moderate improvement was defined as 20% ≤ efficacy index < 59%. No improvement was defined as efficacy index < 20% . EffectiAdverse reactions were recorded by inquiring the subject and observation during the follow-up visits.α = 0.05. Power (1-β) was 80%. In our preliminary studies, we found the effective rate of two-week treatment was 76.7% and 39.5% in Qingpeng ointment group and control group, respectively. The ratio between the two groups was 1:1. The sample size was 30 in each group based on the normal approximation method.The significant level  t-test and repeated measures analysis of variance were used for intragroup comparison of continuous variables. Categorical variables are presented as rates. Intragroup comparison of categorical variables was analyzed by χ2 test. Multivariate analysis of variance was used for intergroup comparison. P < 0.05 was considered statistically significant.The statistical software SPSS 16.0 was used for data analysis. Continuous variables are presented as mean ± standard deviation (SD). PairwiseParticipant flow diagram is presented in P < 0.001). The placebo control group also showed a significant reduction in the VAS score 10 minutes after the first treatment compared with the score before treatment, but did not decrease further at 30 minutes after the first treatment. Intergroup comparison demonstrated that VAS scores of the Qingpeng ointment group were significantly lower than those of the placebo control group at 10 minutes  and 30 minutes  after the first treatment. In addition, VAS scores of the Qingpeng ointment group at the one-week and two-week follow-up visits were also significantly lower than those of the placebo control group (All P < 0.0001). These data suggest that Qingpeng ointment may attenuate eczema-pruritus effectively.The VAS scores before and after treatment are displayed in  P < 0.05). However, the placebo control group showed no such reductions. Intergroup comparison revealed that the total score and scores of pruritus severity, frequency, and duration were similar in the two groups before the treatment whereas were significantly lower in the Qingpeng ointment group than in the control group at the end of week 1 and week 2 of the study . The scores of lesion morphology before the treatment and at the end of week 1 of the study were not significantly different between the two groups, but the Qingpeng ointment group showed significantly lower score than the placebo control group at the end of week 2 of the study (P = 0.011). These results indicate that Qingpeng ointment-mediated attenuation in pruritus may occur earlier than the drug-mediated reduction in skin lesions.The scores of pruritus symptoms and lesion morphology before and after treatment are presented in For the Qingpeng ointment group, the percentage of EASI at the end of week 1 and week 2 relative to the EASI before the treatment was 64.22% and 29.75%, respectively; the percentage of VAS score relative to the baseline score was 46.06% and 28.63%, respectively. For the placebo control group, the EASI% was 78.72% and 65.96%, respectively, and the VAS score% was 98.61% and 82.71%, respectively. The extent of reduction in EASI and VAS score in the Qingpeng ointment group was greater than that in the placebo control group . These rP < 0.0001). The effective rate of the Qingpeng ointment group was 43.33% and 76.67% at the end of week 1 and week 2 of the study, respectively (The EASIs at week 0 and week 1 were similar in the two groups . At the ectively . The effNo adverse event was observed in the study.The current study found that Qingpeng ointment significantly reduced pruritus severity, frequency, duration, and lesion severity. Qingpeng ointment-mediated attenuation in pruritus occurred earlier than the reduction in lesions because the significant reduction in VAS score of pruritus occurred one week earlier than the significant reduction in EASI. These results suggest that Qingpeng ointment can inhibit eczema-associated pruritus without relying on the reduction in lesions. The effective rates of 43.33% at the end of week 1 and 76.67% at the end of week 2 of the study are consistent with the results of previous studies –16. No aThe molecular and cellular mechanism underlying the beneficial effects of Qingpeng ointment may be associated with the drug-mediated inhibition of the signal transduction pathway involved in pruritus development. Furthermore, vacuum homogenizing emulsification technology, super Micro-grinding technology, and unique oil-in-water cream form was used to manufacture Qingpeng ointment. Thus, the ointment can moisturize the skin. Compared with other traditional Tibetan medicine ointment, Qingpeng ointment can more effectively moisturize the skin and facilitate the drug absorption. The matrix of Qingpeng ointment consists of liquid paraffin, glycerin, water, and other moisturizing ingredients. Moisturizing the skin can improve skin barrier function by reducing KLK7 expression and thus effectively alleviate pruritus .The current study showed that in the placebo control group, the VAS score of pruritus reduced significantly 5 minutes after the first administration of the sham ointment, suggesting the sham ointment also relieves pruritus at the first administration. The possible reason for this transient antipruritic effect from the sham ointment may be related to the cooling effects caused by evaporation of the water component of the ointment. This cooling effect might transiently mask the itching sensation. Thus, the VAS score of the placebo control group rose to the baseline levels at the end of the first week of the study. In contrast, VAS score of the Qingpeng ointment group reduced further at 10 and 30 minutes after the first treatment and remained at low levels during the study. The VAS score of Qingpeng ointment group reduced significantly at the end of the first week of the study, indicating a significant relieve of pruritus at the end of the first week, but lesion reduction occurred later. The lesion score reduced significantly at the end of the second week of the study and so did the EASI. Therefore, we believe that Qingpeng ointment may rapidly alleviate pruritus first and then reduce skin lesions by inhibiting inflammation and improving skin barrier function. Oxytropis falcata Bunge and anti-inflammatory and antiallergic ingredients from Chebulae Fructus, Fructus Terminaliae Billericae, and musk.The current study showed that the magnitude of reduction in the VAS score was greater than that in the EASI, which suggests that the drug-mediated alleviation of pruritus had a greater magnitude than the effects of the drug on lesion reduction and that Qingpeng ointment can inhibit pruritus without depending on lesion reduction. Qingpeng ointment contains antipruritic ingredients fromThe limitations of this study include a relatively small sample size and short follow-up time. Long-term follow-up is required to determine the long-term efficacy and the effects of Qingpeng ointment on eczema recurrence. The molecular and cellular mechanism underlying the benefit of Qingpeng ointment remains to be further investigated.


Recently, humidity sensors have been investigated extensively due to their broad applications in chip fabrication, health care, agriculture, amongst others. We propose a capacitive humidity sensor with a shielding electrode under the interdigitated electrode (SIDE) based on polyimide (PI). Thanks to the shielding electrode, this humidity sensor combines the high sensitivity of parallel plate capacitive sensors and the fast response of interdigitated electrode capacitive sensors. We use COMSOL Multiphysics to design and optimize the SIDE structure. The experimental data show very good agreement with the simulation. The sensitivity of the SIDE sensor is 0.0063% ± 0.0002% RH. Its response/recovery time is 20 s/22 s. The maximum capacitance drift under different relative humidity is 1.28% RH. In addition to daily applications, such as air conditioners and humidifiers, humidity sensors are widely used in industrial process control, medical science, food production, agriculture, and meteorological monitoring ,6,7,8,9.Electronic humidity sensors can be divided into resistive and capacitive . ResistiVarious materials can be used as humidity sensing materials, such as electrolyte , ceramicCapacitive humidity sensors have two basic structures: parallel plate (PP) capacitance a and intIn PP sensors, the upper plate is perforated by an array of holes or parallel stripes to allow water molecules from the air to reach the sensing material underneath. Since the sensing area of the PP capacitor is sandwiched between two parallel plates, the change in the relative dielectric constant of the sensing material in the PP sensors affects the overall capacitance change. Unlike PP sensors, IDE sensors usually only affect the change in the upper capacitance of the IDEs, which makes them less sensitive than PP sensors. However, the exposed sensing area of the PP sensors is smaller than for IDE sensors, which causes a slower response than for IDEs.The IDEs are fabricated on an inert solid or flexible substrate as parallel comb electrodes that overlap each other ,33. IDE It is clear that there are advantages and disadvantages of these two types of sensors. There has been a significant effort to improve the sensor structures. For example, Zhao et al. used RIE (Reactive Ion Etching) and ICP (Inductively Couple Plasma) to etch sensing materials between parallel plates of the sensors to obtain a larger contact area with the tested environment to reduce response time from 35 s to 25 s , but thiInspired by combining the advantages of PP and IDE structures, this paper proposes a novel IDE humidity sensor with a shielding electrode under the IDEs, namely, SIDE. On the SIDE, the capacitance of the lower half of the IDEs is shielded by an additional electrode underneath the IDEs, which effectively raises the relative capacitance change as it becomes exposed to moisture. Thus, a SIDE humidity sensor combines the high sensitivity of PP sensors and the fast response (20 s) as the IDE ones.In this work, we first verified the feasibility of the SIDE structure in the simulation software. Secondly, the thickness of the sensing layer with different electrode gaps and the dielectric thickness between the shielding electrode and the IDEs were optimized regarding the sensitivity and response speed. The SIDE sensor with optimized parameters was fabricated. The sensitivity, response time, recovery time, and stability of the sensor were measured.COMSOL Multiphysics®  is applied to simulate the SIDE and IDE structure. 0) of SIDE and IDE under different relative dielectric constant of PI representing the humidity conditions. C0 is the total capacitance when the relative dielectric constant of the sensing layer is 2.9. ΔC is the capacitance difference between any other relative dielectric constant of PI and 2.9. It can be seen that under the same conditions, ΔC/C0 of the SIDE structure, is about 4 times bigger than that of the IDE structure, which implies that the SIDE will have much higher sensitivity than IDE with the same parameters.max/C0 is also simulated by COMSOL Multiphysics® . We define that ΔCmax/C0 equals to ΔC/C0 with the relative dielectric constant of PI at 2.9 (C0) and 3.7 (Cmax), which indicates the sensitivity of the sensor.The effect of the thickness of the sensing film on ΔCmax/C0 increases as the thickness of the sensing film increases, but flattens at higher thickness. To optimize the sensing film thickness, two facts should be taken into account. On the one hand, it is clear that when the sensing film thickness is equal to the gap between the IDEs  A 2.5 µm-thick negative photoresist is patterned. (b) An e-beam-evaporated Ti/Au layer is deposited and selectively removed by a lift-off process to form the bottom shielding electrode. (c) A layer of 1 µm silicon dioxide is deposited by PECVD . (d) IDEs are fabricated on the silicon dioxide by the same sequence of lithography, e-beam evaporation, and lift-off. (e) A 5 µm-thick PI is spin-coated. Subsequently, the device is baked at 120 °C for 1 h, 180 °C for 1 h, and 250 °C for 6 h to cure the sensing layer. The completed sensor and cross-section of the SIDE structure under scanning electron microscope (SEM) are shown in The setup for the humidity measurement is shown in Cx can be directly measured using the circuit shown in pn . Cx is the sensing capacitance proportional to the humidity. Cp1 and Cp2 are the capacitances between the shielding electrode and the IDEs. Cf is the fixed capacitance of the IC chip. U1 and U2 are the potentials before the humidity sensor and after the IC chip that both can be measured. Therefore, Cx can be calculated using Equation (1).The capacitance above the shielding electrode Before the test, each device is placed in an oven at 100 °C for 10 min to get rid of the effect of the previous measurement.1 − C0, C0 is the capacitance measured at the RH, which is 23.7% ± 0.8%, and C1 is the capacitance measured when the RH is 73.0% ± 0.8%. Δ(% RH) is the difference between the relative humidity values when measuring C1 and C0.The sensitivity (S) can be expressed as Equation (2):The response and recovery dynamics are among the most important characteristics for evaluating the performance of humidity sensors. The response time for RH increase and the recovery time for RH decrease are usually defined for a sensor as the time taken to reach 90% of its total capacitance variation. The response and recovery curves are measured by exposing the SIDE sensor to alternate levels of humidity between 2.0% ± 0.8% and 77.0% ± 0.8% RH.In order to evaluate the functioning of the humidity sensor over long periods of time, we measured the sensor’s capacitance over the duration of 20 h at 25 °C with relative humidity levels of 25.7% ± 0.8%, 34.4% ± 0.8%, 45.0% ± 0.8%, 57.0% ± 0.8%, and 73.5% ± 0.8% RH.R2 of 0.996 and 0.991, respectively. The slopes of the line, i.e., S of SIDE and IDE are 0.0063 and 0.001,65, respectively. Taking the uncertainty of HC2-S into consideration, the S of SIDE and IDE are 0.0063 ± 0.0002 and 0.001,65 ± 0.000,05, respectively. Hence, the sensitivity of the SIDE structure is 3.82 times bigger than that of the IDE. These results show the significant improvement of sensitivity brought by the shielding electrode, that minimizes the large constant capacitance of the substrate. Indeed, whatever substrate the IDE is built on, the relative dielectric constant of the substrate is larger  or close to  the relative dielectric constant of PI (2.9–3.7). The experimental result and simulation data verify the effects of the shielding electrode and shows high agreement as well. It is clear that our proposed SIDE structure can provide an effective way to measure relative humidity more sensitively and accurately. Another advantage of the shielding electrode is that it can effectively suppress the external electromagnetic interference and reduce the noise in the measurement process.A sensitivity test is carried out on the SIDE and IDE structure. max is the maximum measured capacitance after the sensor is exposed to different RH atmosphere, and Cmean is the average capacitance of all recorded values at a certain relative humidity, C0 is the capacitance measured when the RH is 23.7% ± 0.8%. The maximum drift value (D) obtained from In summary, we propose a novel shielded interdigitated electrode structure for humidity sensing. We perform a comprehensive simulation of this structure to optimize the parameters for the sensor fabrication. In simulation and actual testing, we find that the sensitivity of the SIDE structure is much higher than that of the IDE structure because of the effect of the shielding electrode on the capacitance change rate. Since the surface structure of the SIDE structure is still the same as IDE, the SIDE sensor combines the high sensitivity of the parallel plate sensors and fast response of the IDE sensors. The sensitivity of SIDE is 0.0063% ± 0.0002% RH, and the response/recovery time is 20 s/22 s. The stability of the SIDE sensor was also characterized. The maximum drift value under different relative humidity is 1.28% RH. Meanwhile, since the basic operating principle of many capacitive sensors is the same, the SIDE structure can even be applied to capacitive gas sensors, such as volatile organic compound (VOC) sensors which are used to monitor toxic gases. This shows that SIDE can replace IDE in various sensors that are more sensitive to the accuracy and response speed.


Ultra-high exfoliation in water of a nanosized graphite (HSAG) was obtained thanks to the synergy between a graphene layer edge functionalized with hydroxy groups and a polymer such as chitosan (CS). The edge functionalization of graphene layers was performed with a serinol derivative containing a pyrrole ring, serinol pyrrole (SP). The adduct between CS and HSAG functionalized with SP was formed simply with a mortar and pestle, then preparing water dispersions stable for months in the presence of acetic acid. Simple casting of such dispersions on a glass support led to carbon papers. Aerogels were prepared through the freeze-dry procedure. Exfoliation was observed in both these families of composites and ultra-high exfoliation was documented in aerogels swollen in water. Carbon papers and aerogels were stable for months in solvents in a wide range of solubility parameter and in a pretty wide range of pH. By considering that a moderately functionalized nanographite was straightforwardly exfoliated in water in the presence of one of the most abundant biobased polymers, the obtained results pave the way for the simple and sustainable preparation of graphene-based nanocomposites. HSAG–SP/CS adducts were characterized by wide angle X-ray diffraction (WAXD), scanning and transmission electron microscopy , Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy. Thermal stability of the composites was studied by thermogravimetric analysis (TGA) and their direct electrical conductivity with the four-point probe method.  According to the nomenclature for graphene and graphene related materials -linked 2-acetamido-2-deoxy-D-glucopyranose and 2-amino-2-deoxy-D-glucopyranose. This polymer was selected as it can be easily obtained (through deacetylation) from chitin, the second most abundant natural biopolymer in the world. Moreover, chitosan can be dissolved in acidic water and a polycation is formed. A working hypothesis was that the exfoliation of the graphitic aggregates could be favored by the interaction between a polycation, such as chitosan treated with acetic acid, and the aromatic layers. In the scientific literature, the covalent functionalization of graphene sheets with chitosan has been reported r, where x and y are fractions of repeating units with free amine and acetylated amine, respectively. The chitosan used in this work has a deacetylation degree of 30%, and therefore x = 0.3 and y = 0.7. It appears that the atomic ratios measured for both HSAG–SP/CS aerogel and paper are very similar to the ones calculated for pure chitosan. Considering that XPS analyses probe only the outer layer of a sample (maximum ca. 100 Å), these results suggest that CS is at the surface of both the aerogel and carbon paper and covers HSAG–SP.XPS analyses were performed on HSAG–SP and on HSAG–SP/CS 1:1 aerogel and paper. For all samples, wide scan spectra, reported in N-acetylglucosamine rings; ii) C3, C4 and C5 in the glucosamine segment and in the N-acetylglucosamine segment; iii) C1 atoms in both segments; iv) C7 in the N-acetylglucosamine unit. Taking into account this classification, the relative amount of the C1s components for the chitosan used in this work  are 35.1%, 44.8%, 14.9% and 5.2%, respectively. These values are very close to the signal area obtained for each C1s contribution for both HSAG–SP/CS aerogel and paper  reflections that indicate the order in the direction orthogonal to structural layers: (002) at 26.6°, which corresponds to an interlayer distance of 0.338 nm, and (004) at 54.3°. The in-plane order is proven by the (100) and (110) reflections at 42.5° and 77.6°, respectively. By using (002) reflection and applying the Scherrer equation (Equation (1)), the out of plane correlation length (D┴) was calculated and about 29 stacked layers were estimated. The two reflections at 10.0° and 19.9° as 2θ angles demonstrate the CS crystallinity a. The paIn the pattern of HSAG–SP/CS 1:1 carbon paper c, the  reflection was observed in the latter sample but not in the composite with 20% by mass of HSAG–SP (1:4 HSAG–SP/CS). Such finding can be attributed to the pronounced exfoliation of HSAG but could also be due to its low relative amount (20% by mass). Water was added to the aerogel with 50% HSAG–SP  are shown in −3. The walls of the cavity are of chitosan, in the presence or in the absence of HSAG–SP, which is in most cases present in large aggregates. A continuous filler network is visible in some areas. This structure accounts for the electrical conductivity of the aerogel, discussed below in the text. Aerogels exhibit a spongy like structure. The highly porous structure is confirmed by the low density, equal to 0.026 g cmThe four-point probe method was used to measure the electrical conductivity of HSAG–SP/CS aerogels . Table 25 μS/cm). A critical value of HSAG–SP/CS ratio exists for having values of electrical conductivity in line with those reported in the literature for carbon papers and aerogels. HSAG–SP/CS 1:1 aerogel shows conductivity compared to the paper with the same HSAG–SP/CS ratio. By increasing the quantity of graphite (4:1 aerogel) conductivity, as expected, increases , n-hexane (b) and DMF (c), as shown in Resistance of HSAG–SP/CS aerogels and carbon papers to solvents was preliminarily investigated. Vials containing specimens of the HSAG–SP/CS 1:1 sample were kept for two months in H3COOH, NaHCO3 and KOH to modulate pH values. The stability of HSAG–SP/CS aerogels in a water solution having different pH was investigated, using substances such as HCl, CHThis paper reports a novel and simple approach to achieve ultra-high exfoliation of graphitic aggregates: An adduct between edge functionalized graphene layers and cationic chitosan was formed in water and subjected to mild sonication. Few-layer graphene was obtained, and the structure of the layers was not appreciably perturbed. In previous reports it was shown that the sonication in water of either the same graphitic material (HSAG), edge functionalized with the same pyrrole compound , or of aCarbon papers and aerogels based on graphene layers and containing high amounts (up to 50% by mass) graphitic material were successfully prepared. Moreover, only few of the graphene layers were stacked or their exfoliation was so extensive that the X-ray pattern of the aerogel swollen in water did not reveal the typical (002) reflection of graphitic aggregates. The procedure here reported for obtaining few-layer graphene and highly exfoliated graphene in polymer matrices is sustainable in many aspects. A polymer, such as chitosan, was used. An HSAG–SP adduct was prepared with a high yield and thus almost no wastes , with the help of only thermal energy. HSAG–SP was mixed with chitosan in water, in the presence of only acetic acid, obtaining stable suspensions. Carbon papers and aerogels were obtained by simple casting and lyophilization of HSAG–SP/CS suspensions, respectively. Moreover, the nanocomposites were shown to have interesting properties: They were electrically conductive and stable for months with unaltered properties if stored in different solvents and at different pH.