PD-L1 DNA methylation (mPD-L1) in prostate tissue samples and to evaluate its potential as a biomarker in prostate cancer (PCa).The rapid development of programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibitors has generated an urgent need for biomarkers assisting the selection of patients eligible for therapy. The use of PD-L1 immunohistochemistry, which has been suggested as a predictive biomarker, however, is confounded by multiple unresolved issues. The aim of this study therefore was to quantify mPD-L1 compared to tumor tissue. High mPD-L1 in PCa was associated with biochemical recurrence (BCR) in univariate Cox proportional hazards (hazard ratio (HR)=2.60 [95%CI: 1.50-4.51], p=0.001) and Kaplan-Meier analyses (p<0.001). These results were corroborated in an independent validation cohort in univariate Cox  and Kaplan-Meier analyses (p=0.029). Although mPD-L1 and PD-L1 protein expression did not correlate in the validation cohort, both parameters added significant prognostic information in bivariate Cox analysis .In the training cohort, normal tissue showed significantly lower levels of mPD-L1 was analyzed in a training cohort from The Cancer Genome Atlas (n=498) and was subsequently measured in an independent validation cohort (n=299) by quantitative methylation-specific real-time PCR. All patients had undergone radical prostatectomy.mPD-L1 is a promising biomarker for the risk stratification of PCa patients and might offer additional relevant prognostic information to the implemented clinical parameters, particularly in the setting of immune checkpoint inhibition. We further analyzed the prognosis of PCa patients stratified according to their mPD-L1 status (high vs. low) in combination with the dichotomized PD-L1 protein expression status. Figure mPD-L1 present with shorter BCR-free intervals compared to patients with simultaneous low PD-L1 protein expression and low mPD-L1. Patients with either high PD-L1 protein expression and low mPD-L1 or low PD-L1 protein expression and high mPD-L1 showed an intermediate BCR-free interval. The finding that PD-L1 protein expression and methylation seem to be unrelated in our cohort prompted us to investigate additional mechanisms of post-transcriptional regulation of PD-L1.High PD-L1 protein expression determined semi-quantitatively mPD-L1, suggesting that mPD-L1 was associated not only with decreased mRNA expression but also with lower levels of mRNA destabilizing miR-197 and three of the miR-200 family members miR-200a-c . In particular miR-197, miR-200, miR-570, miR-34a, and miR-513 have been shown to play a key role in the regulation of PD-L1 expression (reviewed by ). MiRs aTargeting the immune checkpoint pathway PD-1/PD-1 has emerged as a promising therapy providing significant clinical activity and durable benefits with minimal toxicities in several solid tumors . The rapPD-L1 promoter methylation in PCa. The aim of this study therefore was to quantify PD-L1 DNA methylation in prostate tissue samples and to evaluate its potential as a biomarker in PCa.Aberrant DNA methylation of certain loci has been reported to play a major role in carcinogenesis . Such epDNA methylation of promoter regions is often associated with epigenetic silencing . In normPD-L1 gene were analyzed. In this training cohort, mPD-L1 levels were increased significantly in PCa compared to normal tissue, indicating that PD-L1 might be differentially methylated in prostatic malignancies. Further investigations revealed that two of the five examined beads (cg19724470 and cg15837913) showed a significant correlation of mPD-L1 with BCR in univariate Cox proportional hazards analysis and Kaplan-Meier analysis. Interestingly, differential methylation was found in the border regions and not the centre of the CpG-dense promoter area. One of the two beads (cg19724470) further showed an inverse correlation with PD-L1 mRNA expression and an association with prognostic clinicopathological variables including the ISUP grading group, pT, pre-surgical prostate-specific antigen (PSA), resection margin, and ERG status.In the present study, five Illumina Infinium HumanMethylation450 BeadChip beads taken from the TCGA dataset and located in the promoter region of the PD-L1 DNA methylation, analyzed as a continuous variable and as a dichtomized variable using an optimized cut-off, was significantly associated with an increased risk for BCR in patients in univariate Cox proportional hazards analysis and its prognostic value was further confirmed by Kaplan-Meier analysis.In order to validate these findings with an additional technology in an independent cohort, a quantitative methylation qPCR assay was designed within the region of the best performing bead (cg19724470). In this validation cohort, high PD-L1 promoter methylation and protein expression in PCa. This may point at a complex post-transcriptional and post-translational regulation of PD-L1. According to our findings, mPD-L1 is inversely correlated with mRNA transcription and associated with miR as cellular component, which modify the downstream processing of PD-L1 mRNA. Differential expression of miR may therefore potentially interfere with the linear translation of PD-L1 mRNA into PD-L1 protein. Thus, further studies are warranted to unravel the complex interplay between DNA methylation and protein expression in PCa. In addition, post-translational modifications which affect the stability of the protein (e.g. glycolization) and in turn alter the affinity of the anti-PD-L1 antibody to membranous PD-L1 might further be causative for ostensibly conflicting results =100%*2ΔΔCTThe DNA methylation of PD-L1 methylation, expression, and clinicopathological variables were analyzed using the Spearman's rank correlation, Kruskal-Wallis (≥ 3 groups) or Mann-Whitney-U (2 groups) tests. BCR-free survival analyses were conducted by Kaplan-Meier and Cox proportional hazards regression analyses. P-values < 0.05 were considered statistically significant.The statistical analyses were performed using SPSS, version 22 . Associations/correlations between


Furthermore, the chirality of the QHE edge channels can be changed by a top gate. These results demonstrate that basic QHE physics are experimentally accessible in graphene for a fraction of the price of conventional setups using superconducting magnets, which greatly increases the potential of the QHE in graphene for research and applications.We have observed the well-kown quantum Hall effect (QHE) in epitaxial graphene grown on silicon carbide (SiC) by using, for the first time, only commercial NdFeB permanent magnets at low temperature. The relatively large and homogeneous magnetic field generated by the magnets, together with the high quality of the epitaxial graphene films, enables the formation of well-developed quantum Hall states at Landau level filling factors  Experimentally demonstrated for the first time in 1980This very peculiar state of matter has been the object of an extremely vast corpus of fundamental research on various materials. Indeed, quantum Hall systems host a great variety of unique states and phenomena, such as fractal quantum Hall states345Experimentally, the QHE is obtained in materials such as gallium arsenide heterostructures, under a strong magnetic field (of at least a few Tesla) and at low temperatures. These constraints, which require the use of cryogenic refrigerators containing superconducting magnets to generate the magnetic field, stem from the small energy gap between Landau levels at low magnetic fieldsGraphene can circumvent those constraints because, owing to its unique band structure, the energy gap is more than one order of magnitude larger than in gallium arsenide heterostructures at typical magnetic fields of a few Tesla.Indeed, shortly after the first experimental observation of the QHE in graphene10 cm−2 can be attained, making the QHE at the Landau level filling factor v = 2 reachable for magnetic fields lower than 1 T13152Fe14B alloy, which have remanence fields of up to 1.4 T. Replacing the expensive, cumbersome and fragile superconducting solenoid magnets with affordable permanent ones would considerably expand efforts to probe and exploit the properties of the QHE.The ability to tune the density of carriers, and even their type, in graphene by the field effect implies that the constraint of high magnetic fields could also be lifted for clean enough samples, in which carrier densities lower than 5 × 10In this paper, we show that the QHE can be induced in a graphene sample using NdFeB permanent magnets instead of the usual superconducting magnets.v = 2 QHE plateau with respect to both gate voltage and magnetic field2021222425Several fabrication techniques yield clean enough graphene samples so as to reach the QHE at sub-tesla magnetic fields. While boron nitride-encapsulated graphene12 cm−2), the top gate dielectric, made of hydrogen silsesquioxane , decreases the carrier density to around 5 × 1011 cm−2 at zero top gate voltage Vgate, leading to high mobilities (up to 16000 cm2/Vs at Vgate = 0 V). This, combined with the ability to further decrease the density by changing Vgate, allows us to reach the QHE regime at magnetic fields as low as 0.5 T, which is easily attainable with permanent magnets. This is clearly shown in H) and longitudinal (Rxx) resistances under the magnetic field generated only by permanent magnets as a function of Vgate at the base temperature of our He3 fridge (300 mK).We used this setup to perform QHE measurements in an epitaxial graphene Hall bar. An optical micrograph of the sample is shown in H (red squares) at the value 0.5 × h/e2, accompanied by a zero value of Rxx (black circles), for Vgate between −33 and −39 V. This is the hallmark of the QHE at filling factor v = 2. As Vgate is further swept towards negative values, the v = 2 plateau vanishes and the charge neutrality point (CNP) is reached at Vgate ≈−45 V. This is indicated by the local maximum in Rxx and the change in the sign of RH. For Vgate between −50 and −55 V, Rxx again reaches zero while RH becomes quantized at −0.5 × h/e2, characteristic of the QHE at filling factor v = -2. This change in sign, corresponding to a change in the chirality of charge carriers, is a signature of the ambipolarity of charge transport in graphene.The results plotted in v = ±2 in epitaxial graphene, we now focus on the robustness of the QHE phase against temperature. H (red squares) and Rxx (black circles) on the v = ±2 plateau at up to T = 2 K. A sevenfold increase in the temperature only leads to a deviation smaller than one-thousandth of the expected values for RH. Note that the low-temperature value of RH is slightly smaller than 0.5 × h/e2, which is due to an error in the estimated gain of the room-temperature amplifiers of about 0.04%.Having shown that our setup enables us to reach the QHE regime at gate dependence of Rxx is plotted for several values of temperature up to 30 K. Note that fluctuations arise in Rxx around the CNP below 7 K, indicating the presence of residual electron/hole puddles at the CNP. In the explored temperature range, the minimum longitudinal conductivity σxx = Rxx /(Rxx2 + RH2), plotted in the main panel of VRH ∝1/T × exp[(TM/T)1/3]. This model is similar to the one presented in ref. xx in epitaxial graphene samples immersed in magnetic fields generated by a superconducting magnet.The deviations become larger as the temperature is increased, as shown in the inset of e.g. one cannot reverse in-situ the direction of the magnetic field) can be mitigated by implementing a mechanical displacement of the magnets. In addition, it is possible to combine it with a small coil to induce small changes in the applied magnetic field30In summary, we have combined the low carrier densities attainable in epitaxial graphene with the relatively large magnetic fields generated by NdFeB permanent magnets to obtain a robust quantum Hall phase without the use of either superconducting or normal electro-magnet. In those conditions, the QHE could be reached in various types of graphene samples. For fundamental physics, using this setup with encapsulated monolayer and bilayer graphene2 insulating layers, allows us to tune the density and the type of the charge carriers. At zero gate voltage, the sample has n-type carriers with a density of about 5 × 1011 cm2, due to the doping from the SiC substrate and the HSQ.The epitaxial graphene wafers were prepared by thermal decomposition of a 6H-SiC(0001) substrate, annealed at around 1800 °C in Ar at a pressure of less than 100 Torr. Metallic contacts are made of Cr/Au. An Au top gate, separated from the graphene film by a 100-nm-thick hydrogen silsesquioxane (HSQ) and a 60-nm-thick SiOThe measurements were performed using standard lock-in techniques at roughly 30 Hz with NF LI-75A room-temperature amplifiers.How to cite this article: Parmentier, F. D. et al. Quantum Hall effect in epitaxial graphene with permanent magnets. Sci. Rep.6, 38393; doi: 10.1038/srep38393 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


PD‐1/PD‐L1 immunotherapy is viewed as having clinical benefits in advanced cancers but is effective in only a few patients, suggesting that an efficient combination approach is needed to improve efficacy. Immunohistochemistry analysis indicated that PD‐L1 expression was correlated with the E6 expression in tumors from 122 lung cancer patients. The poorest survival occurred in PD‐L1‐positive/E6‐positive tumor. PD‐L1 expression was increased by the expression of E6, but not the E7, oncoprotein in lung and cervical cancer cells. PD‐L1 expression was responsible for E6‐mediated colony formation and soft agar growth. Therefore, PD‐L1 secreted from tumor cells may directly promote tumor progression, particularly in E6‐positive tumors. Immune deficiency nude mice were used to test the possibility that combining anti‐PD‐L1 mAb with Lm‐LLO‐E6 vaccine could have a higher antitumor activity compared with anti‐PD‐L1 mAb or Lm‐LLO‐E6 vaccine alone. A greater antitumor activity was obtained with anti‐PD‐L1 mAb + Lm‐LLO‐E6 vaccine than with anti‐PD‐L1 mAb or Lm‐LLO‐E6 alone in subcutaneous and metastatic tumors induced by TL‐1 and SiHa cells. The longest survival time for nude mice was observed in the anti‐PD‐L1 mAb + Lm‐LLO‐E6 vaccine group. In conclusion, an anti‐PD‐L1 mAb + Lm‐LLO‐E6 vaccine may be an efficient treatment for suppression of tumor growth and metastasis induced by HPV‐infected cells. Program death ligand‐1 (PD‐L1) acts as an inhibitor of human T‐cell responses by binding to its receptor PD‐1 to create the tumor microenvironment. This, in turn, results in tumor progression due to tumor immune surveillance Poor prognosis in NSCLC is associated with the epithelial‐mesenchymal transition (EMT), a key process that drives cancer metastasis Human papillomavirus (HPV) 16/18 infection is associated with lung cancer development in Taiwan Antibody‐mediated blockade of PD‐L1 can induce a durable tumor regression and prolonged stabilization of disease in patients with NSCLC Lung tumor specimens were collected from 122 patients who underwent primary NSCLC surgical resection at the Department of Thoracic Surgery, Taichung Veterans General Hospital  between 1998 and 2004. Patients were asked to submit written informed consent; the study was approved by the Institutional Review Board (TMUH No. 201301051). The tumor type and stage of each collected specimen were histologically determined in accordance with the World Health Organization classification system. Cancer relapse data were obtained from chart review and confirmed by thoracic surgeons. Clinical parameters and OS and relapse‐free survival (RFS) data were collected from chart review and the Taiwan Cancer Registry, Department of Health, Executive Yuan, ROC.TL‐1 and TL‐4 cells were cultured from the plural effusions from lung adenocarcinoma patients μm sections, mounted on glass, and dried overnight at 37°C. All sections were then deparaffinized in xylene, rehydrated through alcohol, and washed in phosphate‐buffered saline. This buffer was used for all subsequent washes. Sections were heated in a microwave oven twice for 5 min in citrate buffer (pH 6.0) and then incubated for 60 min at room temperature following a conventional streptavidin peroxidase method . Signals were developed with 3, 3′‐diaminobenzidine for 5 min and the sections were counterstained with hematoxylin. Negative controls were obtained by leaving out the primary antibody. The signal intensities were evaluated independently by three observers. Immunostaining scores were defined as the cell staining intensity  multiplied by the percentage of labeled cells (0–100%), leading to scores from 0 to 300. A score greater than or equal to 150 was defined as “high” immunostaining, whereas a score <150 was defined as “low”.Immunohistochemistry was performed to evaluate PD‐L1 protein expression. PD‐L1 antibody was purchased from Genetex . HPV E6 antibody was purchased from Santa Cruz Biotechnology . Formalin‐fixed and paraffin‐embedded specimens were cut into 3 The HPV 16 E6 cDNA plasmid and HPV 16 E6/E7 small hairpin (sh)RNA were described previously 2. Colonies were visualized and quantified with a microscope after 14 days of cultivation, and the colonies larger than 100 μm in diameter were counted.Anchorage‐independent growth was assayed as the ability of cells to form colonies on soft agar plates. The bottom agar consisted of growth medium containing 10% fetal bovine serum and 0.5% agarose in six‐well plates. A total of 300 cells were resuspended in growth medium containing 10% fetal bovine serum and 0.4% agarose and placed on top of the bottom agar. The cells were grown at 37°C in a humidified incubator in 5% CO6 CFU/mouse. The anti‐PD‐L1 mAb obtained from BioLegend  was injected intravenously at a dose of 25 μg/mouse. The aim of the experiment was to determine the tumor growth changes induced by different treatments when compared with the control group. Briefly, mice were subcutaneously implanted with 500,000 TL‐1 cells/mouse in the right flank on day 0. On day 7 (tumor size ~3–4 mm in diameter), the mice were injected with Lm‐LLO‐E6 or Lm‐LLO‐E7 intraperitoneally and/or with anti‐PD‐L1 mAb intravenously. Mice were treated with anti‐PD‐L1 mAb an additional three times, on days 14, 21, and 28 after tumor implantation. The control group of mice was only injected with TL‐1 or SiHa cells. Tumors were measured every 3–4 days using digital calipers, and tumor volume was calculated using the formula V = (W2 × L)/2, where V is volume, L is length (longer diameter), and W is width (shorter diameter). A flow chart is presented in Figure Six‐ to eight‐week‐old female BALB/c nude mice and C57Bl/6 mice were purchased from National Laboratory Animal Center . Mice were cared for under protocols approved by the NLAC Animal Care and Use Committee. Lm‐LLO‐E6 (E6) vaccine and Lm‐LLO‐E7 (E7) vaccine were injected intraperitoneally at doses of 5 × 106 CFU/mouse. The anti‐PD‐L1 mAb was injected intravenously at a dose of 25 μg/mouse. The aim of the experiment was to determine the changes in tumor growth and survival induced by the different treatments. Briefly, mice were injected with 100,000 TL‐1 or SiHa cells/mouse by tail‐vein injection on day 0. On day 14, the mice were injected with Lm‐LLO‐E6 or Lm‐LLO‐E7 vaccine intraperitoneally and/or with anti‐PD‐L1 mAb intravenously. Mice were treated with anti‐PD‐L1 mAb an additional three times, on days 21, 28, 35 after tumor cell injections. Mice continued to eat normally until death. A flow chart is presented in Figure Lm‐LLO‐E6 (E6) and Lm‐LLO‐E7 (E7) vaccines were injected intraperitoneally at a dose of 5 × 10P < 0.050 was considered statistically significant.All statistical analyses were conducted using the SPSS statistical software program, as described previously  P = 0.039; Table P = 0.042; Table We collected 122 surgically resected lung tumors from NSCLC patients to examine whether E6 expression could be associated with PD‐L1 expression. The data for E6 expression were obtained from previous studies P = 0.001 for OS, P = 0.003 for RFS; Fig. P = 0.017 for OS, P = 0.121 for RFS; Figure P = 0.026 for OS; HR, 1.55, 95% CI, 0.97–2.48, P = 0.045 for RFS; Table The possible association between E6 and PD‐L1 expression, singly and combined, and overall survival (OS) and relapse‐free survival (RFS) was examined in NSCLC patients. Kaplan–Meier analysis showed that shorter OS and RFS were observed in patients with positive PD‐L1 tumors than with negative PD‐L1 tumors RNA and for comparison with HPV16‐negative TL‐4 cells transfected with the E6 or E7 expression vector. This comparison would verify whether the E6, but not the E7 oncoprotein, is responsible for PD‐L1 expression in lung cancer cells Fig. A. WesterWe next examined whether manipulation of PD‐L1 expression by E6 could attenuate the abilities for colony formation and soft agar growth in lung cancer cells. The representative colony growth in agar plates and soft agar growth colonies in soft agar plates are shown in Figure The anti‐PD‐L1 mAb may provide durable tumor suppression and a clinical benefit in advanced NSCLC We also used a tail‐vein animal model to verify whether the anti‐PD‐L1 mAb + Lm‐LLO‐E6 combination can suppress metastatic lung tumor nodule formation induced by TL‐1 cells in nude mice. The experiments were halted at 77 days. The representative metastatic lung tumor nodules induced by TL‐1 cells in each group were compared with those induced by SiHa cells. Tumors were confirmed by H & E staining Fig. A. RepresP = 0.004 for anti‐PD‐L1 mAb + Lm‐LLO‐E7 vaccine, P = 0.037 for Lm‐LLO‐E6 vaccine, P = 0.043 for anti‐PD‐L1 mAb, Fig. The body weights of the control mice injected with TL‐1 cells were significantly lower than those of mice injected with anti‐PD‐L1 mAb, Lm‐LLO‐E6 vaccine, anti‐PD‐L1 mAb + Lm‐LLO‐E6 vaccine, and anti‐PD‐L1 mAb + Lm‐LLO‐E7 vaccine, but they did not differ from those that received Lm‐LLO‐E7 vaccine treatment Fig. . SimilarThe prevalence of HPV16/18 DNA and E6 oncoprotein expression were approximately 55% and 28% κB inhibitor BAY11‐7082, but unchanged by PI3K/AKT inhibitor wortamannin .Figure S4. Changes in body weight in mice injected with TL‐1 or SiHa cells and treated with PD‐L1 mAb, Lm‐LLO‐E6 vaccine, or Lm‐LLO‐E7 vaccine.Figure S5. Tyrosine Kinase inhibitor (Gefitinib), MEK/ERK inhibitor (AZD6244), PI3K/AKT inhibitor (Wortamannin), and NF‐κB inhibitor (BAY 11‐7082) were treated with TL1 and SiHa cells for 48h.Figure S6. Lm‐LLO‐E7 vaccine alone and the combination of anti‐PD‐L1 mAb+Lm‐LLO ‐E7 vaccine markedly suppress tumor growth induced by TC‐1 cells in C57B/L6 mice compared with other treatments. The mice were subcutaneously injected with HPV16‐infected mouse TC‐1 cells.Figure S7. Mature infiltrating T lymphocytes in tumor of each group of nude mice were not detected in nude mice with different treatments.Click here for additional data file.


Understanding the mechanism of humidity transduction calls for experimental data and a theory to interpret the data and design new experiments. A comprehensive theory of humidity transduction must start with agreement on what humidity parameters are measured by hygroreceptors and processed by the brain. Hygroreceptors have been found in cuticular sensilla of a broad range of insect species. Their structural features are far from uniform. Nevertheless, these sensilla always contain an antagonistic pair of a moist cell and a dry cell combined with a thermoreceptive cold cell. The strategy behind this arrangement remains unclear. Three main models of humidity transduction have been proposed. Hygroreceptors could operate as mechanical hygrometers, psychrometers or evaporation detectors. Each mode of action measures a different humidity parameter. Mechanical hygrometers measure the relative humidity, psychrometers indicate the wet-bulb temperature, and evaporimeters refer to the saturation deficit of the air. Here we assess the validity of the different functions by testing specific predictions drawn from each of the models. The effect of air temperature on the responses to humidity stimulation rules out the mechanical hygrometer function, but it supports the psychrometer function and highlights the action as evaporation rate detector. We suggest testing the effect of the flow rate of the air stream used for humidity stimulation. As the wind speed strongly affects the power of evaporation, experiments with changing saturation deficit at different flow rates would improve our knowledge on humidity transduction. Insects are continually challenged to achieve and maintain optimal functions as ambient humidity changes. In addition to metabolic adaptations, most insects improve their survival abilities by humidity preference and avoidance responses. These behaviors make the existence of hygroreceptors very likely. The structural and the electrophysiological properties of hygroreceptors have been studied in only a few insect species. These hygroreceptors are associated in antagonistic pairs of a moist and a dry cell in the same sensillum with a thermoreceptive cold cell. The mechanism by which humidity stimulates the moist and dry cells remains controversial. Three main models of humidity transduction have been proposed, namely in which hygroreceptors operate as mechanical hygrometers, psychrometers or evaporation detectors  of water evaporating from its surface. Rising air temperature (sensible heat) increases both the wet-bulb and dry-bulb temperatures Figures , but theThe loss of water by evaporation is directly proportional to the saturation deficit of the air. Rising air temperature increases the saturation deficit Figure  when the2 surface area; Apodaca, Thus, transduction may involve chemical or mechanical mechanisms. Nonetheless, an evaporation controller that relies on concentration sensors might be unstable because chemical concentration is affected by multiple factors. Evaporation acting by fluid shear stress, pressure across, or tension within the dendritic membranes may create mechanical forces that deform membranes—as has been suggested for the mechanical hygrometer model. By contrast, the mechanosensory transduction stimulated by evaporation requires fine-tuning of the transducing ion channels. Because the moist cell and the dry cell discharge continuously during upward and downward changes in the saturation deficit, a receptor current must continuously flow when the evaporation rate is in equilibrium with ambient humidity. The current will be modulated between maximum and minimum values, and the sign will be opposite in the moist and dry cell. Conductivity of both antagonists must account for this symmetric change in receptor current as well as for its maintenance. A simple mechanism described for mechanosensitive channels is based on variation of the membrane capacitance (Petrov and Usherwood, Electrophysiological studies on the cockroach revealed a dependence of the responses of the moist and the dry cell to slowly fluctuating changes in vapor pressure on the temperature level at which the humidity changes are carried out Figures . The posAll authors contributed to data analysis, interpretation of results, writing the manuscript and designing the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The pooled relative risk (RR) value for objective response rate (ORR) was 1.53, . Further, subgroup analysis based on PD-L1 expression indicated that pooled HR for OS was significant with 0.66 for PD-L1≥1%. However, PD-L1 < 1% had HR value of 0.79 . Our study concluded that advanced NSCLC patients benefited more with PD-1/PD-L1 antibodies than docetaxel in the second line treatment. PD-L1≥10% in tumor tissues is sufficient to show significant improvement in patient's outcome with PD-1/PD-L1 antibodies compared to docetaxel. Moreover, PD-1/PD-L1 antibodies treatment showed significant decrease in conventional chemotherapy adverse events, but increased immune-associated adverse effects.Meta-analysis was conducted to systematically assess the effectiveness and safety of programmed cell death protein-1 or ligand-1 (PD-1 or PD-L1) antibodies versus docetaxel alone in advanced non small cell lung cancer (NSCLC). In addition, the prognostic significance of PD-L1 expression in advanced NSCLC was also investigated. 5 eligible studies including 3579 patients were identified through comprehensive search of multiple databases. The results showed that pooled hazard ratios (HR) for overall survival (OS) and progression free survival (PFS) were 0.69 (95% CI: 0.63-0.75;  Lung cancer ranks first in term of causing cancer associated deaths worldwide, with an estimation of 221,200 new cases and 158,040 deaths alone in the year 2015 . Lung caApparently the cost of immunotherapy for advanced NSCLC has been enormous  and is det al. had analyzed two dosage (2 and 10 mg/kg) of pembrolizumab. Furthermore, the Cochrane risk of bias tool was used to measure the quality of the included studies, and the results are shown in Figure Based on the search criteria, we initially identified 1011 studies from our database search. Among these, 858 studies were excluded because they did not fit our selection criteria. Of the remaining 153 studies, only 5 randomized controlled trials (RCTs) passed the inclusion criteria, and others were excluded because of repetitive or insufficient information Figure . Out of 2 = 0%). Further subgroup analysis of OS based on PD-L1 expression again revealed statistically significant advantage for PD-1/PD-L1 therapy as compared to docetaxel, with pooled HR values of 0.79  in subgroups with PD-L1 expression of < 1%,0.66  with PD-L1 expression of ≥1%, 0.55  with PD-L1 expression of ≥5%, 0.41  with PD-L1 expression of ≥10%, and 0.49  with PD-L1 expression of ≥50%. However, the pooled HR values were not statistically significant in subgroups with PD-L1 expression of < 5% [0.86], and < 10% [0.86]. In addition, we found very little overall heterogeneity for OS in all studies , but the heterogeneity at the PD-L1 expression subgroup levels was different. For instance, PD-L1 expression of ≥1%, 5%, 10%, 50% and < 1 %, displayed I2 values of 0% (p = 0.740); 10.0% (p = 0.343); 0% (p = 0.537); 0% (p = 0.811);18.5% (p = 0.298). respectively, and represented less heterogeneity. However other subgroups based on PD-L1 expression like, < 5% and < 10% showed I2 values of 56.1% (p = 0.131) and 56.5% (p = 0.129), respectively, and suggested high heterogeneity . The subgroup analysis for PFS based on PD-L1 expression showed different levels of heterogeneity, ranging from no to significant levels. For instance, PD-L1 expression with ≥1%, 5% ,10%, 50% and < 1% showed I2 values of 0% (p = 0.567); 6.2% (p = 0.362); 0% (p = 0.748); 0% (p = 0.993) and 42.8% (p = 0.155), respectively. But PFS analysis of PD-L1 subgroups with expression < 5% and < 10% showed I2 values of 83% (p = 0.015) and 84.8% (p = 0.010), respectively, and represented high heterogeneity.Similarly, forest plot analysis of PFS indicated better results with PD-1/PD-L1 antibodies than docetaxel in advanced NSCLC patients Figure . The PD-2 = 59.2%) in favor of PD-1/PD-L1 antibodies in NSCLC patients. Further subgroup analysis of ORR according to PD-L1 expression also indicated that the pooled RRs in subgroups were in favor of PD-1 antibodies treatment. For example subgroups displaying PD-L1 expression of ≥1%, 5%,10% and 50% had RR values of 1.70 ; 2.08 ; 2.75 ; and 3.55 , respectively. However, the pooled RRs of subgroups with PD-L1 expression of < 1%, < 5% and < 10% were 0.81 ; 0.79; and 1.01 , respectively, and were not statistically significant. The heterogeneity among other subgroups except overall RR was between 0 to 50% and was thus not a major factor effecting RR values.The comparison of overall response rate in all four RCTs also demonstrated that PD-1/PD-L1 antibodies have significantly better effect than docetaxel Figure . The ORRAs compared with standard second line docetaxel chemotherapy, the advanced stage NSCLC patients receiving PD-1/PD-L1 antibodies showed significant increase in the incidence rate of grade 1-4 adverse events (AEs). The overall RR value for AE was 0.77 , and specific RRs based on each event also showed significant effect, like for rashes, the RR value was 2.01, 95% CI: 1.14-3.51, P = 0.02; for hypothyroidism, the RR value was 23.36, 95% CI: 8.04-67.90; P < 0.001; for hyperthyroidism, the RR value was 5.1, 95% CI: 2.23-11.68, P < 0.001; for pneumonitis, the RR value was 3.19, 95% CI: 1.9-5.34, P < 0.001; and for colitis, the RR value was 4.99, 95% CI: 1.45-17.11, P = 0.011. But, we did not observe any significant difference for the incidence rate of interstitial lung disease between both groups . However the incidence rates of other grade 3-4 AEs including: nausea, rash and colitis, did not show any significant difference between PD-1 antibodies and docetaxel therapy  with nivolumab, for patients with NSQ tumors by 67%, and led to 40% reduction in the incremental costeffectiveness ratios (ICER) (from US$ 176K to 105K). In case of pembrolizumab, the use of 50% instead of 1% cutoff, helped to increase the incremental QALY by 18% and reduced ICER by 15% (from US Dollar163K to 138K) . ConsistHowever, in our meta, the OS of the patients was significantly elevated with PD-1/PD-L1 antibodies than docetaxel in subgroups with PD-L1 expression of < 1%, which is consistant with the rusults of several some studies –32. BrahThe adverse events due to immunotherapy have been because of disruption in immune tolerance. The immune-related adverse events (irAEs) are usually defined as any AE associated with drug exposure and consistent with an immune-mediated mechanism of action , 42. OurThus, in conclusion, our meta-analysis study indicated that PD-1/PD-L1 antibodies treatment indeed has beneficial effects on advanced NSCLC patients in comparison to docetaxel monotherapy, along with displaying few adverse events. In addition, the PD-L1 expression of more than 10% on tumor tissues can serve as a biomarker to identify the NSCLC patient populations that might respond positively to PD-1/ PD-L1 antibody therapy, and thereby helping many patients to make a informed decision about this high cost immunotherapy.All the random controlled trials with information about NSCLC, docetaxel, PD1 and PD-L1 antibodies, from January 1990 to January 2017, were searched using the following databases, Cochrane library, Embase, PubMed, China hospital knowledge database, China National Knowledge Infrastructure, Wangfang Data and Weipu Data. The medical subject heading (MeSH) terms included for searching the relevant studies were: lung neoplasm, pulmonary neoplasm, lung carcinoma, pulmonary carcinoma, lung cancer, pulmonary cancer and PD-1 or PD-L1, PD-1 or PD-L1 inhibitors and chemotherapy or docetaxel.The studies with the following information were selected for inclusion in our meta-analysis; (1) Phase II/III (randomized controlled trails) RCTs with primary endpoints as OS or PFS; (2) histological confirmed SQ and/or NSQ non small cell lung cancer; (3) having the information about OS, PFS and ORR, AEs, and PD-L1 expression; (4) published in English language; and (5) having some similarity between experimental design and methods across different studies. However, the studies were excluded if they were: (1) reviews, duplicate reports, letters, unfinished studies, or conference reports; (2) studies conducted with cell lines, animal models or other types of non-lung cancers; (3) studies where HR and 95% CI could not be determined due to insufficient survival data; (4) papers in other laungages except English; (5) methods or experimental design were substantially different from other selected RCTs; and (6) sample size was smaller than 100.Two reviewers (Qiang Su and Yanli Hou) independently searched all the relevant studies and read the titles, abstracts and full texts of the identified studies. Cases of disagreement were resolved through discussion with the third reviewer (Hongchao Zhen). The following information was extracted from the selected studies; year of publication, name of the journal, author's name, methods of randomization, OS, PFS, ORR, PD-L1 expression rate and adverse events (AEs) with grades 1-4 and 3/4.2 test was used to assess the heterogeneity among the RCTs. When I2 value was < 50%, the fixed-effects model (FEM) was employed for analysis, and if I2 value was ≥50%, random-effects model (REM) was used. The Begg's and Egger's tests were used to analyze the publication bias between different RCTs.In our meta-analysis, Risk of bias analysis was prepared using Review Manager 5.3 software . Two reviewers (Q.S. and C.G.Z.) independently assessed the quality of the included studies according to the Cochrane risk of bias tool, which assesses the following six domains: selection bias , performance bias, detection bias, attrition bias, reporting bias and other bias. The Stata version 12.0 statistical software  was used to conduct the meta-analysis. The HR and 95% CI values were collected and merged to estimate overall OS and PFS. The HR value of < 1.0 indicated reduced progression or death in the PD-1/PD-L1 antibody group. The RR value was used to estimate ORR and AEs for grade 1-4 /3-4, and RR value of >1.0 represented higher ORR or the incidence of grade 1-4 and 3/4 AEs in the PD-1/PD-L1 antibodies group. In addition, the Cochran's χ


Graphene, the thinnest, strongest, and stiffest material with exceptional thermal conductivity and electron mobility, has increasingly received world‐wide attention in the past few years. These unique properties may lead to novel or improved technologies to address the pressing global challenges in many applications including transparent conducting electrodes, field effect transistors, flexible touch screen, single‐molecule gas detection, desalination, DNA sequencing, osmotic energy production, etc. To realize these applications, it is necessary to transfer graphene films from growth substrate to target substrate with large‐area, clean, and low defect surface, which are crucial to the performances of large‐area graphene devices. This critical review assesses the recent development in transferring large‐area graphene grown on Fe, Ru, Co, Ir, Ni, Pt, Au, Cu, and some nonmetal substrates by using various synthesized methods. Among them, the transfers of the most attention kinds of graphene synthesized on Cu and SiC substrates are discussed emphatically. The advances and the main challenges of each wet and dry transfer method for obtaining the transferred graphene film with large‐area, clean, and low defect surface are also reviewed. Finally, the article concludes the most promising methods and the further prospects of graphene transfer. Besides, the methods of directly synthesizing graphene on target substrates are very immature now. So, the LAG films synthesized by both CVD method and the epitaxial growth method through SiC have to be transferred from the growth substrates to the target substrates in the applications of LAG films. Up to now, various methods have been developed to transfer the graphene film, many of which are of advantages for industrial applications. No doubt the development of the transfer methods will promote the study and application of LAG films. This work aims to give a comprehensive review of the method for transferring graphene from various growth substrates to the target substrates.TableIn this review, we first discuss the transfer methods of graphene grown on different kinds of metal substrates and focus on the transfer of graphene grown on Cu substrate. Then, the progress in each wet or dry transfer method is reviewed along with the properties of the transferred graphene film, respectively. In the third part, the transfer of epitaxial graphene grown on SiC is discussed. The properties of the transferred graphene film by different methods are listed in 24) at high temperature and then deposit the decomposition products on metal substrate to form single‐layer or few‐layers graphene. Graphene synthesized by CVD method has been realized on many metal substrates, such as Fe,Chemical vapor deposition method is believed to be an efficient method to synthesize LAG. The mechanism of CVD method is described as follows: decompose the hydrocarbon precursors  stamp as the supporting layer. Various sizes of graphene film can be transferred to arbitrary substrate. For graphene growing on Co film, Wang et al.3 solution as the Co etchant. After the removal of Co film, the graphene film was transferred to the target substrates.An et al.Among all of these methods for growth graphene, CVD synthesis of graphene on Cu foil is of many advantages. For example, the C solubility of Cu is low, the graphene layers can be controlled easily, and it can also be transferred easily with relatively low cost. Therefore, Cu has been often used as the metal substrate. Here, we mainly focus on the transfer of graphene synthesized on Cu substrate. The transfer methods for CVD graphene can be classified as wet and dry transfer, depending on the environment where graphene touches the target substrate.2.12.1.12 plasma in order to etch the Cu substrate; 2) PMMA layer is coated on the surface of the graphene/copper substrate and then cured; 3) the underlying copper substrate is etched away by the etchant, then washed by the deionized (DI) water; 4) transfer the PMMA/graphene sample to the target substrate; 5) the PMMA layer is removed by acetone, then washed by the DI water and dry.Generally, taking advantage of the polymer layer such as PMMA as a supporting layer is a necessary step for wet method of transferring graphene grown by CVD. PMMA has many prominent features, such as the relatively low viscosity, excellent wetting capability, flexibility, and good dissolubility in several organic solvents.Besides using PMMA as the polymer supporting layer, poly(bisphenol A carbonate) (PC)2.1.2Figure2/Si substrate to SiC substrate.The transfer of carbon nanotubes Figure137 hasLi et al.2/Si substrate.FigureIn order to make the graphene to be better contacted with the substrate, Li and co‐workers developed an improved method by adding a second PMMA coating after the PMMA/graphene was placed on the SiO2.1.3−1).PMMA layer was removed by acetone in the wet‐transfer method. Due to the high molecular weight and high viscosity of PMMA, it is inevitable to leave PMMA residues on the graphene surface without further annealing. However PMMA residues may trigger charged impurity scattering and unintentional graphene doping thus affect the property of graphene.FigureIt is believed that annealing in high temperature (250 °C–350 °C)For the above improved methods, the subsequent processes were added to remove the PMMA residues. However, the adding steps will make the manipulation more complex, which may introduce defects to the graphene and increase the cost. Many simple improved methods have been reported to reduce the PMMA residues. Taking advantage of the excellent solubility of the organic small molecular in the solvent, Han et al. inserted an organic small molecular buffer layer between the PMMA and graphene.22H14) thin film as the supporting layer had transferred the graphene to 6 in. Si wafer with clean surface and almost no crack. The 200 nm thick pentacene was thermally evaporated onto CVD‐grown graphene on a copper foil to be used as a supporting layer. After transferring the pentacene/graphene onto a target substrate, pentacene was removed by thermal evaporation (250 °C and 300 °C for 1 h) and chemical solvent (tetrahydrofuran (THF)). They also tried other solvent such as benzene, toluene, and chlorobenzene (CB). As a result, the THF was believed to be the best solvent for removing pentacene layer. The graphene transferred by this method showed excellent electrical properties with the hole and electron mobility observed in FETs were 8050 and 9940 cm2 V−1 s−1, respectively. These values were almost two times higher than the field‐effect mobility of the graphene transferred by the PMMA‐assisted method.Scientists also tried to look for the suitable replacer of PMMA layer to get the clean and doping‐free transfer of LAG graphene. Kim et al.Figure−1 and the mixed water/IPA solution was simultaneously injected at the same rate. The surface tension for graphene in the solution could be controlled by this way. The solution was pulled out with the syringe to lower the graphene onto the substrate when the etchant was replaced by the mixed solvents. Compared with the graphene transferred by the conventional polymer‐assisted methods, the graphene transferred by this polymer‐free method showed advanced electrical properties. The mobility of the polymer‐free monolayer graphene is as high as 63 000 cm2 V−1 s−1, which is 50% higher than the similar graphene transferred by the conventional method. But the shape and size of the transferred graphene films are limited by the graphite holder.Transferring graphene without using the polymer supporting layer may be the best way to reduce the polymer residues. These kinds of methods are called the “polymer‐free” methods.To transfer graphene with unrestricted shape, Pasternak and co‐workers used a waterproof marker‐frame to transfer graphene by “polymer‐free” method.2/Si or PET). The negative charges formed on the target substrates. The graphene was thus attracted to the target substrate by the electrostatic force during the transfer process. After the metal substrate removal, graphene was transferred onto the target substrate. Graphene can be transferred layer by layer in this way. These methods did not use the polymer supporting layer, which enabled the clean transfer of CVD‐grown graphene.Wang et al. developed a unique technique called the “clean‐lifting transfer” method by using the electrostatic force to transfer graphene onto target substrates.The “polymer‐free” methods that direct transfer the graphene from the growth substrate to the target substrate provide a way to transfer graphene without polymer residues. The interfacial bonding condition between the graphene and the target substrate is critical for transfer success when direct transfer the graphene film to the soft substrate. A key factor for high quality graphene transfer is the wettability of the target substrate. Martins et al.Etchants are efficient to remove the copper substrate, and the commonly used etchants are aqueous solutions of iron nitrate,2O/H2O2/NH4OH (5:1:1) solution; (2) the second step is oxide strip using a diluted H2O/HF (50:1) solution to remove a thin silicon dioxide layer where metallic contaminants may have accumulated as a result of step 1; (3) in this step, ionic and heavy metal atomic contaminants are removed by using a solution of H2O/H2O2/HCl (5:1:1).A cleaning technology called “RCA cleans” is used for Si wafer cleaning in the semiconductor manufacturing. The traditional RCA cleans involve three sequential steps: (1) the first step is intended for the removal of insoluble organic contaminants with a H2.1.4The traditional wet transfer methods that use the etchants to etch the metal substrates have many disadvantages, such as time‐consuming, high cost , the metal particulates residues and serious environmental pollution. These disadvantages will no doubt restrict the industrial scalability application of the wet transfer methods.FigureThe electrochemical delamination methodm NaOH aqueous solution under a constant current of 1 A, with the applied voltage of 5–15 V. Graphene film of 1 × 3 cm2 was separated from the Pt substrate in 30 s. What is more, the Pt substrate can be repeatedly used for graphene growth. But when taking the PMMA/graphene/Cu stack as the anode electrode,m H2SO4 electrolyte with a potential of 0.5 V and a current of about 0.03 A. It was enough to etch away the Cu foil rapidly. The whole process ended within several minutes, which was much faster than the conventional wet transfer methods. But the Cu substrate cannot be reused.Gao et al.The electrochemical delamination methods in aboveThe wet transfer method is an important way to transfer graphene. However, as we have mentioned above, it has many problems, for example, it is easy to form defects, easy to induce impurity residues on the graphene surface, time consuming, high cost, environment pollution. Etchant and chemical solvents are used in the wet transfer methods. The solvents will introduce the unwanted doping to graphene,2.1.5−1) and 2D peak (≈2700 cm−1) are the typical peaks for graphene  will emerge in the Raman spectrum  can be used to estimate the graphene layers and the doping level of graphene.I2D/IG indicates the increase of the graphene layers, while the increase of the I2D/IG usually indicates the lower doping level of graphene. Since D peak is associated with the defects of the graphene film, the lower ID/IG indicates the higher quality of graphene film. So, the values of I2D/IG and ID/IG can give us information of graphene quality. Raman spectroscopy is a very powerful tool to test the quality of graphene film. The development and application of the Raman technology will notably promote the study of the graphene film.Raman spectroscopy is usually used to characterize the graphene layers, defects, and the doping concentration of graphene. The G peak , atomic force microscope (AFM) and scanning electron microscopy (SEM) are also efficient tools to characterize the surface morphology of the transferred graphene. These images can give direct information to determine whether there are contaminants or wrinkles on the graphene film. And AFM can also be used to determine the number of graphene layers from the film thickness. But when taking for practice, it is only possible to distinguish between one and two layers by AFM if the graphene contain folds or wrinkles.2.2As we mentioned in the beginning of the section Figure−1 without additional doping. They also fabricated graphene‐based capacitive touch‐screen devices, which demonstrated the potential application of graphene as electrode in the capacitive multitouch devices installed in the most sophisticated mobile phones.  The adhesion of subsequent graphene and dielectric layers on flexible substrates is very poor, so it is difficult to fabricate graphene‐based flexible devices where more than one graphene layer is need. Bointon et al.The “Roll‐to‐roll” method of synthesizing and transferring the 30 in. graphene has been reported in 2010 Figure7a.28 FFigure−2). The TRT was also used as the supporting layer in this process. After the removal of Cu substrate, graphene on the TRT was inserted into two rollers to attach the target substrates (PET or SiO2/Si). The TRT lost the adhesion force after the process of hot pressing for less than 10 s, then graphene was transferred onto the target substrate. The improved method can transfer graphene to the flexible or rigid substrates. Compared with “Roll‐to‐roll” method, graphene transferred by the “hot pressing” method forms relatively less defects  and the graphene is very strong, so it is inevitable to leave adhesive residues on the graphene surface. In order to get a clean surface of the transferred graphene, Chen et al.−2 after etching away the copper substrate. The PSAF layer was peeled off from the graphene/target substrate with the speed and angle with respect to substrate surface were 2 mm s−1 and about 90°, respectively. The graphene transferred by this method had cleaner surface than transferred by the PMMA and TRT. The PSAF‐assisted transfer method is applicable to both rigid and flexible substrates without difficulty in scaling up for industrial application.Kim et al.In the dry transfer methods discussed above, the etchants have also been used to remove the Cu substrates, so Cu foil cannot be reused to grow graphene. Graphene is grown on both side of the Cu foil by CVD method. One side of the graphene must be removed in advance before etching away the Cu substrate. This process causes a lot of waste. So there are still many problems that restrict the industrial scalability application of these methods. Zaretski et al.−2. And they used the dry transfer method to transfer the graphene to the polyimide (PI) by the assistance of an epoxy layer. The Cu etchant was avoided in this process, and Cu substrate can be reused to grow graphene.To realize the real dry transfer process, the adhesion energy between the graphene and substrate should be measured for the precise control of the delamination process. Yoon et al.2 was transferred to the target substrate in short time. Juang et al.It is very important to achieve highly conformal contact between the graphene and the target substrate to obtain high adhesion energy between them. Jung et al. developed a mechano‐electro‐thermal method,FigureN‐ethylamino‐4‐azidotetrafluorobenzoate (TFPA‐NH2) was deposited on the polymer surface after plasma treatment. This process made the adhesion between the graphene and polystyrene to be much higher than that between the graphene and metal substrate. Graphene can be separated from the metal substrate by the assistance of the nanoimprinter. The whole process took less than 3 h. This method is also suitable for the industrial scalability application. The properties of the transferred graphene film by dry transfer methods are listed in Table Besides using the adhesives as the above methods done, introducing covalent bonds between the graphene and the substrates has also been adopted to increase the adhesion energy. Graphene can be transferred with high quality without using any chemical etchant. Lock et al.Figure2/Si substrate was pretreated by N2 plasma to convert the surface of the substrate to silicon oxynitride phases. This step was demonstrated to be helpful for facilitating the formation of capillary bridges. The “Face‐to‐face” method realized both the growth and transfer of graphene on one wafer, and this process did not have to be done by hand, which were different from the wet and dry transfer methods. The quality of the transferred graphene was better than the conventional transfer method.In above, we review the wet transfer and dry transfer method of graphene grown on metal substrates. Since the “Roll‐to‐roll” dry transfer method has succeeded transferring the 30 in. graphene, it seems to be a more available way for industrial application. But many defects are emerged after transferring. And the disadvantages we have mentioned above suggest that the wet transfer method is difficult to scale up. Gao et al. developed a “Face‐to‐face” transfer method Figure10.36 D3We have discussed the transfer methods of graphene grown on the metal substrates by CVD method in the part 2. CVD method is believed to be an efficient way to synthesis LAG. But the surface of the Cu substrate is rough, so graphene grown on Cu substrate is easy to form wrinkles. Besides, the orientation of the graphene cannot be controlled easily. Epitaxial growth of graphene on SiC surface is also an important way to synthesis LAG. High‐quality graphene can be grown on SiC wafer by self‐limiting sublimation of Si. And the synthesized graphene has a single orientation because of the self‐limiting growth process. SiC substrate is insulated, so in some cases it can be used directly without transfer. But the cost of SiC is very high and sometimes the graphene is needed to be transferred to other substrates. It is also necessary to develop the method for transferring graphene grown on SiC wafer. However, SiC is resisted to the chemical etchants. The traditional wet transfer method is not available here.2/Si substrate by the assistance of the adhesive tape. But the size of the transferred graphene was limited to be about 1 μm2 or less.Graphene grown on SiC substrate can be transferred onto SiOFigure2/Si substrate successfully. The PI and the metal layer were removed by O2 plasma reactive ion etching and chemical etchant (Transene), respectively. This method can transfer the graphene film layer by layer. It is also suitable to transfer LAG. But the results of Raman spectrum showed that more defects were formed after transferring  have been estimated by Hamada and Otani.2/Si substrate and then the PVA was dissolved by water. No chemical solvent was used during this process. The doping‐free graphene can be obtained by this method.The metal layers have been used in the above transfer methods. When the graphene was transferred to the target substrates, chemical etchants were used to remove the metal layer. Defects were introduced during the etching process easily. The residue metal particles and the doping of graphene introduced by the chemical etchants will influence the property of graphene. Tanabe et al.In above, we have reviewed the transfer methods of the epitaxial graphene grown on SiC substrate. The “Etch‐free” transfer method seems to be an efficient method because it does not use any chemical solvent. This method is a more environment friendly and economical way to transfer the graphene for applications. The dry transfer method4The transfer of graphene acts as the bridge between the production and application of graphene. In this work, we have reviewed the transfer methods of graphene grown on metal (mainly on Cu) and SiC substrates. All of these methods are aimed at transferring graphene with clean surface and little defect. Many transfer methods have achieved these goals. The success of the graphene transfer is of great significance for the application of graphene film, making it possible for the industrial scalability application of graphene. The “Roll‐to‐roll” dry transfer method can transfer the graphene film as large as 30 in., so it seems to be the efficient method for industrial application. Other methods also have their own advantages. Some of them are hopeful for the practical industrial application. Despite that, the transfer methods are still need to be optimized to transfer graphene with little defect and large area. There are still many challenges to transfer LAG films. (1) The first challenge is avoiding the crack during the transfer process while achieve large area transfer at the same time. Monolayer graphene itself can be easily cracked during the transfer process without the assisted of the supporting layer. The improper operation during the transfer process can also induce cracks on the graphene. (2) The second challenge is obtaining LAG film with clean surface. The contaminants on the graphene surface are also big problems to be solved because they may act as scattering centers and induce unintentional graphene doping thus affect the graphene property. It is necessary to obtain LAG film with clean surface when fabricate graphene‐based devices. (3) Simplifying the transfer process to reduce the cost is also a main challenge. The cost of the transfer process must be taken into consideration in the practical industrial application. The complex and time‐consuming transfer methods are unsuitable for transferring the LAG film. Besides, both the transfer of graphene and the synthesis methods of graphene are very important for the application of graphene. It is necessary to optimize the methods to product LAG films with high quality. In recent years, the transfer‐free growth methods where graphene grew directly on target substrates have been reported.


GII/GI, leading to lower adhesion toughness. For silicon oxide substrate and pressure loading, mode mixity decreases from 232% for monolayer films to 130% for multilayer films, causing the adhesion toughness Gc to decrease from 0.424 J m−2 to 0.365 J m−2. The mode I and II adhesion toughnesses are found to be GIc = 0.230 J m−2 and GIIc = 0.666 J m−2, respectively. With point loading, mode mixity decreases from 741% for monolayer films to 262% for multilayer films, while the adhesion toughness Gc decreases from 0.543 J m−2 to 0.438 J m−2.Interface adhesion toughness between multilayer graphene films and substrates is a major concern for their integration into functional devices. Results from the circular blister test, however, display seemingly anomalous behaviour as adhesion toughness depends on number of graphene layers. Here we show that interlayer shearing and sliding near the blister crack tip, caused by the transition from membrane stretching to combined bending, stretching and through-thickness shearing, decreases fracture mode mixity  The reason why the surface adhesion of a graphene monolayer is much greater than that of graphene multilayers remains unclear. Here, the authors build a model to show interlayer sliding and fracture mode mixity cause the decrease in adhesion toughness of multilayer graphene. Multilayer graphene films may conform less well to the substrate than monolayer ones. Koenig et al.1 made roughness measurements on the top surfaces of graphene films and found a large drop in roughness from monolayer to two layer; however, they also found a large drop from two layer to three layer. This suggests that the roughness of the substrate surface is unable to explain the large decrease in adhesion toughness. To investigate the effect of interface roughness further, Gao and Huang2 argued that the rough surface of silicon oxide causes graphene films to bend; hence, the total adhesion energy consists of both van der Waals interaction energy and a negative contribution of bending strain energy. By assuming the substrate to have a sinusoidal rough surface, they attempted to calculate the adhesion energy. They concluded that the large decrease in adhesion toughness from monolayer to multilayer graphene films is due to the increase in bending strain energy caused by the large increase in the bending stiffness. Jiang and Zhu3 measured the van der Waals interaction energy between monolayer graphene films and silicon oxide substrate using atomic force microscopy. Their measurements show, however, that the roughness increases the interaction energy. In contrast, He et al.4 studied the large decrease in adhesion toughness from another perspective. They proposed that the total adhesion energy consists of both van der Waals interaction energy and residual in-plane strain energy due to lattice mismatch strain at the graphene film-silicon oxide interface. Their results show that the van der Waals interaction energy remains nearly the same for graphene films with any number of layers, but that the residual in-plane strain energy and Young’s modulus decrease sharply from monolayer to multilayer graphene films. Koenig et al.1, however, reported convincing experimental results that show a constant Young’s modulus. This observation provided a solid foundation for their subsequent adhesion toughness calculations using a continuum mechanics approach.Koenig et al.1 also suggested possible sliding between graphene layers in multilayer graphene films. The present work follows Koenig et al.’s1 continuum mechanics approach but with consideration for the interlayer shearing and sliding effect. Furthermore, the present work considers the effect of shearing and sliding on the fracture mode mixity. This is an important consideration, since interface adhesion toughness is not a purely intrinsic material property, but instead also depends on the mode mixity.Koenig et al.10 and we argue that this has caused confusion when calculating adhesion toughness. Cao et al.12 did, however, recently report studies on adhesion toughness between photoresist films and copper substrates using blister tests and the finite element method. Two types of film are considered: One is pure photoresist film and the other is combined photoresist film and a monolayer graphene. Mode mixity is considered by using cohesive zone modelling.Note that the fracture mode mixity and the interlayer shear and sliding effect are not considered anywhere in the current analytical mechanical modelsGII/GI, consequently reducing the adhesion toughness Gc. By considering the interlayer shearing and sliding effect, the mode I and mode II toughnesses are shown to be independent of the number of graphene layers. Accounting for the interlayer shearing and sliding effect on the fracture mode mixity explains the behaviour reported in the literature1, where adhesion toughness measurements seemingly depend on the film thickness . Once the mode I and mode II adhesion toughnesses have been found, the linear failure criterion can accurately determine the adhesion toughness under general loading conditions for real-world applications of graphene film-substrate systems.The present work shows that adhesion toughness is mode mixity dependent, and that interlayer shearing and sliding near the blister crack tip, caused by the transition from membrane stretching to combined bending, stretching and through-thickness shearing, decreases the mode mixity RB, the thickness of the monolayer graphene is t, n represents the number of graphene layers and the Young’s modulus of graphene is E. In Fig. 1. According to Jensen14, the deflection δ at the centre of the blister in the membrane limit isp is the pressure load and f(ν) is given by Storåkers15 asf(1/3) = 0.645 obtained by Jensen13 since Storåkers’ formula15f(ν) = [3(1−ν)/(7−ν)]1/3 is approximate. The bending moment per unit width MB, in-plane force per unit width NB, and shear force per unit width PB, at the blister crack tip14 can be expressed in the following forms, respectively  isFigure entclass1pt{minimaentclass1pt{minima20ρ = GII/GI asλ parameter in Eqs. , which is assumed to take the following form:At this stage, the effect of interlayer shearing and sliding on the fracture mode mixity can be introduced. An introduction to mixed-mode partition theory is given in Supplementary Note mentclass2pt{minimsing Eq.  in conjuλ is given in Supplementary Note A more thorough and detailed explanation for the origin of GI in Eq. (GII in Eq. (Gc can now be determined by using the mode I and mode II adhesion toughnesses and a linear failure criterion in which Gc = (1 + ρ)/(1/GIc + ρ/GIIc). Note that GIc and GIIc are intrinsic interface material properties but Gc is not. One major aim of the present study is to determine values for GIc and GIIc based on Koenig et al.’s1 experimental results. Once these two properties are known, the adhesion toughness under other loading conditions can be readily calculated.The total ERR is simply the sum of the mode I ERR I in Eq.  and the I in Eq. . The modMB in Eq. , then Eqs. (f(0.16) = 0.6907, φ(0.16) = 0.3099, ζ(0.16) = 0.4502, respectively. Then, the essential equations above, namely Eqs. , 1 found tely Eqs. , 17),  = GJ(1 + η), thenNote that Koenig et al.e to Eq.  in the pe to Eq.  and 15)1 used Ge to Eq. , 21) an an1 usedp, the central deflection δ and the radius RB of the multilayer graphene film blisters are taken from figures in Koenig et al.’s1λ, η and ρ, respectively, for monolayer and multilayer graphene films are plotted based on the measured values of p and δ from Koenig et al.1. In Fig. Gc is plotted vs. the fracture mode mixity ρ. In Fig. p, δ and RB, and the present mechanical model for graphene films with different numbers of layers. Note that the ‘Theory’ curve in Fig. p; then for Fig. RB and δ. There is generally very good agreement between the present mechanical model and the experimental measurements1.In the following, the pressure 1, results are calculated using the pressure p and the central deflection δ meaning that GJ = 0.6517 pδ and λ parameter, based on Koenig et al.'s1 measurements, are recorded in Supplementary Tables λ parameter are very close to each other. This shows the typical interlayer shearing and sliding behaviour.The numerical data for Fig. rom Eqs.  and 20)1, resultGc = 0.424, 0.362, 0.389, 0.348 and 0.359 J m−2 for the mono-, two-, three-, four- and five-layer graphene film blisters, respectively, which correspond to the following mode mixities ρ = GII/GI = 2.319, 1.400, 1.259, 1.263 and 1.272. There is a large decrease in mode mixity for two-layer graphene film blisters in comparison to monolayer films, which results in a large decrease in the adhesion toughness. For higher numbers of graphene layers, the adhesion toughness does not change significantly from the two-layer case as there are no significant changes in mode mixity. An overall average adhesion toughness for multilayer graphene films blisters is Gc = 0.365 J m−2 with ρ = GII/GI = 1.299. These results are shown in Table The average adhesion toughnesses are GIc and GIIc, are considered. He et al.4 showed that the van der Waals interaction energy remains nearly the same for graphene films with any number of layers at 0.266 J m−2. This suggests that GIc and GIIc are the same for interfaces between monolayer graphene films and silicon oxide substrates, and between multilayer graphene films and silicon oxide substrates. As adhesion toughness is generally very small, a linear failure criterion can provide an accurate representation of the fracture mechanics in question18.Now the mode I and mode II adhesion toughnesses, Let subscripts ‘1’ and ‘2+’ represent values for monolayer and multilayer graphene films, respectively. Substituting the monolayer and multilayer results from Table GIc = 0.230 J m−2 is very close to He et al.’s4 theoretical calculation of the van der Waals interaction energy at 0.266 J m−2. In fact, the van der Waals interaction energy is essentially the same in concept as the mode I adhesion toughness. The mode I adhesion toughness GIc can be determined using atomic force microscopy measurements3 and JKR model asFadh = 378 nN is the van der Waals interaction force and Rtip = 405.4 nm is the radius of the microsphere tip used in the atomic force microscopy measurements. It is seen that the measured GIc = 0.198 J m−2 is very close to the present value of GIc = 0.230 J m−2.It is interesting to note that GIc = 0.230 J m−2 and GIIc = 0.666 J m−2 will be used to predict adhesion toughness under point loading in order to examine the validity of the approach.In the following section, the theory developed above for the circular blister test under pressure loading and the determined values of P (refs. 13) is shown in Fig. 13 Fig. 15 gives φ(ν) asA blister under a point load f(ν) now becomesThe function p can now simply be replaced everywhere with 20The pressure load  in Eqs.  to 5), , p can nIn addition, Eqs.  and 15)15) becomν = 0.16, then Eqs. (φ(0.16) = 0.4636, f(0.16) = 1.497, ζ(0.16) = 0.3743, respectively. Equations , ν = 0.16,quations –31) the theν = 05 in which they used nanoparticles to create a point load on five-layer graphene membrane blisters. The blisters typically possessed a radius RB in the range 250–300 nm and central deflection δ in the range 50–70 nm. They used the formula Gc = 0.0625nEt(δ/RB)4 with E = 0.5 TPa and nt = 1.7 nm. Note that Zong et al.’s5 value for E is half of that used by Koenig et al.1, and that n ≈ 5. Zong et al. reported the adhesion toughness as Gc = 0.151 J m−2 meaning that δ/RB = 0.2309. When using Koenig et al.’s1 value of E = 1.0 TPa, then Eq. , Boddeti et al. reported further studies on the adhesion toughness between monolayer graphene and silicon oxide substrates. The adhesion toughness was found to be Gc = 0.24 J m−2, which is significantly smaller than Gc = 0.45 J m−2, reported by Koenig et al.1. Boddeti et al.21 suggest that the difference arises from the differences in interface properties such as roughness and chemical reactivity between the samples in ref. 1 and the samples in refs. 22. In line with this suggestion, the present work suggests that the reduction is caused by reduction of the mode I and mode II adhesion toughnesses at the interface, GIc and GIIc, which are now estimated. Taking the Young’s modulus and Poisson’s ratio still as E = 1 TPa and ν = 0.16, Eq.  to determine the mode I and mode II adhesion toughness of thin films by using blister tests. The analytical predictions agree very well with the experimental results reported by Cao et al.It should be noted that a general methodology has been presented, and the substrate should not be restricted to silicon oxide substrates. Furthermore, the ‘adhesion energy’ commonly used in the literature is generally different from the adhesion toughness unless the mode I adhesion toughness is equal to mode II adhesion toughness, which is not generally the case. It is the adhesion toughness that matters for the design of graphene film-substrate material systems.The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information file.Supplementary InformationPeer Review File


Graphene aerogels have many advantages, such as low density, high elasticity and strong adsorption. They are considered to be widely applicable in many fields. At present, the most valuable research area aims to find a convenient and effective way to prepare graphene aerogels with excellent properties. In this work graphene/carbon nanotube aerogels are prepared through hydrothermal reduction, freeze-drying and high temperature heat treatment with the blending of graphene oxide and carbon nanotubes. A new reducing agent-ascorbic acid is selected to explore the best preparation process. The prepared aerogels have compression and resilience and oil absorption properties due to the addition of carbon nanotubes as designed. To take advantage of these properties, the monolayer graphene must be assembled into macroscopic materials, such as graphene films, graphene fibers and 3D graphene aerogels (GAs). GAs have attracted wide attention from scientists due to their ultra-low density blow 10 mg/cm0 mg/cm3 3)  and mech3) ,5,6,7,8,However, GAs are formed by self-assembly processes, which are mainly affected by Van der Waals forces and π–π bonds  between Carbon nanotubes (CNTs) have good mechanical properties ,15 and iIf CNTs can be added to GA, CNTs and graphene can play a synergistic role through their π–π bonds . Large lIn this paper, the two aspects are focused on: (1) CNTs are mixed in the solution of GO so that the aerogels prepared are homogeneous mixtures containing graphene and CNTs; (2) Ascorbic acid is chosen as a reducing agent to make the hydrothermal reduction reaction of GO take place under mild reaction conditions. The lamellar graphene layers are close to each other, and the hydrogel is formed on the macro surface. The water solvent is removed from the hydrogel to become an aerogel through a freeze-drying process. Then the structure and properties of graphene/CNTs aerogels are provided. Test results are consistent with the design results.The preparation process of graphene/CNT aerogels is shown in With the increase of hydrothermal reaction time, the volume shrinkage of the hydrogel becomes more and more serious. With the increase of ascorbic acid amount, the volume shrinkage is also more and more serious. Then graphene/CNTs hydrogel is dialysed for 24 h using ethanol/deionized water solution in order to prevent the frost heave phenomenon in the subsequent freeze-drying process which will make the aerogels crack and destroy the macrostructure integrity of the aerogels. Such a destruction would inevitably lead to a decline of the mechanical properties of the aerogels. Therefore, the dialysis process for graphene/CNTs hydrogels is a key step in the whole preparation process. After dialysis, the freeze-drying is used to remove water to guarantee that there is no volume reduction, structural deformation or collapse during drying. Then the aerogels are heated at 500 °C under an argon atmosphere. Through the process of heat treatment, the graphene is further reduced so that the six element ring structure is further repaired, and the superfluous ascorbic acid is decomposed at the same time. Finally, the pure reduced graphene/CNTs aerogels are obtained.3 and the density of aerogels after heat treatment is 3 mg/cm3. The density of aerogels after heat treatment is reduced by more than half compared with that before heat treatment. This is because the heat treatment makes the residual ascorbic acid decompose and some functional groups of the reduced graphene can be degraded at the same time. Therefore, to a great extent, the quality of aerogels is reduced. The pore structure of the aerogels is uniform and well-arranged under the compression of ice crystal structure during the freezing process. Therefore, the perfect 3D pore structure of aerogels with the joint action of the graphene and CNTs can withstand the high temperature without volume reduction or structural deformation. Therefore, the heat treatment can greatly reduce the density of graphene/CNTs aerogels.The density of aerogels has a very important impact on their properties and applications. Therefore, this paper explores the factors that influence the aerogel density during the preparation process. Firstly, the influence of final heat treatment on aerogel density during the preparation process is discussed and the result is illustrated in 3 to 8.5 mg/cm3, and the density after heat treatment increases from 3.0 mg/cm3 to 4.0 mg/cm3. The reason is that the increase of the reducing agent leads to the rate and degree of the hydrothermal reduction reaction increase and the force between the lamellae of graphene is increased, therefore, the porosity and pore size of hydrogel and aerogel and the density of the aerogel increases.In addition, as for the preparation process, the smaller the volume shrinkage during the hydrothermal reduction step, the greater density of the resulting aerogels. Therefore, we need to find a balance point that can form a complete hydrogel and guarantee a slight volume contraction. By controlling the reaction time and the dosage of ascorbic acid, the optimum reduction degree is finally found. The effect of ascorbic acid on aerogel density during preparation of graphene/CNTs aerogels is investigated and the result is shown in The macrostructure of graphene/CNTs aerogels is shown in The microstructure of graphene/CNTs aerogels was observed by scanning electron microscope (SEM) and the results are shown in The prepared aerogels have excellent compression and resilience due to the introduction of CNTs, as shown in During the compression process, the pore network structure of pure graphene aerogels is extruded into almost dense parallel structures. The storage energy is very low. From the macro structure viewpoint, there is an unrecoverable destruction. In the structure of graphene/CNTs aerogels, graphene is wrapped by CNTs wrapped around each other. There is a synergistic effect between graphene and CNTs during the compression process. Therefore, the external force can be transferred to the carbon nanotube through the graphene. In the process of compression, the slip of the graphene layer and the fracture of the connection point can be avoided by CNTs so that the porous network structure is very stable. Meanwhile, CNTs also store a lot of energy to ensure that the aerogels can restore the original shape when the external force is removed.3 as an example. In order to verify the stability of the elastic properties of aerogels, the compression fatigue tests of 1, 5 and 10 times are repeated at the compression speed of 10 mm/min. As is shown in Taking the aerogel with density of 5 mg/cm3 as an example. The maximum recoverable elastic deformation of aerogels with different CNTs content is investigated, and the result is shown in In order to study the influencing factors of the elastic properties of graphene/CNTs aerogels, taking the aerogel with density of 5 mg/cmThe excellent elastic properties of aerogels are due to the synergistic effect of graphene and CNTs. Therefore, in addition to the influence of the content of CNTs, the interaction force between graphene and CNTs also has a great affect. The greater the binding force between graphene and CNTs, the better this synergistic effect is, and the better the elastic properties of the aerogel. It can be seen from The hydrophilic properties of the prepared aerogel are tested, and the water contact angles of aerogels with different CNTs content are shown in The oil affinity test of the aerogels is carried out, and the results are that aerogels showed super strong affinity to oil, as shown in In order to explore the adsorption ability of aerogels to different oils, several different oil products are selected, and their basic properties are listed in We take aerogels of graphene and CNTs mass ratio is 3:1 as an example to explore the oil absorption of different oil products. The result is shown in As an oil absorbing material, aerogels have good compression resilience performance. After the adsorption of oil, the oil pollution can be discharged simply by extrusion, and the shape and structure of the aerogels are not destroyed during the extrusion process. Therefore, aerogels as an oil absorbing material can be recycled, and the amount of oil contaminated by extrusion can reach 63%, and the recovery of low density oil, such as gasoline, can be over 80%. The recovery and efficiency of adsorption materials are achieved.3 so they can be considered ultralight materials. However, when the aerogel with 50% CNTs content is compressed to its own height of 90%, it can still quickly rebound to its original height without structural collapse or permanent deformation, showing excellent compressibility. The oil absorption capacity reaches 100 g/g, showing excellent oil absorption properties. Graphene/CNTs aerogels are also hydrophobic so that they can be used can be used as materials for oil and water separation.In this paper, a method of preparation of graphene/CNTs aerogels is developed. Ascorbic acid is chosen as a reducing agent for hydrothermal reaction, and then aerogels undergo the process of freeze drying and heat treatment. When the mass ratio of graphene and ascorbic acid is 1:2, the volume shrinkage degree is minimum and the aerogel density is the smallest. When the CNTs content is 50%, aerogels have the best performance. The resulting graphene/CNTs aerogels show a density as 3 mg/cm


There are several issues in this study that must be commented.In a recent study, Satar et al.  have reported “the synthesis and characterization of graphene for the immobilization of  Moreover, it exhibited neither high thermal conductivity nor high-specific surface area as graphene. In addition, in Fig. 2, the scheme is related to graphene oxide not graphene. In Fig. 1, one can find “Transmission electron microscopy revealing the size of graphene”. There are two problems: (1) it is highly improbable to achieve such perfect surface of graphene oxide (it is not a TEM picture) and (2) this picture does not reveal the size of graphene. In our opinion, the presented graphene sheet is generated in silico.At first, it is necessary to address the problem of nomenclature, i.e., graphene and graphene/graphite oxide. Authors use these terms interchangeably which are highly misleading. Authors use “graphene”; however, they synthesized graphene oxide (in fact graphite oxide). Already in “Introduction”, graphene properties are described instead of quite different materials which were synthesized, i.e., graphene oxide (GO). The discrepancy lies in the fact that GO does not possess only spMisrepresentation of the experimental parts has become common in recent years, so it is not worth mentioning that the synthesis cannot be performed as it is described. Nevertheless, in the cited reference, the method of GO production proposed by Zhao et al.  is complHowever, more important shortcomings are presented further in this section:How is it possible to force ethanol to dissociation through simple bath in GO solution? Even if so, authors did not prove this. Moreover, what is the sense of incorporation of –OH into the material which possesses lots of such functionalities?The concentrations of GO at the level of few mg/mL cause that the solution possess very high viscosity. Thus, reasonably seems to be using 1 mg of GO. Nevertheless, it would be helpful to know how much enzyme was used, as well as the volume of the solution.β-Galactosidase was stirred slowly in modified GO (1.0 g) and kept for overnight”—the authors provide no information about the concentration/amount of protein added, no immobilization yield (was the whole protein adsorbed? How much enzyme was unbound and discarded?)“The yield calculated by authors (from equation) is nowhere mentioned in the manuscript. Why?Another chemical problem appears in “enzyme immobilization”. Authors unnecessarily describe it  as covalent bonding.The principle of direct alkylation of amines with alcohols, leading to the formation of the secondary amines from primary ones, is known in the literature e.g., . HoweverExtremely huge problem is with Fig. 3. In fact, this figure shows completely nothing and it was the inspiration for this comment. Let us first start from “Experiment”, and actually the lack of it. It is not so important to write the name of FTIR apparatus, as the obtained spectrum should be independent on it. More important is to describe the used technique, ATR, DRIFT, transmittance, etc. From “The nondestructive analysis”, readers know essentially nothing.− 1”: (1) cannot be attributed to –OH and (2) there is not such a band in Fig. 3. Similarly, “the broadening of peaks from 1200 to 1700 cm− 1”: (1) there are no such a peaks and (2) even if so, such effect cannot be attributed to protein attachment to the GO.The spectra in Fig. 3 are of extremely weak quality. Skipping the fact that there is no scale, and usually, it is impossible to obtain in transmittance mode the up-going bands . The bands “present at 3105 cmProblem with Figs. 4 and 5 and their description is that authors did not realize the real size of the graphene sheet. Omitting the fact that it is not a graphene oxide, the size of modelled sheet is extremely too small.β-galactosidase (IβG) showed improved stability against various physical and chemical denaturants” was not proved here; authors tested only the influence of the temperature and (2) these are not the conclusions, rather a summary.Summarizing, the analysis of catalytic activity was done on graphene oxide, not activated GO nor graphene. The material was not characterized and “The size of synthesized graphene was observed to be 25 nm by TEM analysis while interaction of enzyme with the nanosupport was observed by FTIR spectroscopy.” This sentence from Abstract is untruth. As we fully agree with authors’ statements written in the “Conclusion”, it is necessary to mention that (1) claim “Immobilized