This study investigated an association between schizoid tendencies and aggressive behaviors in Chinese adolescents, and explored the underlying mechanism.The data of 3094 adolescents aged 12 to 16 years were collected from an epidemiological survey in China. All the subjects or their parents completed the Achenbach’s Child Behavior Checklist, the Childhood Trauma Questionnaire-Short Form, and the Adolescent Self-rating Life Event Checklist (ASLEC).male = 0.620, βfemale = 0.638, both P < 0.001) and ASLEC , and by childhood trauma score . The ASLEC and childhood trauma scores mediated the association between scores for schizoid tendencies and aggressive behaviors, specifically, emotional neglect  in males . In females, the interaction between scores for childhood trauma and ASLEC affected the aggressive behaviors score (P = 0.023).Relative to the non-schizoid group, adolescents with schizoid tendencies  showed significantly higher scores for aggressive behaviors, emotional abuse, and ASLEC. Regarding females only, those with schizoid tendencies had significantly higher (lower) scores for physical abuse and emotional neglect . The aggressive behaviors score was predicted by scores for schizoid tendencies (βSchizoid tendencies are associated with aggressive behaviors among Chinese adolescents. Recent life events and childhood trauma mediated an association between schizoid tendencies and aggressive behaviors. The interaction between childhood trauma and recent life events affected aggressive behaviors in females. Aggressive behaviors in adolescents may be ameliorated by reducing childhood trauma and life events. Schizophrenia is a psychotic disorder characterized by hallucinations, delusions, and cognitive deficits, accompanied by impaired executive functions, memory, and speed of mental processing . The preBased on these studies, we hypothesized that increased aggressive behaviors in the general adolescent population may partly result from experiences that are like psychotic symptoms, such as hearing voices or seeing things that do not exist. To explore the association between aggressive behaviors and psychotic symptoms in adolescents, the present study utilized schizoid and aggressive behavior factors of the Achenbach’s Child Behavior Checklist (CBCL). We hypothesized that schizoid tendencies may be closely associated with aggressive behaviors in Chinese school adolescents.While an association between schizophrenia and aggressive behaviors has been shown, the underlying mechanism remains unclear. Understanding the mechanism in general adolescent populations has significance for earlier intervention of aggressive behaviors. Some previous similar studies have made attempts in this field. For example, several studies investigated the mediating factors between schizotypy  and aggChildhood trauma is defined as childhood adverse events such as emotional and physical neglect, or physical, emotional, and sexual abuse . ChildhoThere are also links between childhood trauma and aggression. For example, individuals with childhood trauma had a higher risk of perpetrating violence, according to a systematic review and meta-analysis of prospective studies by Fitton et al. . In ChinGiven the above studies, our second hypothesis is that childhood trauma may mediate the association between schizoid tendencies and aggressive behaviors. This exploratory model may help us better understand the underlying mechanism between schizophrenia and aggressive behaviors, and guide early intervention to reduce aggressive behaviors of adolescents with schizoid tendencies.Recent life events such as loss of family member or exam failure may also be an important mediator between schizoid tendencies and aggressive behaviors. Prior studies support this hypothesis. For example, Raine et al.  observedSome studies reported that childhood trauma may influence the cognition of recent negative stimulation. For example, Aas et al.  found thThere is evidence of gender differences in aggression. For example, men demonstrated more physical aggression than did women –31. In aThe Ethics Committee of Second Xiangya Hospital of Central South University approved this study. All subjects participated voluntarily. The subjects and their parents or guardians provided signed informed consent.n = 3465), CBCL-negative students were randomly selected and matched according to the 1:1 principal rule. Finally, 3465 CBCL-positive students and 3465 CBCL-negative students completed a further questionnaire survey. The questionnaire included the Childhood Trauma Questionnaire-Short Form (CTQ-SF) and the Adolescent Self-rating Life Event Checklist (ASLEC). Considering the age limitation of the CTQ-SF ), relatively wealthy (452 [14.61%]), or financially constrained (177 [5.72%]).P = 0.005).In males, the scores for aggressive behaviors, emotional abuse, and ASLEC were significantly higher in the group with schizoid tendencies compared with the non-schizoid tendencies group Table . In femaP < 0.001; Table P < 0.001).In both the males and females, the score for schizoid tendencies positively correlated with, respectively, the scores for aggressive behaviors, ASLEC, and the 4 dimensions of the CTQ-SF  were included for the linear regression analysis (Table P < 0.001), emotional neglect score , and ASLEC score .Based on the results of the correlation analyses, only statistically significant variables , emotional abuse , and ASLEC . However, the sexual abuse score  was significant negative predictor of the aggressive behaviors score.In females, the positive predictors of the aggressive behaviors score were the scores of the following: schizoid tendencies  did not include zero Table . The scoP = 0.023).This study further tested the effect of interaction between childhood trauma score and ASLEC score to moderate the association between the schizoid tendencies score and aggressive behaviors score Table . The resThis study determined that both the female and male adolescents in the schizoid tendencies group had suffered more emotional abuse and recent life events, compared with adolescents without schizoid tendencies (control). In addition, the females in the schizoid tendencies group were more likely to have experienced physical abuse and emotional neglect. These results are consistent with other reports in which individuals with schizoid personalities were more likely to experience adverse life events  and chilThe results of the present study also indicated that childhood trauma differed between genders in the schizoid tendencies group, and occurred more frequently in females. This is consistent with previous researches, which found that childhood maltreatment appeared to be more profound in the female general population , 46 and In the present study, adolescents in the schizoid tendencies group achieved higher aggressive behaviors score compared with the control group, and linear regression analysis suggested that schizoid tendencies score could predict the aggressive behaviors score. This is consistent with most other similar studies, in which aggression was common in people with schizotypy , 13. SchIn the present study, the linear regression analysis revealed that the life events score could positively predict the aggressive behaviors score. Those who experienced recent life events were more likely to display aggressive behaviors. These results are consistent with previous articles , 54. EttIn the present study, emotional neglect was a positive predictor of aggressive behaviors in males, and emotional abuse was a positive predictor of aggressive behaviors in females. This suggests a difference in gender response to childhood trauma that affects aggressive behaviors. A study conducted in Ontario Canada found that adolescents exhibited aggressive behaviors because of neglect, specifically when their caregivers no longer played a caregiving role . AnotherInterestingly, sexual abuse was negative predictor of aggressive behaviors in female adolescents, which is inconsistent with another study . The reaIn accord with previous studies , 65, theAdolescents with schizoid features are more likely to experience childhood maltreatment and adverse life events , which iReducing childhood maltreatment and life events in adolescents with schizoid tendencies may be crucial to reduce their aggressive or violent behaviors, whereas parental love may be important protective factors. For example, Family Attachment Narrative Therapy was used to heal the bad effect of childhood maltreatment . If adolThe present results showed that the association between schizoid tendencies and aggressive behaviors was not influenced by an interaction between childhood trauma and recent life events. There was an interaction effect between childhood trauma and recent life events that influenced aggressive behaviors in the females. This result is in accord with Beckmann’s study , which iFunctional magnetic resonance imaging studies have shown that maltreatment could lead to changes in the amygdala of females . These cThere are several limitations in this study. First, the CTQ-SF and ASLEC are retrospective questionnaires, which can cause recall bias. Second, this was a cross-sectional study, so it is impossible to infer the causal association between schizoid tendencies and aggressive behaviors. In addition, there were overlaps between childhood maltreatment and recent life events. The ASLEC evaluated the past 12 months, which may coincide with the existence of childhood trauma in an investigation of adolescents. There are also childhood adversities that were not included in this analysis, such as witnessing community violence . FinallyThis is the first study to investigate the association between childhood trauma, recent life events, schizoid tendencies, and aggressive behaviors among Chinese adolescents. It was found that adolescents with schizoid tendencies were more likely to experience childhood trauma, recent life events, and have aggressive behaviors, compared with those without schizoid tendencies. Recent life events, childhood trauma , and schizoid tendencies independently increased aggressive behaviors. Recent life events and childhood trauma mediated the association between schizoid tendencies and aggressive behaviors in male and female adolescents, and the interaction between childhood trauma and recent life events affected the aggressive behaviors of females. Therefore, reducing childhood trauma  and adverse life events may ameliorate aggressive behaviors among adolescents with schizoid tendencies.


GTPase and KRAS) within 2 h of exposure. Furthermore, the PCNA (proliferating cell nuclear antigen) gene was upregulated after exposure to graphene at all concentrations. Ingenuity pathway analysis revealed that STAT3 and VEGF signaling pathways (known to be involved in cell proliferation and growth) were upregulated. Graphene exposure (10 µg/mL) for 24 h significantly increased levels of pro-inflammatory cytokines IFNγ, IL-8, IL-17, IL-6, IL-9, MIP-1α, and Eotaxin. Collectively, these results indicated that graphene may activate the STAT3–IL23–IL17 response axis. The findings in this study provide information on toxicity evaluation using a human-relevant ex vivo colon model and serve as a basis for further exploration of its bio-applications.Toxicology studies on pristine graphene are limited and lack significant correlations with actual human response. The goal of the current study was to determine the response of total colonic human tissue to pristine graphene exposure. Biopsy punches of colon tissues from healthy human were used to assess the biological response after ex vivo exposure to graphene at three different concentrations . mRNA expression of specific genes or intestinal cytokine abundance was assessed using real-time PCR or multiplex immunoassays, respectively. Pristine graphene-activated genes that are related to binding and adhesion ( Graphene, a single layer of monocrystalline graphite with spications . Graphenications . Graphenications ,6,7,8,9.hia coli . On the hia coli , flowerihia coli ,12.Human exposure to graphene can occur through dermal, ingestion, inhalation, or intravenous routes. Although the majority of published articles have evaluated the toxicity of graphene from an occupational health perspective and have focused mostly on the impact of graphene on the respiratory system ,14, reseConsidering the positive impact of graphene on plant cell growth and proliferation, as well as other in other medical uses (drug delivery), it is logical to question if the longer retention time of graphene in the colon could have any impact on the growth or excessive proliferation of cells in the lining of the colon. Thus, the current study investigated the impact of graphene exposure through molecular profile (mRNA expression), as well as through levels of key cytokines/chemokines in the colonic tissue, to reveal the mechanisms by which graphene could interact with the human intestinal tract. We have previously shown that pristine graphene can affect the gastrointestinal system through modulation of the rat intestinal microbiome at doses equal to or higher than 100 µg/mL . Here, tThis study evaluated changes in human colonic tissue gene expression and immune response after 2 and 24 h of exposure to pristine graphene. The 2 and 24 h time points were chosen to mimic the time for which processed food can remain in the colon before it is removed from the human body. The 2 h time was chosen to mimic the interaction of test compounds  in an empty/partially filled stomach state. The 24 h time point was chosen because it is the approximate time of orally administered test compounds for interaction with the intestinal mucosa ,27. ColoTaking into account both time points and all graphene concentrations tested, the gene expression analysis showed that out of the 86 total genes investigated in this study, 13 genes were significantly downregulated, and 24 genes were significantly upregulated after exposure to graphene .CRP, KDR, NOTCH1, EGFR, RRM2, ZWINT, PCNA, VIM, CXCR4, VDR, FN1, IGF1R, PTG85, and PPARG (PCNA (proliferating cell nuclear antigen) gene was shown to be highly upregulated in graphene-treated colon tissue during the first 2 h of exposure  [TYMS, PTEN, SP1, TGFB1, and HIF1A (TYMS (thymidylate synthase), which is involved in cell proliferation [PTEN (phosphatase and tensin homolog), which prevents cell growth and overly rapid proliferation [Sp1 [tgfb1 (transforming growth factor beta-1 proprotein). This gene exhibited a decrease in mRNA expression between 2 and 24 h. However, its expression remained significantly higher than the control (p < 0.05). The third category of genes included genes that were altered at 24 h and did not exhibit any changes during the first 2 h of exposure. These genes included GSTP1, IL6, BIRC5, CDKN2A, CXCL12, IFNG, IGF1, KRAS, TERT, SPP1, AKT1, and COL1A2  . The damse (LDH) . Apart fnd HIF1A . The conferation , along wferation , furtherferation . Transcrion [Sp1 . Anotherd COL1A2 . The fund COL1A2 . The gend COL1A2 . In factKRAS, TERT, SPP1, BIRC5, and TGFB1. In our study, the binding or adhesion of graphene to epithelial or immune cells could have led to the downstream modulation of molecular and catalytic activity, as shown in an earlier study [E. coli and reduced their viability [Binding or adhesion of graphene to the surface of colonic cells was detected at 2 h and sustained a gene expression response for key genes including er study . Furtherer study ,41. For er study . On the iability . Thus, tp-values obtained following the raw data gene expression analysis were then mechanistically investigated using IPA software (Qiagen). Pathway analysis was then carried out with 86 genes that fulfilled the threshold cut-off of -log2 fold change and p  <  0.05 in the graphene-treated colon tissue and compared to the control non-treated colon tissue. Canonical pathways that had activation z-score values were taken for further analysis and pathways that showed no activity patterns were omitted.The fold change and p = 14.348) and VEGF  signaling pathways (-log p = 9.692) were the most significant canonical pathways. Both signaling pathways showed higher z-scores. The STAT3 pathway in colon tissue treated with graphene at 10 µg/mL showed the highest positive activation z-score of 1.15 among other graphene doses, indicating overall upregulation of the pathway, whereas the highest dose of graphene at 100 µg/mL showed a lower z-score of 0.577, indicating upregulation of the pathway compared to controls  signaling pathway  showed early (2 h) and lasting activation (24 h) of different biofunctions  was characterized at the Nanocore facility, National Center for Toxicological Research, Jefferson, Arkansas, and at the Center of Integrative Nanotechnology Sciences, University of Arkansas at Little Rock, Arkansas, as previously described ,52. Befo2 and 37 °C for 2 h. Next, pristine graphene nanoparticles were added to the upper compartment at final concentrations of 1, 10, or 100 µg/mL. The selection of concentration was based on the risk of low versus high concentration plus a medium concentration, as defined elsewhere [The use of colon tissue samples was reviewed and approved by the U.S. Food and Drug Administration’s Research Involving Human Subject Committee (RIHSC #12-062T exemption). The inclusion and/or exclusion criteria for the human subjects included in this study were that the colon samples should be from de-identified subjects aged 18 years or older who do not have inflammatory bowel disease or pathological conditions involving the colon. Colon tissues from three subjects  were obtained from the Cooperative Human Tissue Network—Southern Division. Sectioned intestinal tissues were shipped in RPMI media with overnight delivery. Upon receipt, tissues were immediately transferred into a cocktail of 50% MACS tissue storage solution  and 50% RPMI-1640  media containing 1% fetal bovine serum and 5% streptomycin/penicillin . Tissue biopsy punches 6.00 mm in size were collected and placed into the upper compartment of transwells  filled with 200 µL of growth media, as described previously . The lowlsewhere ,54. A neThe total RNA of colonic tissues was extracted using an RNeasy Mini kit . Obtained RNA was treated with DNAse. This DNAse-treated RNA was reverse-transcribed using a PrimeScript ™ RT reagent kit . After cDNA synthesis, real-time PCR was performed on PrimePCR plate Intestinal neoplasm Tier 1 apparatus using SYBR green to amplify PCR products. Real-time PCR was conducted at the optimized annealing temperature of 60 °C, as described previously . The relp value; as compared to control) were further analyzed by Ingenuity Pathway Analysis  software to identify the pathways and functions that were significantly affected due to graphene exposure The IPA software analyzed the real-time gene expression data based on algorithm predictions, providing a list of potential up regulators organized based on their z-scores (significance). A detailed description of IPA is available on the Ingenuity Systems website . It calculates a probability value for each pathway/function according to the fit of users’ data to the IPA database using the right-tailed Fisher’s exact test. Pathways with p < 0.05 were considered significantly affected.Data generated by real-time PCR . Statistical analysis was conducted using SPSSThis study was conducted to address the knowledge gap related to the risk/safety assessment during the interaction of pristine graphene with the human intestinal barrier using an ex vivo model (a human relevant translation model). Our results highlighted the pathways affected by graphene upon tissue exposure. We have shown that graphene can stimulate the mRNA expression of genes involved in cell proliferation and growth upon binding/adhering to epithelial tissue. This interaction is coupled with the dose-dependent activation of a pro-inflammatory response through many pathways. The correlation between real-time PCR and protein data showed that the IL-23–IL-17 axis signaling and its interaction with STAT3 emerged as principal pathways by which graphene could impact human epithelial tissue . Additio


The results also showed that patients with high levels of sPD-L1 in serum had shorter cancer-specific survival compared with patients with low levels (p = 0.002). The results indicate that sPD-L1 most significantly reflects tumor progression in RCC.CD163+ M2 macrophages have been suggested to counteract tumor immunity by increasing immunosuppressive mechanisms including PD-L1 and IL-10 expression. Soluble levels of PD-L1, IL-10, and CD163 have been reported as potential biomarkers in various cancers, although the prognostic value in renal cell carcinoma (RCC) has to be further elucidated. In the present study, we measured the levels of sPD-L1, sIL-10, and sCD163 in 144 blood samples from patients with RCC. The levels were determined by using enzyme linked immunosorbent assays. Soluble PD-L1 and CD163 were detectable in 100% of the serum samples, and sCD163 in 22% of the urine samples, while only a minority of the samples had detectable sIL-10. Significantly higher serum levels of sPD-L1 and sCD163 were observed in patients with metastatic disease ( Renal cell carcinoma (RCC) has the highest mortality rate of all genitourinary cancers, and constitutes approximately 2% of all malignancies. The clinical outcome in RCC patients varies considerably, but the prognosis for patients with localized disease is generally good, with a five-year survival rate of approximately 90% after tumor removal by partial or radical nephrectomy . UnfortuExtensive data support the hypothesis that the immune system plays an important role in hampering tumor initiation and progression. Thus, evasion of the immune system is now considered as a hallmark of cancer . By creaProgrammed death ligand 1 (PD-L1) is an inhibitory molecule that when interacting with its receptor PD-1, acts as a negative regulator of the T cell-mediated anti-tumor immunity. The prognostic value of membranous PD-L1 expression has been extensively investigated in a number of malignancies, including several urological malignancies such as renal, bladder, prostate, and penile cancer. For the majority of cancer types investigated, high PD-L1 expression was associated with more advanced tumors and poorer prognosis ,6,7,8,9.Until now, IL-10 has been considered an immunosuppressive cytokine due to its association with multiple regulatory or suppressive immune cells, including regulatory T-cells, myeloid-derived suppressor cells, and M2 macrophages. In vitro experiments have shown that IL-10 has the capability to impair proliferation and cytotoxic activity of anti-tumor T-cells, thereby stimulating tumor growth . ConversPrevious evaluations of the prognostic value of CD163, PD-L1, and IL-10 have reported associations between increased expression and poor clinical outcome in several different malignancies ,18,19,20In the present study, we investigated the feasibility to measure the levels of sCD163, sPD-L1, and sIL-10 in prospectively collected preoperative serum and urine samples from patients diagnosed with RCC. We also assessed associations between the levels and clinical parameters related to worse outcome.n = 24, angiomyolipoma n = 6, kidney cyst n = 5, leiomyoma n = 1, and metanephric adenoma n = 1) and 144 patients with malignant tumors. The patients diagnosed with RCC had a median age of 68 years (range: 30–86 years) and a median BMI of 27.1 (range: 21.1–39.9). Their clinical and pathological characteristics are summarized in In this study, we included preoperatively collected serum and urine samples from 181 patients undergoing partial or radical nephrectomy for suspected RCC, where 37 patients were diagnosed with benign tumors (oncocytoma p = 0.044). A trend for higher levels in patients with malignant tumors was also observed for sPD-L1 in serum, 87.2 pg/mL (64.9–348.2 pg/mL) versus 80.2 pg/mL (72.5–123.8 pg/mL) (p = 0.054). Investigation of the relationship between preoperative serum levels of sCD163 and sPD-L1 in RCC patients showed a low correlation between sCD163 and sPD-L1 .When analyzing the serum samples from the total cohort, sCD163 and sPD-L1 were detectable in all samples and sIL-10 in 11.6% (21/181). Similar rates were observed when including only RCC-patients, sCD163 and sPD-L1 were detectable in 100% (144/144) of the samples and sIL-10 in 13.2% (19/144). The median serum levels of sCD163, sPD-L1, and sIL-10 were 741.4 ng/mL (range: 618.9–890.8 ng/mL), 87.2 pg/mL (79.1–96.7 pg/mL), and 0.9 pg/mL (0.9–1.4 pg/mL), respectively. In urine samples, sPD-L1 was detectable in 22.1% (40/181) of the samples. The median urine level of sPD-L1 was 68.2 pg/mL (63.8–91.2 pg/mL). Significantly higher serum levels of sCD163 were observed in patients with malignant tumors compared to patients with benign tumors, 747.4 pg/mL (339.2–2386.7 pg/mL) versus 674.3 pg/mL (303.3–1538.5 pg/mL)  and pT stages (p = 0.001) (There was a significant difference in serum levels of sPD-L1 between Fuhrman grades (= 0.001) . No assop = 0.065) and pT stage (p = 0.066)  .p = 0.041) (p = 0.011) (p = 0.023)  versus 99.4 pg/mL (99.4–99.4 pg/mL). To analyze possible associations between preoperative levels of sCD163 and sPD-L1 and aggressive RCC, we assessed the relationship in respect of metastatic disease at diagnosis. The results showed that RCC patients with metastatic disease at diagnosis had significantly higher serum sCD163 levels than patients presenting without metastatic disease (= 0.041) . A signi= 0.011) . The dat= 0.023) . Howeverp = 0.001) (p = 0.219), or between sPD-L1 levels in urine and RCC-specific death (p = 0.115)  , where t= 0.115) .p = 0.002)  . HoweverRenal tumors evade eradication by the immune system through a number of mechanisms, including creating an immunosuppressive TME comprising increased infiltration of immunosuppressive cells and increased expression of immune inhibitory mediators. In the present study, we investigated the feasibility to measure soluble levels of CD163, PD-L1, and IL-10 in blood samples from 144 RCC patients. We also investigated associations between the levels and clinical parameters related to adverse outcome. Soluble PD-L1 and sCD163 were detectable in all serum samples while sIL-10 was detectable in only a minority of the samples. In urine, approximately 20% of the patients had detectable levels of sPD-L1. The results further showed a positive association between sPD-L1 and adverse clinical outcome, especially sPD-L1 in serum, and it appears the urine levels of sPD-L1 are higher in RCC patients presenting with metastatic disease at diagnosis.Tumor-associated macrophages represent a major part of the leukocyte infiltrate in the TME, where the dominant phenotype is CD163+ M2 macrophages with tumor-promoting functions. Among other things, these macrophages have the capability to produce immunosuppressive cytokines such as IL-10 and express immune response inhibitory molecules such as PD-L1. Analysis performed on TAMs isolated from RCC tumors revealed frequent infiltration of CD163+ M2 macrophages associated with increased production of IL-10 and enhanced expression of PD-L1. A substantial number of investigations have reported high infiltration of tumoral CD163+ M2 macrophages and increased PD-L1 expression as independent prognostic factors for poor clinical outcome in various malignancies, including RCC. However, measurement of tissue-based prognostic markers requires an invasive sample procedure, thus finding prognostic markers demanding a less invasive sampling technique is preferable. Given the relationship between the abovementioned markers and poor prognosis, it may be reasonable to speculate that the soluble level of each marker would also be of prognostic significance in RCC patients. In the present study, a low positive correlation was observed between levels of sCD163 and sPD-L1, suggesting that both CD163-mediated and PD-L1-mediated pathways contribute to immunosuppression in RCC. A significant correlation between serum levels of sCD163 and sPD-L1 was also observed in patients diagnosed with hepatocellular carcinoma ,22.High serum levels of sPD-L1 are related to poor prognosis in various cancers, such as hepatocellular carcinoma, esophageal cancer, lung cancer, gastric cancer, rectal cancer, and lymphoma . In lineA recent proof-of concept study provided data showing that sPD-L1 can also be detected in urine samples. Patients with urinary bladder cancer were found to have significantly higher urinary levels of sPD-L1 compared to patients with non-malignant urological diseases . The preIncreased serum levels of sCD163 have been reported previously as an independent marker for predicting poor prognosis in patients diagnosed with B-cell chronic lymphocytic leukemia, myeloma, melanoma ,31,32. HPrevious investigations of sIL-10 as a prognostic marker in RCC have produced limited data with inconclusive results. Wittke et al. detected high levels of sIL-10 in 25% of the serum samples collected from 80 patients diagnosed with metastatic renal carcinoma, and found that a high level was an independent predictor of unfavorable clinical outcome . On the Since different histological subtypes in RCC have different prognoses, we also performed a subgroup analyses including only patients diagnosed with clear cell RCC, revealing the same results as when including patients with all histological subtypes (Data not shown).This study has some limitations. The relatively small number of participants and outcome and the short follow-up period limited the possibility to draw definite conclusions concerning the value of sPD-L1 and sCD163 as predictive biomarkers for shorter cancer-free survival in RCC. However, the data presented here motivate further evaluations using larger cohorts with longer follow-up periods. It can also be viewed as a limitation that we did not evaluated correlations between sPD-L1 and sCD163 and PD-L1- and CD163- expression in tumor tissue from the patients.Between February 2014 and November 2017, patients were recruited to the Renal Cancer Blood Urine Study (REBUS), a prospective cohort study in which all patients referred to the Department of Urology, Örebro University Hospital, Sweden, with suspected RCC planned for surgery were asked to participate. On the day of study inclusion, demographic data were collected from each patient as well as preoperative blood and urine samples. All patients underwent partial or radical nephrectomy according to current guidelines. Tumor grade and clinical stage were determined according to the Fuhrman system and the TNM classification, respectively. The study cohort was followed for cancer-specific mortality until March 2021.g for 10 min at 4 °C. The urine samples were centrifuged at 2000 rpm for 10 min. After centrifugation, both serum and urine samples were aliquoted and stored at −80 °C until further analyses.Blood samples were allowed to clot for 30 min at room temperature and serum was obtained by centrifugation at 3000× Soluble CD163 in serum was measured using a commercially available human CD163 enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions . The detection range was 1.56–100 ng/mL. Each sample was analyzed in duplicate along with protein standards and controls with known concentrations . The optical density was measured using a Multiskan Ascent plate reader  at 450 and 540 nm. The coefficient of variation  was calculated for each sample, and all samples with a value above 10% were analyzed a second time. The mean values of absorbance versus concentration were plotted and a log/log curve-fit was applied.Soluble PD-L1 in serum and urine was measured using a commercially available human PD-L1 ELISA according to the manufacturer’s instructions . The detection range was 25–1600 pg/mL. Each sample was analyzed in duplicate along with protein standards and controls with known concentrations . The optical density was measured using a Multiskan Ascent plate reader (Thermo Fisher Scientific) at 450 and 540 nm. The CV was calculated for each sample, and all samples with a value above 10% were analyzed a second time. The mean values of absorbance versus concentration were plotted, and a four-parameter logistic non-linear regression model fit was applied.Soluble IL-10 in serum was measured using a commercially available human IL-10 ELISA according to the manufacturer’s instructions . The detection range was 0.78–50 pg/mL. Each sample was analyzed in duplicate along with protein standards and controls with known concentrations . The optical density was measured using a Multiskan Ascent plate reader (Thermo Fisher Scientific) at 490 and 650 nm. The CV was calculated for each sample, and all samples with a value above 10% were analyzed a second time. The mean values of absorbance versus concentration were plotted, and a four-parameter logistic non-linear regression model fit was applied.All samples were analyzed retrospectively, and the technician performing the CD163, PD-L1, and IL-10 measurements was blinded to the pathological features and outcome of the patients analyzed.All patients were informed about the study before giving written consent. The study was approved by the Ethical Review Board in Uppsala-Örebro, Sweden (2012/161).p < 0.05.In order to test the data for normality, a Shapiro-Wilks test was used. A Spearman’s correlation test was used to investigate correlations between the two soluble markers. Differences in levels of sCD163 and sPD-L1 between groups were determined by using the non-parametric Wilcoxon–Mann–Whitney and Kruskal-Wallis tests. Box plots were created in which the horizontal boundaries of the boxes represented the first and third quartile and the vertical line in between indicated the median. The Kaplan–Meier method was used to estimate survival probabilities and the log-rank test was used to compare groups. The data were dichotomized as high or low (over and under median). A Cox regression model was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). All analyses were performed in SPSS version 22.0 . Statistical significance was set at In the present study, sPD-L1 and sCD163 were detectable in all 144 serum samples analyzed as opposed to sIL-10, which was detectable in only a minority of the samples. Soluble PD-L1 most significantly reflected tumor progression, since patients with high levels of sPD-L1 presented metastatic disease at diagnosis and had shorter cancer-specific survival. Identifying new biomarkers to predict outcomes and for therapeutic monitoring of RCC patients are urgently needed. Using soluble biomarkers would be preferable, since sampling a blood sample is less invasive than collecting a tissue biopsy. Additional studies in larger cohorts with longer follow-up periods are needed to further investigate the role of sPD-L1 and sCD163 as prognostic biomarkers in RCC.


Lidocaine, a commonly used local anesthetic, has recently been developed into a number of ointment products to treat hemorrhoids. This study examined its efficient delivery to the dermis through the pharmaceutical improvement of hemorrhoid treatment ointments. We attempted to increase the amount of skin deposition of lidocaine by forming a nanoemulsion through the self-nanoemulsifying effect that occurs when glycerol monostearate (GMS) is saturated with water. Using Raman mapping, the depth of penetration of lidocaine was visualized and confirmed, and the local anesthetic effect was evaluated via an in vivo tail-flick test. Evaluation of the physicochemical properties confirmed that lidocaine was amorphous and evenly dispersed in the ointment. The in vitro dissolution test confirmed that the nanoemulsifying effect of GMS accelerated the release of the drug from the ointment. At a specific concentration of GMS, lidocaine penetrated deeper into the dermis; the in vitro permeation test showed similar results. When compared with reference product A in the tail-flick test, the L5 and L6 compounds containing GMS had a significantly higher anesthetic effect. Altogether, the self-nanoemulsifying effect of GMS accelerated the release of lidocaine from the ointment. The compound with 5% GMS, the lowest concentration that saturated the dermis, was deemed most appropriate. Hemorrhoid disease is the third leading outpatient gastrointestinal diagnosis, accounting for 3.3 million office and emergency department visits in the United States . The selLidocaine is a widely used local anesthetic that relieves pain, burning, and itching caused by hemorrhoids ,12. SpecLocal delivery can be defined as the application of a drug-containing formulation to the skin in order to directly treat cutaneous disorders, such as acne, or the cutaneous manifestations of a general disease, such as hemorrhoids . Local dThe use of fatty acids in local treatments enhances skin permeability, mainly due to disturbance of the stratum corneum ,24,25. MThis study aimed to improve lidocaine ointments to relieve hemorrhoid pain using a petrolatum base ,43. Alth® ointment, was obtained from Ildong Pharm. Co., Ltd. . Water was purified by filtration in the laboratory. High-performance liquid chromatography (HPLC)-grade solvents were used for analysis. HPLC-grade ethanol and acetonitrile were purchased from Honeywell Burdick & Jackson . Sprague Dawley® rats (SD rats) and Institute of Cancer Research mice (ICR mice) were purchased from Samtako Corporation .The lidocaine base was a gift from DONG-A Pharm Co., Ltd. . GMS was obtained from Gattefosse Corporation . Petrolatum was purchased from Wonpoong Pharm Co., Ltd. . Medium-chain triglyceride oil (MCT oil) was obtained from KLK OLEO . Other excipients were supplied by DONG-A Pharm Co., Ltd. . Reference Product A (Ref. A), PrepainThe ointments containing lidocaine were prepared using a fusion method ,46. EachThe composition of each ointment is listed in The viscosity test was performed in an RV-7 viscometer  at 25 °C and 50 rpm for 3 min. The minimum extrusion force was determined with a TA-XTplusC texture analyzer . After the tube was completely filled with the ointment, the minimum force at which the ointment started to flow when compressed was measured. Each experiment was conducted in triplicate.The pH measurement was conducted by pH meter  with a 10-fold diluted ointment using distilled water.To determine the droplet size and polydispersity index the Portal ELSZ Zeta-size analyzer  was employed. The measurements were made at 90° at room temperature (25 °C). One milliliter of the sample was placed in the cuvettes and the lidocaine ointments were diluted . Each experiment was conducted in triplicate .The thermal properties of the lidocaine, other excipients, and prepared lidocaine ointments were analyzed using a DSC Q2000  thermal analyzer system. The samples were weighed, loaded into an aluminum pan, and analyzed at a heating rate of 10 °C/min over a temperature range of 40 °C to 120 °C. The thermal responses of the prepared samples were calculated using the TA Advantage/Universal Analysis software .X-ray diffraction (XRD) patterns of lidocaine, other excipients, and lidocaine ointments were analyzed using a D8 Discover with GADDS  at a wavelength of 1.54 Å. The 2θ scans were conducted between 5° and 60°.Raman spectroscopic measurements were performed using a RAMANtouch device  equipped with a CCD camera and a diode laser. The spectra of the individual components and formulations were acquired by 10 scans for a 10 s exposure time. Measurements were carried out with a 532 nm laser with a power level of approximately 1 mW at a slit width of 50 μm in the point measurement mode. Lidocaine, petrolatum, MCT oil, GMS, and prepared ointments were analyzed. In addition, raw SD rat skin and ointment-treated SD rat skin were analyzed. Ointment-treated SD rat skin was treated with ointment for 6 h, and the remaining ointment was removed using an alcohol swab, then analysis was performed. w/v) phosphotungstic acid solution for 10 s. The sample was dried for approximately 3 h at 25 °C. Images were obtained with an accelerating voltage of 200 kV using a LIBRA 120 instrument  [The morphology and structure of the diluted ointment were characterized using transmission electron microscopy (TEM). First, the diluted ointment was loaded onto a copper grid coating  by soaking for 1 min. Next, the samples were dyed using 1% (Germany) .® mini dialysis devices 10 K MWCO, ThermoFisher Scientific Inc., Waltham, MA, USA). Precisely, 1 g of ointment was added to the device, and the device was placed into a 50 mL conical tube containing the dissolution medium, 45 mL of PBS. The conical tube was gently shaken at 100 rpm and maintained at 37 ± 1 °C in a water bath . At a defined time, 200 μL of the medium was added, and the same volume of fresh PBS was added. Drug content was quantified using HPLC. The HPLC system  was operated at 265 nm with an L1 packing 250 mm × 4.60 mm, 4 μm column . The mobile phase, consisting of acetonitrile and buffer  at a 20:80 (v/v) ratio, was eluted at a flow rate of 1.0 mL/min. The column temperature was maintained at 30 °C, and the volume of each injected sample was 20 μL.The in vitro dissolution behavior of the lidocaine ointments was evaluated using a dialysis membrane (Slide-A-Lyzerv/v) was used as the receptor phase. The prepared lidocaine ointments and the Ref. A (3% w/w lidocaine) was applied to the donor compartment (approximately 2.0 cm2). An ex vivo skin test (n = 4) was performed over a 6 h period, after which the excess ointment was removed with an alcohol swab. Adhesive tape  was used to separate the stratum corneum. The epidermis and dermis were cut into six pieces [The ex vivo skin deposition test and Raman mapping of lidocaine ointment  were conducted using SD rat skin. Tests of L4, L5, L6, and Ref. A was performed using the human cadaver skin. The skin was hydrated with PBS and mounted in a Franz diffusion cell. PBS (pH 7.4) containing ethanol (50% x pieces . The pien = 3), the skin was cut into 50 µm slices from top to bottom using a cryomicrotome . Up to approximately 300 µm of the skin was obtained and cut vertically to determine the difference in lidocaine by depth. The skin slice was placed on a glass slide  and examined by Raman mapping . In each experiment, the laser light  was focused on the sample. The magnification of the sample was 20. The acquisition time was 0.1 sec and the slit size was 50 µm. The point-by-point mode was used for mapping. The analysis was performed at 1670 cm−1. The view size was a square with 100 µm × 100 µm. The pixel size was 1.0 µm × 1.0 µm. In the Raman mapping, a high concentration of lidocaine is presented as red color, whereas a relatively low concentration of lidocaine was displayed as blue color.Raman mapping was performed to determine the distribution of lidocaine throughout the skin depth. After the ex vivo skin permeation test for 6 h (n = 4). The mice were housed at a temperature of 20 °C and relative humidity of 40% during the experiment. The mice were acclimatized for 7 days before the tail-flick test. To perform the test, a 1.5 cm portion of the tail was heated by a radiant heat source. Tail flick latency (TFL) time was measured by turning on the heat source to the flicking of the tail. The mean time of four measurements before the administration of ointment was performed as the baseline latency. Mice were selected for testing with a mean baseline latency time of 2–4 s. A 10 s cutoff time was applied to avoid thermal damage to the tail at all points in the experiment [The Chungbuk National University Institutional Animal Care and Use Committee approved the experimental protocols and animal care methods used in this study. In the present study, 20 male ICR mice were divided into five groups (periment . The five groups of mice were treated with either 50 mg of L4 ointment (L4 group), 50 mg of L5 ointment (L5 group), 50 mg of L6 ointment (L6 group), 50 mg of Ref. A (Ref. A group), or normal saline . The time points of the tail-flick test were 0, 30, 50, 60, 75, 90, and 105 min after drug administration. The area under the receiver operating characteristic curve (AUC) was calculated from the TFL-time profile of each formulation using the linear trapezoidal rule. p < 0.05.Statistically significant differences were evaluated using one-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) post hoc test using SPSS version 23 . Statistical significance was set at Physicochemical characterization of lidocaine ointment was performed. The droplet size and polydispersity index (PDI) of the diluted lidocaine ointment were determined . The exaDSC thermograms of the raw materials and lidocaine ointment are shown in The correlation of XRD data  and the −1 . 2, while L2 released 815.1 μg/cm2 and the L3 ointment released 1021.6 μg/cm2. Additionally, the kinetics of the release data showed the best fit with the zero-order release model . The correlation between the flux and GMS ratio was very high, with a value of R2 0.999. In vitro permeation tests were performed on human cadaver skin using the compositions L4, L5, L6, and Ref. A . L5 and ectively B. L5 delp-values < 0.005. There was no statistically significant difference in the AUC between L5 and L6. The effect folds were calculated for a relative comparison of anesthesia effects [ effects . CompariAs shown in The thermograms of the lidocaine ointments were most affected by the presence of petrolatum and GMS . The theThe sharp and narrow peaks of lidocaine disappeared in the XRD patterns of L1, L2, and L3 . Based o−1 peaks correspond to methyl antisymmetric stretching, CH2 antisymmetric and symmetric stretching, and the C=O bond of the amide bond [−1 did not overlap with other excipients and were not found in raw SD-rat skin, but were found in ointment-treated SD-rat skin. Excipient-derived peaks were also confirmed in ointment-treated SD rat skin, as they were derived from the excipients merged into the skin. In addition, 1670 cm−1 peaks, which were not visible in the ointment, were identified in ointment-treated SD rat skin. This is because the intensity of the 1670 cm−1 peak increased even after removal of the ointment, as lidocaine migrated from the ointment to the skin. Since the excipient did not migrate to the skin, the intensity of the excipient-derived peak decreased after the ointment was removed. Therefore, the intensity of the peak at 1670 cm−1 was relatively higher in ointment-treated SD rat skin. This wavelength was used for Raman mapping and subsequent experiments (In ide bond . The peaide bond ,57. Theside bond . Next, Reriments B.The TEM image in As the dissolution progressed, L2 slowed down, while L1 and L3 maintained their dissolution rates A. This iAs shown in the Raman mapping results , L1 was The permeation tests using human cadaver skin also showed that the influence of the GMS ratio was the largest A. SimilaIn all formulations, latency tended to increase up to approximately 60 min overall and then decreased. However, L5 and L6 maintained a high latency of up to 105 min, with a relatively small decrease . As showHowever, there was no difference between L4 and Ref. A at 90 min. L5 and L6 showed higher latencies than those in Ref. A at all-time points B,C. The We conducted a pharmaceutical study to take advantage of the self-nanoemulsifying effect of GMS, allowing lidocaine to be released rapidly from the petrolatum ointment and rapidly penetrate the dermis. The self-nanoemulsifying effect of GMS was confirmed through an in vitro dissolution test, which resulted in rapid release from the ointment. Due to these properties, in vitro skin permeation also shows a rapid transition to the receptor phase with GMS, while a larger amount of lidocaine was deposited in the dermis. In the in vitro permeation test, the penetrated amount in the receptor phase and flux increased as the amount of GMS increased, and the profile showed a zero-order form with no lag time, which had a linear correlation with the ratio of GMS. However, there was no difference in the amount deposited in the dermis, as confirmed by Raman mapping. In the case of L1 without GMS, the drug permeated to a depth of 200 μm, which is the position that seems to be the top of the dermis, while in L2 and L3, which contained GMS, the drug was distributed to a deeper layer of the dermis. After saturation of the dermis above a certain amount, the drug shifts to the receptor phase, so it is considered that there is no significant difference in the amount deposited in the dermis between L2 and L3, beyond that which was necessary for saturation. Furthermore, similar results were obtained from the in vivo tail-flick test. In the case of the Ref. A and L4 ointment without GMS, the latency between stimulus and tail-flick was higher than that in the normal saline group, but there was no significant difference between Ref. A and L4. However, L5 and L6 had significantly higher latency and AUC, and were more effective than Ref. A, L5 at 1.38 times, and L6 at 1.52 times. Additionally, improvement of skin deposition was confirmed through in vitro Raman mapping, and the superiority of local anesthetic effect was confirmed through an in vivo tail-flick study. In conclusion, the self-nanoemulsifying effect of GMS was applied to improve local anesthetic ointment, primarily for the treatment of hemorrhoids.


Microbial communities normally comprise a few core species and large numbers of satellite species. These two sub-communities have different ecological and functional roles in natural environments, but knowledge on the assembly processes and co-occurrence patterns of the core and satellite species in Tibetan lakes is still sparse. Here, we investigated the ecological processes and co-occurrence relationships of the core and satellite bacterial sub-communities in the Tibetan lakes via 454 sequencing of 16S rRNA gene. Our studies indicated that the core and satellite bacterial sub-communities have similar dominant phyla . But the core sub-communities were less diverse and exhibited a stronger distance-decay relationship than the satellite sub-communities. In addition, topological properties of nodes in the network demonstrated that the core sub-communities had more complex and stable co-occurrence associations and were primarily driven by stochastic processes (58.19%). By contrast, the satellite sub-communities were mainly governed by deterministic processes (62.17%). Overall, this study demonstrated the differences in the core and satellite sub-community assembly and network stability, suggesting the importance of considering species traits to understand the biogeographic distribution of bacterial communities in high-altitude lakes. In natural ecosystems, bacteria within a metacommunity could be partitioned into different ecological assemblages, such as abundant or rare sub-communities and core or satellite sub-communities in light of potential importance for the community function . DefininPrevious studies have reported that deterministic processes and stochastic processes play important roles in the regulation of spatial distribution of bacterial communities in natural environments ,b. DeterIn this study, we used 454 pyrosequencing of the bacterial 16S rRNA gene to investigate the diversity and composition of core and satellite bacterial sub-communities in 47 lake water samples of 30 lakes located on the Tibetan Plateau. The Tibetan Plateau has the largest number of plateau lakes group in the world . A most 2  from twenty lakes, respectively. Duplicate samples were collected at the same time from same points from 10 lakes, AGC, BC, BGC, DZC, GZC, NMC, PE, PMYC, Yamdrok, and ZGTC . Water s® Spin kit  according to the manufacturer’s instructions. It was checked for concentration and purity using a NanoDrop Spectrophotometer . The V4-V5 region of the bacterial 16S rRNA genes was amplified using the primer pair 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 907R  is subsequently calculated by comparing empirically observed Bray-Curtis and simulated null distribution. The |βNTI|<2 and RCbray<−0.95 or the |βNTI|<2 and RCbray≥0.95 RCbray were identified as homogenizing dispersal and dispersal limitation, respectively. When the |βNTI|<2 and |RCbray|<0.95 were identified as “Undominated” :jB represents the habitat niche breadth; ijP is the mean relative abundance of OTU j in lake i ; and N is the total number of communities. A high B-values indicate a wide range of OTUs and even distribution, representing wide habitat niche breadth and more metabolic flexibility at the community level  between those OTUs were calculated pairwise by the “Hmisc” package in an R environment. Only robust correlations with Spearman’s correlation coefficients (ρ)>0.6 and false discovery rate-corrected values of p<0.01 were used to construct networks  reads. In contrast, the remaining 809 OTUs , surpassing 2.5% confidence limit line of χ2 distribution, formed core sub-communities and accounted for 90.01% of the total reads .In all taxa, Proteobacteria, Bacteroidetes, and Actinobacteria were the dominant phyla in the core and satellite sub-communities, together accounting for 71 and 78.62% of each sub-community sequences, respectively . Cyanoba indices . Both Chp<0.05; R2<0.1), indicating weak relationship of community dissimilarity with geographic distance for the core and satellite sub-communities. Meanwhile, the slope of DDRs was significant (p<0.01) steeper for the core sub-communities (0.019) than that of the satellite sub-communities (0.004).Distance-decay relationship (DDR) is a fundamental pattern in ecology, in which community similarity decreases as the geographic distance increases. In the current study, although the significant positive DDRs (Mantel B) analysis indicated that the average niche breadth for the core communities (4.11) was significantly wider than that of the satellite communities  than that of satellite sub-communities . Meanwhile, the real network exhibited higher values of average clustering coefficient (0.58 vs. 0.04), average path length (5.99 vs. 2.41), and modularity (0.64 vs. 0.19) than those of the respective Erdős-Rényi random, suggesting the real network was non-random and modular structure  than satellite sub-communities, while DDR controlled by dispersal limitation showed an opposite trend in the core and satellite sub-communities  can be found at NCBI (BioProject ID PRJNA306720).This work was conceived by KL and YL. Field work was done by KL. Laboratory work was done by KL and FW. Analysis was carried out and the manuscript was written by QY and KL. Writing-reviewing and editing were done by JD. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (grant nos. 41771086 and 42006200), the Second Tibetan Plateau Scientific Expedition and Research Program , the National Key Research and Development Program Project (grant no. 2019YFC1509103), and the Strategic Priority Research Program (A) of the Chinese Academy of Sciences (grant no. XDA20050101).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The lowest graphene content delays the degradation of filmswith time and light irradiation and leads to enhanced photovoltaicperformance and stability of the solar cells, with relative improvementover devices without graphene of 15% in the power conversion efficiency,PCE. A higher graphene content further stabilizes the perovskite filmsbut is detrimental for in-operation devices. A trade-off between thepossible sealing effect of the perovskite grains by graphene, thatlimits ionic diffusion, and the reduction of the crystalline domainsize that reduces electronic transport, and, especially, the detectedincrease of film porosity, that facilitates the access to atmosphericgases, is proposed to be at the origin of the observed trends. Thiswork demonstrated how the synergy between these materials can helpto develop cost-effective routes to overcome the stability barrierof metal halide perovskites, introducing active layer design strategiesthat allow commercialization to take off.The relatively lowstability of solar cells based on hybrid halideperovskites is the main issue to be solved for the implementationin real life of these extraordinary materials. Degradation is acceleratedby temperature, moisture, oxygen, and light and mediated by halideeasy hopping. The approach here is to incorporate pristine graphene,which is hydrophobic and impermeable to gases and likely limits ionicdiffusion while maintaining adequate electronic conductivity. Lowconcentrations of few-layer graphene platelets (up to 24 × 10 Moisture, air, UV light, thermal stress(heat), light soaking, and electric fields, among other agents, degradethe perovskite solar cells,9 especially outdoors,10 and prevent market requirements from being achieved.MHP rapidly evolves to PbI2 or PbOx in the presence of moisture or light irradiation.13 Even in an inert atmosphere, the most representative perovskiteabsorber material, MAPbI3, appears to be thermally unstableas revealed by the observed transformation into PbI2.14 Stability is therefore one of the main barrierspreventing the widespread application of PSCs.16Thegoal of lowering solar electricity costs by improving the efficiency,reliability, and durability of emerging metal halide perovskite (MHP)solar cells (PSCs) is actually an intense area of research. Three-dimensional(3D) MHPs are described by the general formula ABX17 could enhance stability and passivation of traps at the crystallinedomain interfaces, or better energy level alignment18 could improve charge injection/extraction that could leadto more efficient devices. In fact, the incorporation of graphene-relatedmaterials (GRMs) in PSC devices has been demonstrated to be a promisingroute toward applications and improvements in performance and stabilityand, very importantly, for their commercial launch.22 It has been observed that coveringa two-dimensional (2D) perovskite with a graphene monolayer maintainedreasonable optical properties after exposure to high temperature andhumidity.23 Note that pristine graphene(sp2 C) and functionalized graphene  have different properties and behaviors relatedto the presence of oxygen (or other) functional groups.Most of the advances in the performance of PSCs have resultedfromimprovements in device architecture and material composition. Theexcellent properties that MHPs provide on the one hand, and thoseof graphene (G) on the other, can envision great enhancement of deviceproperties. The hydrophobic character of G24 Nitrogen-doped rGO, N-rGO,has been added to obtain bigger crystal domains to passivate the surfaceand to delay the recombination of charges.26 Eventually amine-functionalized-G27 andGQDs29 have been added to improve charge-extractionefficiency and facilitate charge transport. Chen et al.30 simultaneously incorporated N, S codoped GQDsin the ETL-perovskite interface and into the perovskite active layerof the PSC acting as a nucleating template, easing charge extractionand defect passivation, obtaining efficiency and stability improvementsof the device. Also, electrospun graphene nanofibers31 added into the perovskite improved nucleation and crystallizationat the nanofiber interface, favoring charge transport and resultingin improved photovoltaic performance and stability of the final device.Recently, Lou et al.32 used a graphene/halogenated-polymercomposite dispersion as an antisolvent in the perovskite synthesis,observing that halogen elements (Cl/F) in the polymer can p-dope thegraphene and endow it with higher hole-transport selectivity. However,it should be noted that, to our knowledge, the effect of only pristinegraphene platelets on the active perovskite layer has not been investigatedand until now they have been hardly used in the charge-transport layers.Wang et al.33 and Agresti et al.34 tested nanocomposites of G and TiO2 nanoparticles and doped the mesoporous TiO2 layer withgraphene flakes, respectively, and reported charge-carrier injectionand collection improvements and cell stability. Recently, we havedemonstrated a route for doping poly–poly(styrenesulfonate)(PEDOT:PSS) HTL with pristine G-nanoplatelets, obtaining significantimprovement on conductivity without the loss of transmittance at concentrationswell below percolation.35 The integrationof this G-doped HTL into inverted MAPbI3 solar cells resultedin more efficient devices due to an enhancement of charge extractionand a reduction of charge accumulation at the graphene–PEDOT:PSSinterface as well as stability improvement under environmental conditions.Theliterature describes how GO or rGO is used to dope or substitutethe hole transport layer (HTL) and the use of G, GQDs, Li-GO, andrGO in the electron transport layer (ETL), with a positive effecton both charge extraction rates and stability of PSCs. However, thecombination/incorporation of GRM into the active layer is less frequentand extremely scarce are the works that incorporate pristine graphene.Morphology, grain size, and thickness are critical factors in thecharge dynamics and the final performance of the device that can bemodified by the use of this GRM. Among them, carbon nanotubes havebeen used as additives in the perovskite active layer as electrondonors or acceptors to reduce charge recombination, acting as a nucleationtemplate.3, is combined with pristine graphene platelets into the activelayers of the solar cells. For the appropriate amount of graphene(only 0.6 × 10–3 wt %), a significant improvementof 15% for the average efficiency value and increased stability isdemonstrated in non encapsulated devices under high-stress conditions.Impedance spectroscopy reveals that graphene induced an enhancementin recombination resistance, Rrec. Theseresults are explained in terms of the modifications induced by graphenein the analyzed structural, morphological, optical, and electronicproperties of the G-MAPbI3 thin films. This work demonstratesa simple route to obtain graphene/metal halide composites that canpave the way to the development of low-cost solar cells.Here, we act on the hybrid lead iodide perovskite active layer,searching to exploit the synergy between these different materials.Specifically, the most commonly used hybrid halide perovskite, MAPbI22.1Supporting Information). The narrow widths of G and 2D peaks reveal thesuperior quality of this graphene36 comparedto that of GO and rGO materials. For fundamental characterization,MAPbI3 thin films on glass substrates were obtained withdifferent graphene contents in the precursor solution .The samples are named as 0G (reference sample without graphene), 2.5G,5G, 10G, 50G, and 100G, corresponding to 0, 2.5, 5, 10, 50, and 100mg/L of G in the precursor solutions of MHPs, equivalent to 0, 0.6,1.2, 2.4, 12, and 24 × 10–3 wt % graphene contentin the films. The thickness of the films is given in Table S1.The used graphene platelets correspond tofew-layer graphene withdefects mainly related to the domain size according to its Raman spectrum-orientedgrains. All samples contain a minor amount of PbI2 impurity.The tetragonal-perovskite lattice parameters were refined by a profilematching approach  obtainingvalues of 8.865(1) Å and 12.661(4) Å for a and c, respectively. These values do not changewhen graphene is added, showing that, as expected, the perovskitecomposition is not modified.Supporting Information). This methodallows determining the crystal domain size and strain with great precisionsince the whole diffraction pattern is used instead of a single peak.The domain size for the smallest amount of graphene, 2.5G film, showsan increase of approximately 8 nm. Such an increase (∼10%)is reliable considering the very small error  methodology . The degradation of the films canbe followed by the evolution of the intensities of (001) PbI2 and (110) MAPbI3 reflections with time (2 (001) reflection with time may lead to conclude thatthe PbI2 content increases to large proportions and thatthere is a large degradation of the perovskite films. However, ithas to be highlighted that to quantitatively analyze the diffractionpatterns, the almost complete (001) preferred orientation of PbI2, that provides a greatly enhanced intensity of the (00l) maxima, has to be considered. In fact, the degree ofdegradation of the perovskite phase is small even after 2000 hours,as confirmed by the intensity of the (110) MAPbI3 reflectionwhich decreases only a little with time  over time has been followed by XRPD S4. The d2.238 that show a local minimaat 775 nm (1.60 ± 0.05 eV) for all films  that correspondto the direct semiconductor-type transitions at the R point in thepseudo-cubic Brillouin zone.39 The absorption spectrahave been recorded along ∼2200 hours to explore the evolutionof the undoped and G-doped MAPbI3 films under ambient conditions; Figure S5a,b for the evolution of absorption spectra for 5G and 10G). With time,the MAPbI3 absorption edge amplitudes barely decrease,indicating that the degradation is moderate in all cases but is slightlymore pronounced for the 0G film compared to the G-doped films. Theseresults are consistent with the trends of the (110) perovskite diffractionpeak upon G incorporation  studied here are due to the combination oftwofactors: (1) the weakness of the signal that reaches the detectorabove the gap  and (2) the presence of “pinholes”in the film . In Ah = fraction of the film without MAPbI3. The fits for 2.5G and 5G spectra using Ah = 10–60 and 10–28, respectively,are excellent. These are extremely small hole-effective fractionsin the films but unambiguously demonstrate that graphene induces someincreasing porosity. For the 10G film, the equivalent hole fractionis significantly higher, Ah = 10–6.5, and the approximation is not so good since it cannot adequatelyfit the region from 2 to 2.5 eV; however, it shows that the porosityis drastically increased. Therefore, while graphene delays degradation,it also modifies the morphology of the grains and their packing withinthe film increasing progressively its porosity. Scanning electronmicroscopy (SEM) images confirm that porosity increases with the addition of graphene platelets.Moreover, since graphene is an impermeable membrane to gases,40 it may be partly sealing the surface of thegrains and we hypothesize that could hamper ionic diffusion.The plateaus typically observed in the high-energy range of absorbancespectra for MAPbIFigure S6). However, graphene affects the evolutionof PL intensity with irradiation time (2) for samples 0G–10G is shown in 41 The presence of defects, particularlyhalide vacancies, photoinduced defects and, in general, trap statesand their evolution with the factors described above, plays an importantrole in the nonradiative processes influencing the recombination dynamics.43 In an earlier work,11 we identified fourmain stages under ambient conditions that MAPbI3 evolvesunder laser irradiation by measuring the PL and the concomitant thin-filmRaman spectra. A decrease in the PL intensity under ambient conditionscorresponded to the appearance of Raman modes associated with PbOx, that therefore, is indicative of the beginningof degradation that eventually results in the total fading of thePL emission.Regarding photoluminescence (PL), the addition of G does not inducesignificant changes in the emission of the samples; all present thecharacteristic band at an identical energy: 1.60 eV (775 nm) S6. HowevLow and intermediate graphene concentrations provoke lessand slowerquenching of the PL emission with time 3b, retai2.33 thin films with temperatureacross the tetragonal to cubic structural transition. All measurementsexhibit Ohmic behavior  anda value at 303 K of 2.2 ± 0.2% MΩ·cm for the 0G sample,within published values for MAPbI3,: 6.6,44 51,45 or 38 MΩ·cm46 for polycrystalline samples and 13 MΩ·cm46 in single crystals.We have studied theeffect of G-platelet incorporation on the darkdc resistivity, ρ, of MAPbI3 thin film shows slightly lower resistivityvalues for the tetragonal phase, T < 333 K. Thetemperature of the observed turning points in resistivity (at ∼323K) is very close to the structural, tetragonal (I4mcm) to cubic (Pm3̅m), transition temperature, Tc = 328 K,47 pointing out to a correlationbetween the resistivity behavior and the crystalline phase transition.The impact of structural transitions on optical,50 thermal,51 dielectric,52 andphotovoltaic53 properties of MHPs has beeninvestigated and the electronic structures for both crystalline structuresof MAPbI3 were calculated.54 In general, experiments show relatively minor effects at the cubic–tetragonaltransition; however, previous works58 reported a significant conductivityincrease at the tetragonal to orthorhombic low-temperature transition,around 160 K, related to the disorder-order character of the transition,due to MA cation ordering.47 The tetragonalto cubic transition has also been described as a disorder-order transitionin several perovskites;60 so, a similar effecton resistivity cannot be ruled out. In fact, this uncommon behaviorfor a metal-semiconducting crossover with temperature was observedby Głowienka et al.61 for dark conductivity,σ, in MAPbIx3–Clx thin films between 280 and 340 K and by Sveinbjörnssonet al.62 between 293 and 353 K under illuminationin MAPbI3 crystals.The variation of resistivityin the 25–100 °C (298–373 K) temperature rangeis shown in Ea) can be obtainedfor the cubic phase from the expression for thermally activated conduction, 0 is a pre-exponentialfactor and kB and T arethe Boltzmann constant and the temperature of the sample, respectively. Ea values of 0.28, 0.24, and 0.46 eV for undoped0G and doped 2.5G and 5G MAPbI3 thin films are obtained,respectively. In the tetragonal phase, the resistivity increases withtemperature (from 298 to 320 K), which is the opposite trend to thatof a semiconductor. The structural transition may have an impact onthe electronic conduction in a temperature range so close around Tc, but ionic conduction has to be consideredsince it has an important role in MHPs. The degree of ionic contributionto the charge transport in MHPs is controversial. In all-inorganichalide perovskites, it is commonly accepted that halide-ion conductionvia the vacancy diffusion mechanism is the dominant conduction process,with reported activation values in the range of 0.2–0.9 eV.65 For MAPbI3, Ea calculatedfrom first principles is reported to be 0.58 eV for iodine vacancies,66 quite larger than the Ea values obtained here in the cubic phase, so electronic conductionseems to be dominating dc transport. Very low graphene concentration(2.5G) lowers the hopping activation energy Ea, while larger amounts hinder this hopping transport mechanism(Ea increases). The increase of the crystallinedomain size is induced by the addition of low graphene concentrationin the 2.5G sample, while maintaining the fraction of (110) orientedcrystals . The photovoltaic parameters, thatis, short-circuit photocurrent (JSC),open-circuit voltage (VOC), fill factor(FF), and PCE, were measured under ambient conditions and AM 1.5Gsolar simulator with a light intensity of 100 mW/cm2. Thestatistical distribution of these parameters and representative J–V characteristics are shown in Figure S8, and the data of the average and championdevices in reverse and forward scans are collected in Table S2. Undoubtedly, the lower amount of G(2.5G) in the MAPbI3 active layer leads to an improvementof ∼6.6% in the final PCE of 2.5G-based solar cells comparedto that without graphene (0G). The graphene content above 10G in theactive layer induces important photocurrent losses, probably associatedto the observed poor crystalline quality of the samples69 and the increasing porosity of the films with the G content. Interestingly,the VOC value does not decrease indicatingthat energy level matching for charge extraction does not suffer significantalterations.18 To summarize, a low graphenecontent in the active layer (2.5G: 0.6 × 10-3 wt %G/PVK) induces, as previously indicated, larger crystalline domains,high preferential orientation, and an increased conductivity, allcontributing to an improvement in the final efficiency of the device.To evaluate the influence of the G-plateletcontent (0G–100G)in the active layer of solar cells on the photovoltaic performance,different concentrations of G-platelets in the MAPbI2/MAPbI3:G/spiro-OMeTAD/Au, . Comparison of solarcell parameters, JSC, VOC, FF, and PCE, of photovoltaic devices using activeMHP layers with and without graphene is shown in Table S3. Graphene additionincreases the reproducibility and averaged values of all the photovoltaicparameters, especially VOC. There is arelative increase in the average PCE of 15% for the 2.5G sample overthe reference cell without graphene. J–V curves of champion devices without graphene, 0G, and 2.5Gare plotted in Figure S9a. There is a goodagreement between the photocurrent measured in the J–V and calculated from incident photon currentefficiency measurements . Weobserved again that the average crystal domain is increased with grapheneaddition, as shown in Figure S10.Based on these results, we have optimized the devices with the2.5G graphene content in the structure: ITO/SnOVOC time evolutionfor both samples is almost identical, see Figure S11, with a small decrease during the first minutes and a stabilizationafterward. However, JSC and FF decreasefaster for the samples without graphene, see Figure S11.Interestingly, graphene addition also has a positive effectonthe stability of the PSCs as expected from the fundamental characterizationcommented above. Devices were tested under high stress conditionsusing non encapsulated samples and constant AM1.5G lighting for 30min under ambient conditions with a relative humidity of 40–60%.The presence of graphene has a positive influence on the stabilityof the solar cell. Figure S12a, according to the modelreported in a previous work,70 consideringthe transport resistance in the perovskite layer negligible in comparisonwith the recombination resistance, Rrec, as it is expected in high-performance PSCs. Two arcs are observedin the Nyquist plot as commonly reported in most of the PSCs, as shownin Figure S12b.70Rrec increases when an optimized amount of grapheneis added, 0.6 × 10–3 wt %, pointing to a decreaseof the nonradiative recombination in the 2.5G PSC, in good agreementwith the observed increase in VOC.Impedance spectroscopy experiments were performedon regular 0Gand 2.5G PSCs, and measurements were fitted using the equivalent circuitof –3 wt %) on the structuraland optoelectronic properties of MHP thin films under ambient conditionsand found a double effect that, properly balanced, can improve thedevice performance and stability of the final solar cell devices.On the one hand, a graphene content of 0.6 × 10-3 wt% G/PVK induces the growth of larger crystalline domains maintaininga high preferential orientation, which increases the stability ofthe MAPbI3 films with time under ambient conditions andthe endurance to illumination. On the other hand, the porosity ofthe films that increases with graphene concentration reduces conductivityand could allow the faster diffusion of oxygen and moisture deep intothe layer. An appropriated balance tuning graphene content to 0.6× 10–3 wt % G/PVK (2.5G) in the precursor solutionproduces an increase of all photovoltaic parameters and consequentlyof the final photoconversion efficiency and delays the degradation,increasing grain size, reducing nonradiative recombination probablyby partly sealing the surface of the grains without significantlymodifying morphology of the grains and their packing within the filmenhancing progressively its porosity. These results open the doorto future studies seeking a balance between the processing and efficiencyof this composite material through cost-effective perovskite metalhalide design strategies aimed at overcoming the stability barrier.We tracked the influence of the small grapheneplatelet content(0–24 × 10


Graphene’s unparalleled strength, chemical stability, ultimate surface-to-volume ratio and excellent electronic properties make it an ideal candidate as a material for membranes in micro- and nanoelectromechanical systems (MEMS and NEMS). However, the integration of graphene into MEMS or NEMS devices and suspended structures such as proof masses on graphene membranes raises several technological challenges, including collapse and rupture of the graphene. We have developed a robust route for realizing membranes made of double-layer CVD graphene and suspending large silicon proof masses on membranes with high yields. We have demonstrated the manufacture of square graphene membranes with side lengths from 7 µm to 110 µm, and suspended proof masses consisting of solid silicon cubes that are from 5 µm × 5 µm × 16.4 µm to 100 µm × 100 µm × 16.4 µm in size. Our approach is compatible with wafer-scale MEMS and semiconductor manufacturing technologies, and the manufacturing yields of the graphene membranes with suspended proof masses were >90%, with >70% of the graphene membranes having >90% graphene area without visible defects. The measured resonance frequencies of the realized structures ranged from tens to hundreds of kHz, with quality factors ranging from 63 to 148. The graphene membranes with suspended proof masses were extremely robust, and were able to withstand indentation forces from an atomic force microscope (AFM) tip of up to ~7000 nN. The proposed approach for the reliable and large-scale manufacture of graphene membranes with suspended proof masses will enable the development and study of innovative NEMS devices with new functionalities and improved performances. Researchers in Sweden have manufactured graphene membranes with attached silicon masses. Graphene is an extremely attractive material for membranes in micro and nanoelectromechanical systems (MEMS and NEMS). However, creating graphene membranes which can support the proof masses used in MEMS and NEMS has proven challenging. Now, a team led by Frank Niklaus at the KTH Royal Institute of Technology developed a technique to manufacture graphene membranes with suspended silicon masses attached. The new method can fabricate membranes ranging from 7 µm to 110 µm on each side, with suspended silicon blocks of up to 16.4 µm x 100 µm x 100 µm. The membranes can withstand forces of up to 7000 nN. The new approach opens the door to the use of graphene membranes in MEMS and NEMS devices, with applications from nanoscale robotics to wearable electronics.  However, the application of suspended graphene in NEMS devices has thus far been limited to resonators19, pressure sensors25, switches28, loudspeakers29, microphones31 and devices for fundamental studies of the material and structural properties of graphene37. The reported suspended graphene structures include doubly clamped graphene beams, fully clamped graphene drums and suspended graphene-based cantilevers. Suspended structures are typically realized by transferring graphene from the original substrate to a prefabricated substrate with trenches15, cavities20 or membranes made of dielectric layers39 or by transferring graphene from the original substrate to a flat silicon dioxide (SiO2)42 or polymer substrate surface44 and then removing parts of the material underneath the graphene by sacrificial etching.The atomically thin structure of graphene (atom-layer distance of ~0.335 nm) and its remarkable mechanical7 to ~1010 µm3 in size) that are suspended on springs in the form of membranes, beams or cantilevers. Graphene, as a robust and intrinsically nanoscale material, could be used to suspend large proof masses, thereby forming spring-mass systems consisting of atomically thin graphene springs for potential applications as ultra-miniaturized transducer elements in future high-performance NEMS devices20. However, the realization of suspended graphene with large attached proof masses is difficult, and to the best of our knowledge, no such examples have been reported in the literature. A previous report of suspended graphene membranes with very small masses included micrometre-sized few-layer graphene cantilevers with diamond allotrope carbon masses  fabricated using focused ion beam (FIB) deposition for the study of the mechanical properties of graphene45. Previous literature also reports a spiral spring, a kirigami pyramid and a variety of cantilevers based on a suspended graphene monolayer supporting 50 -nm thick gold masses attached to suspended cantilevers46. However, these structures had to be kept in a liquid to maintain their mechanical integrity. Suspended graphene membranes with diameters of 3–10 µm, that were circularly clamped by a polymer (SU-8) and that supported a mass made of either SU-8 or gold located at the centre of the membrane, were reported for shock detection caused by ultra-high mechanical impacts47. However, all previous reports involved extremely small masses, and the fabrication methods employed, such as FIB-induced deposition, were slow and typically not compatible with large-scale manufacturing.In contrast to previously reported graphene membranes and beams, MEMS and NEMS devices such as accelerometers, gyroscopes and resonators often employ larger proof masses  proof masses that is compatible with MEMS and NEMS manufacturing processes and that can be utilized for devising NEMS with graphene membranes as structural and functional components. Our approach employs a silicon-on-insulator (SOI) substrate to form silicon proof masses that are etched in the silicon device layer of the SOI wafer. The graphene membranes are formed by transferring a double layer of CVD graphene to the pre-patterned SOI wafer, followed by a combination of dry etching and vapour HF etching of the buried oxide (BOX) layer to release the silicon proof masses and suspend them on the graphene membranes. Static and dynamic mechanical characterization of the manufactured structures shows that they are robust and can potentially be used as spring-mass systems in future ultra-small NEMS, such as resonators and accelerometers.To demonstrate the feasibility of graphene membranes with large suspended proof masses, we fabricated square membranes with different dimensions made of double-layer graphene on which silicon proof masses of different sizes were suspended. A typical device structure is illustrated in Fig. 2 layer  in the Raman spectra indicates the relatively high quality of the graphene. The shape of the 2D band in Fig. 53. For double-layer and multi-layer graphene, the 2D band becomes much broader, as shown in Fig. 53.To verify that double-layer graphene indeed exists in our fabricated structures, we performed Raman spectroscopy. Figure −1 Fig.  and the  −1 Fig. 51,52 dem−5 mbar actively pumped vacuum) by measuring the amplitude of their thermomechanical noise using laser Doppler vibrometry (LDV)  Fig.  that havDV) Fig. . The resDV) Fig. , ~90 kHzDV) Fig. , ~78.8 kDV) Fig. , and ~60DV) Fig. . As expeDV) Fig. . The corDV) Fig.  is estimDV) Fig. . The Q-fTo further confirm the frequency response of the spring-mass system of our structures, we used LDV to measure the frequency response of a device with a trench width of 3 µm and proof mass dimensions of 50 µm × 50 µm × 16.4 µm in air (atmospheric pressure) at room temperature by driving the device with a piezoshaker Fig.  was esti59  and characterizing the resulting structures by SEM. We fabricated twelve chips that comprised 672 structures in different batches, and we obtained similar yields for all chips. Figures 60. During graphene transfer, we used PMMA as a support layer for the graphene, as this allowed easy handling and transfer of the graphene. Even after thorough rinsing with organic solvents such as acetone, PMMA residues (long-chain molecules) remain adhered to the graphene due to the strong dipole interactions between PMMA and chemical groups on graphene60. To remove as many of the PMMA residues as possible, we selected some sample chips with released proof masses for annealing at 350 °C for 2 h. During the annealing process, the temperature was first steadily increased from 100 °C to 350 °C in 1 h and then decreased from 350 °C to 100 °C in 1 h. The surface of the graphene after annealing was cleaner  increased the probability of the masses being detached from the suspended graphene membranes due to capillary forces. Only employing HF vapour to etch the BOX layer required a very long time and increased the risks associated with overetching of the BOX layer. To increase the yields as well as the quality and efficiency of the fabrication process, dry etching followed by vapour HF etching was the preferred approach here.We experimented with several different process flows to fabricate graphene membranes with suspended proof masses and found that wet HF etching was hard to control and often caused graphene displacement, wrinkles or collapse of the graphene due to etching of the SiOWe also evaluated the possibility of transferring monolayer graphene over the trenches using our baseline process Fig. . HoweverIn summary, in this paper, we have reported a robust route to transfer and integrate double-layer graphene membranes onto a silicon substrate. The proposed manufacturing process is based on SOI wafer technology and allows the suspension of large silicon proof masses on graphene membranes. Our approach is scalable and highly compatible with silicon NEMS technology, and complementary metal oxide semiconductor (CMOS) wafers for the integration of NEMS devices with electronic circuits. The ability of the graphene membranes to withstand AFM indentation forces of up to ~7000 nN without failure indicates that the structures are very robust. Thus, the ability to realize graphene membranes with suspended large proof masses offers interesting opportunities for ultra-miniaturized graphene NEMS devices such as accelerometers, gyroscopes and resonators, with exciting applications in nanoscale robotics, autonomous vehicles, wearable as well as consumer electronics and the internet of things (IoT).2 layer on both sides of the wafer  was used to etch the SiO2 layer  , where the copper foil floated on the FeCl3 solution with the graphene side facing away from the liquid. Then, with the help of a silicon-carrier wafer, the PMMA/graphene stack without copper  were used. A standard wet transfer approach was employedher Fig. . The resher Fig. . Then, aher Fig. . Then, c W) Fig. . For wetper Fig.  was firsfer Fig. . The PMMfer Fig.  and subsing Fig. . A layering Fig.  using pring Fig.  and to ting Fig. . The SOIes Figs. , 2c1–c5.2)  from exposing and destroying the graphene, all four sides of the chip were sealed by Kapton tape. Then, an RIE etching process was employed to etch the main part of the BOX layer  Fig. , while m2) Fig. . To prevyer Fig. . To avoitup Fig.  was tempnes Fig. . Despite−5 mbar actively pumped vacuum) and the amplitude of structures that were driven by a piezoshaker in air (atmosphere pressure).Optical microscopy and SEM imaging were used to observe and characterize the morphology of the devices during and after device fabrication. Raman spectroscopy  was used to verify the presence and quality of the double-layer graphene of the manufactured devices. For static mechanical characterization, an AFM  with a cantilever (Olympus AC240TM) and an AFM tip (tip radius = 15 nm) was used to load defined forces at the centre of a proof mass on a graphene membrane to measure the force vs. proof mass displacement and the maximum force that the suspended graphene membrane can withstand without rupture. The spring constant of the AFM cantilever was calibrated to be 5.303 N/m. To measure the resonance frequency and quality factor of the structures, we used an LDV (Polytec UHF-120) with a laser spot size on the order of 2.5 µm to detect the amplitude of the thermomechanical noise of the structures in vacuum (~10Supporting Information


Furthermore, the GaN microrod LED arrays were transferred onto Cu foil by using the chemical lift-off method. Even after being transferred onto the flexible Cu foil substrate, the microrod LEDs exhibited strong emission of visible blue light. The proposed method to enable the dimension- and position-controlled growth of GaN microstructures on graphene films can likely be used to fabricate other high-quality flexible inorganic semiconductor devices such as micro-LED displays with an ultrahigh resolution.This paper describes the fabrication process and characteristics of dimension- and position-controlled gallium nitride (GaN) microstructure arrays grown on graphene films and their quantum structures for use in flexible light-emitting device applications. The characteristics of dimension- and position-controlled growth, which is crucial to fabricate high-performance electronic and optoelectronic devices, were investigated using scanning and transmission electron microscopes and power-dependent photoluminescence spectroscopy measurements. Among the GaN microstructures, GaN microrods exhibited excellent photoluminescence characteristics including room-temperature stimulated emission, which is especially useful for optoelectronic device applications. As one of the device applications of the position-controlled GaN microrod arrays, we fabricated light-emitting diodes (LEDs) by heteroepitaxially growing In In such heterostructures, the GaN microstructures provides a light-emitting active region through radiative recombination, which is significant in fabricating light-emitting devices and displays10. Moreover, when combined with a graphene film substrate, GaN microstructures demonstrate excellent tolerance against mechanical deformation12. Although GaN hybrid-dimensional heterostructures can be applied to prepare several types of flexible devices, the most promising device application pertains to a flexible high-resolution display with a micrometer-sized light-emitting diode (micro-LED) array14. For such applications, the dimensions and positions of the GaN microstructures must be precisely controlled for the individual operation of each micro-LED in the array. Nevertheless, the key criteria for optimizing the device performance, such as the morphology and position control, n- and p-type doping, and formation of quantum structures in the GaN microstructures, have not been extensively studied15. In this study, we realized the dimension- and position-controlled growth of GaN microstructures on graphene films and the formation of their heterostructures with InxGa1−xN/GaN multiple-quantum-well structures for use in flexible micro-LED applications. The precisely controlled growth of GaN microstructure on graphene films can help develop templates for the individual operation of the devices for micro-LED displays.Recently, hybrid-dimensional heterostructures consisting of GaN microstructures with two-dimensional (2D) graphene films have attracted attention for use in transferable and flexible electronic and photonic devices2) layer. To control the positions of the microstructures in the array, a patterned SiO2 growth mask layer with microhole arrays having a diameter and interdistance of 1 µm and 8 µm, respectively, was fabricated on the graphene film, and the microstructures were grown only on the patterned microhole sites. Subsequently, we grew a 0.5-µm-thick ZnO seed layer before the growth of GaN microstructure arrays to enhance the nucleation. The complete ZnO seed layer was deposited on each microhole pattern array  and fast Fourier transform (FFT) techniques. Figure 2); Si and O could be attributed to the SiO2 layer. The higher Ga concentration with respect to N concentration could be attributed to the focused ion beam sample preparation using a Ga ion source.The energy-dispersive X-ray spectroscopy (EDS) maps of the interface between GaN and graphene clearly demonstrated the chemical composition of GaN microstructures grown on graphene. Figure 2. At excitation densities below 350 kW/cm2, a wide emission peak at 367 nm was captured without any remarkable feature shown in Fig. 2, sharp peaks appeared at 367.4, 370.2, 372.9, and 375.8 nm with a regular spacing of 2.8 nm around the near-band-edge emission, which eventually emerged as the dominant feature in the PL spectra. Each waveguide mode underwent Fabry–Perot (FP) oscillations, thereby generating a number of resonance peaks in the lasing spectrum20. The FP resonance of the mode spacing was defined as ∆λ = λ2/2L[1/(n-λdn/dλ)], where ∆λ is the mode spacing, L is the cavity length, n is the effective refractive index, and dn/dλ is the first-order dispersion of the effective refractive index21. ∆λ was calculated to be 2.83 nm, which was consistent with the measured ∆λ. The observation of the lasing characteristics with a low threshold pumping density indicated that the GaN microrods grown on graphene had a high optical quality. It was considered that the GaN microrods having flat side facets, which were naturally grown without etching, enhanced the lasing properties by lowering the diffraction loss.Furthermore, the optical quality of the GaN microstructures grown on graphene was examined using power-dependent micro-photoluminescence (μ-PL) spectroscopy. For the optical characterizations, we used the hexagonal arrays of GaN microrods with a diameter of 3.9 ± 0.2 μm and height of 15.8 ± 0.4 μm  was spin-coated on the heterostructures, and a buffered oxide etchant was used to etch the sacrificial SiO2 from the 300-nm-thick SiO2/Si substrate. The device structure was transferred onto a flexible substrate. The coaxial microrod heterostructures could tolerate the ultimate bending conditions due to the micrometer-sized spacing of the micro-LED arrays. The GaN micro-LEDs were wrapped around a 1-mm-diameter paper clip to test the effect of mechanical deformation, as shown in Fig. The fabrication process of flexible LEDs is illustrated in Fig. p-GaN, InxGa1-xN/GaN MQWs, and n-GaN on CVD graphene films. To fabricate flexible micro-LEDs, thin Ni/Au layers (10/10 nm) were deposited on the surface of coaxial p-GaN microrods. The micro-LEDs on the graphene films were lifted from the sacrificial substrate and easily transferred onto the Cu foil. Subsequently, the flexible LEDs were transferred onto the Cu foil substrate to form an electrode. The current was injected directly through the graphene, thereby generating a vertical structure for the microstructure LEDs.The flexible LEDs on graphene films were fabricated using the coaxial microrod heterostructures consisting of 2 LED devices captured strong blue light emission at applied biases ranging from 0.5 to 2.0 mA. The intensity of emitted light was very strong as visibly seen. It was able to brighten up the room. Figure xGa1−xN MQWs embedded in the LEDs. The EL spectra also showed longer wavelength tail next to the 421 nm peak and a small peak near 550 nm. We believe that the longer wavelength EL emissions are related to the variable composition and thickness of InxGa1-xN/GaN MQWs coated on different facets of GaN microrods which is typically observed in other multifacetted GaN nanostructured LEDs27. In addition, the current–voltage (I–V) characteristic curve of the LED, as shown in Fig. −4 A at –3 V, indicating the formation of a p–n junction LED.We investigated the light-emitting characteristics of the microstructure LEDs through electroluminescence (EL) spectroscopy. As shown in Fig. xGa1−xN/GaN coaxial quantum nanostructures on graphene films, which exhibited strong blue emission; moreover, the diodes exhibited rectifying behavior. The combination of dimension- and position-controlled GaN quantum nanostructures and 2D graphene films can facilitate the realization of other types of flexible device applications with multi-functional characteristics and novel physical properties.We realized the dimension- and position-controlled growth of GaN micro-LEDs on graphene films by using a ZnO seed layer. The use of a ZnO seed layer helped to enhance the nucleation of GaN on graphene and ensured the formation of GaN microrod arrays. The GaN micro-LEDs had a high crystalline structure and high optical quality, as determined by TEM, PL, and EL measurements. Using the dimension- and position-controlled microstructures, micro-LEDs including diverse quantum nanostructures were fabricated on graphene films, leading to the development of high-resolution three-dimensional micro-LEDs and micro devices on graphene films. In particular, we fabricated flexible GaN/InSupplementary Figures.