Smoking is a risk factor for developing rheumatoid arthritis (RA), but the mechanism remains uncertain. We previously demonstrated that smoking lowers the T cell activation threshold by limiting programmed death protein 1 (PD-1) expression.To investigate how smoking influence the levels of soluble PD-1 ligand (sPD-L1).Serum levels of sPD-L1 were measured in 246 RA patients and in 168 healthy subjects. The analysis was done with respect to inflammation, smoking, treatments, and autoantibody status. The effect of therapeutic TNF-inhibiting antibodies (TNFi) on sPD-L1 was studied in 16 RA patients at their first infliximab infusion. The expression of Fcγ-receptor (FcγR) subclass IIB and IIIA was analyzed with quantitative polymerase chain reaction in peripheral blood mononuclear cells (PBMCs) from 12 RA patients and 15 healthy controls, and in healthy PBMC exposed to IgG containing antibodies to cyclic citrullinated peptides (aCCP).p = 0.038). Other covariates in the regression model were serum levels of IL-1β representing inflammation  and aCCP positivity . First infliximab infusion repressed sPD-L1 (p = 0.023) in patients, and low levels of sPD-L1 were found in patients with early RA treated with TNFi (p = 0.018). Treatment with TNFi was associated with higher sPD-L1 in patients with long disease duration (p = 0.041) and restored levels in smokers. In vitro exposure to aCCP+ IgG suppressed sPD-L1 (p = 0.036), but aCCP+ patients with long disease duration had higher sPD-L1 (p = 0.016). High ratio of the inhibitory FcγR subclass IIB over the stimulatory IIIA resulted in low sPD-L1 release (p = 0.029). Smoking was associated with a higher FcγR IIB/IIIA ratio (p = 0.00062) and lower levels of sPD-L1 (p = 0.013).The negative association between smoking and sPD-L1 in RA patients was established by multiple logistic regression (OR = 0.52, In RA, serum sPD-L1 was related to systemic inflammation and aCCP positivity. Smoking altered the expression of FcγRs and limited sPD-L1 in RA patients, permitting inappropriate T cell responses. Differential regulation of sPD-L1 during the early and late RA may indicate transposition from acute to chronic inflammation. Within the past 5 years, the interest in the T cell co-receptor programmed death protein 1 (PD-1) has grown exponentially. Much of this interest is explained by the success of the first therapeutic antibody targeting PD-1 that was approved by The Food and Drug Administration as a new immunotherapy for malignant melanoma in 2014 . In cancRheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints. Several cases of RA flare have been reported among patients treated with PD-1 blocking antibodies , indicatWith the diverse etiology of RA in mind, it is not surprising that the pathogenesis of RA vary between individuals. A common approach to separate patients with different pathogenic ways is by the presence of autoantibodies rheumatoid factor and anti-citrullinated peptide antibodies (aCCP) in serum . The posSeveral reports indicate an association between PD-L1 and RA pathology. Dendritic cells and monocytes typically express PD-L1, but non-hematopoietic cells and malignant cells may also express PD-L1 . In endoIn the present report, we further explore the effect of smoking on the PD-1/PD-L1 inhibitory system in RA with focus on sPD-L1. For this purpose, we analyze the sPD-L1 levels in serum of RA patients and healthy subjects with known smoking status in relation to the expression of FcγRs in leukocytes of the peripheral blood. We also investigate the role of therapeutic TNFα-inhibiting antibodies and RA-specific aCCP antibodies in the regulation of sPD-L1 production.A total of 246 RA patients visiting the Rheumatology units at the Sahlgrenska University Hospital in Gothenburg and the Northern Älvsborg County Hospital in Uddevalla, Sweden were randomly recruited from the methotrexate (MTX) registers to participate in the study between 2011 and 2013. The demographic characteristics and anti-rheumatic treatment of the study participants at enrollment are summarized in Table Clinical activity of RA was calculated at the time of blood sampling based on the number of swollen and tender joints, erythrocyte sedimentation rate (ESR) and global health assessment of the patient, and the disease activity score (DAS28) was constructed . DAS28 aClinicalTrials.gov NCT03449589.All patients gave written informed consent. The study was approved by the Regional Ethical Evaluation Board in Gothenburg, Sweden. Trail registration: Serum samples of 168 healthy controls were randomly selected from the healthy participants of the West Sweden Asthma Study  to matchInformation about smoking habits, medication, and concomitant diseases were collected using a structured questionnaire at visit from RA patients and through a structured interview of the healthy subjects. We considered the current smokers and the individuals who smoked within the past 25 years as smokers, while never-smokers and individuals who stopped smoking longer than 25 years ago were regarded as non-smokers. The use of moist snuff alone or in combination with smoking was also considered smoking.In total 16 RA patients, 13 females and 3 males with disease duration of 1–32 years, participated in the pilot study of short-term serological effects of infliximab. Clinical characteristics of the patients are shown in Figure g for 15 min, aliquoted, and stored frozen at −70°C until use.Blood samples were obtained from the cubital vein using the vacutainer . All blood samples were centrifuged at 800 × IgG was isolated from serum samples of 3 aCCP-negative and 4 aCCP-positive RA patients. All the aCCP-positive patients were also positive for RF. Isolation was performed using HiTrap Protein A HP  according to manufacturer’s protocol. The IgG was eluted in 5 ml 0.1 M citric acid and the pH was neutralized with 180 µl Tris–HCl pH 9 per ml of the eluted citric acid. The IgG was further concentrated using Vivaspin 6 with 50,000 MWCO PES membranes . Concentration of IgG was determined using NanoDrop spectrophotometer . 100 µl of aCCP positive IgG (10 µg/ml in PBS) was then used to coat the bottom of a 96-well cell culture plate in room temperature over night. We used a direct enzyme-linked immunosorbent assay (ELISA) to confirm adherence of IgG to the plate bottom (see below).in vitro stimulation, we collected blood samples from four healthy women (age 45–54 years). Mononuclear cells were isolated immediately after the blood sampling through gradient centrifugation on Lymphoprep . Cells were washed twice in PBS and suspended in RPMI Medium 1640 + GlutaMAX-1 culture media  supplemented with 10% fetal calf serum , 50 µg/ml gentamicin  and 50 µM β-mercaptoethanol (Gibco by Life technologies). Cells were seeded 1 × 109/ml on the IgG-coated plates and cultured at 37°C with 5% CO2 in the presence or absence of 10 µg/ml lipopolysaccharide . After 72 h, the cell supernatant was collected and cells were lysed in RLT/β2-ME buffer .For 6/ml in RPMI and activated for 1 h at 37°C, 5% CO2 with phorbol 12-myristate 13-acetate , and ionomycin  as described (2-ME buffer for quantitative polymerase chain reaction (qPCR) analysis.For transcriptional analysis of FcγR expression, blood samples were collected from 15 female RA patients  and 12 healthy females . Mononuclear cells were isolated from the peripheral blood immediately after the blood sampling through gradient centrifugation on Lymphoprep (Fresenius Kabi). Antigen-presenting cells were prepared by the consequent depletion of CD4+ cells using Dynabeads CD4 positive isolation kit  and CD8+ cells using Human CD8+ T cell enrichment kit . The sorted cells were counted, the concentration was adjusted to 1–2 × 10escribed . The supSerum levels of sPD-L1 were analyzed in the samples diluted 1:2 in PBS supplemented with 1% bovine serum albumin (1% BSA–PBS) using human PD-L1/B7-H1 DuoSet ELISA . The protocol was optimized with respect to antibody concentration. The mouse anti-human B7-H1 capture antibody was diluted 1:90 and the biotinylated goat anti-human B7-H1 detection antibody was diluted 1:60 in 1% BSA–PBS. Streptavidin conjugated horseradish peroxidase and 3,3′5,5′-tetramethylbenzidine (TMB) was used for development. The absorbance was read at 450/650 nm dual wavelengths with SPECTRAmax 340PC384 microplate reader and SoftMax Pro software . The standard curve prepared with human recombinant B7-H1 and ranging from 0.3 to 2500 pg/ml was used for calculation of the absolute PD-L1 levels in the samples.Serum levels of interleukins (IL) IL-6 and IL-1β were analyzed in 1:2 dilutions with PeliPair reagent set  according to the manufacturer’s instructions. The minimal detection level for IL-6 and IL-1b was 0.2 pg/ml.The levels of aCCP antibodies were measured by the automatic multiplex method using the anti-CCP2 kit . The cut-off level above 3.0 U/mL was set positive by the manufacturers and verified on healthy individuals. For cell stimulation, aCCP content of serum was determined by the Immunoscan CCPlus kit according to the manufacturer’s instruction .F(ab′)2 goat anti-human IgG detection antibody .The coating of cell culture plate with human IgG was confirmed by direct ELISA using a biotinylated , to generate a product spanning two exons to avoid unspecific signal from genomic DNA . RNA concentration was measured using NanoDrop. Complementary (c)DNA was then synthesized using the High Capacity cDNA Reverse Transcription Kit . The gene-specific primers were designedNA Table  and syntwww.graphpad.com) was used for graphical representation of the data. Data were plotted as the median with interquartile range. The Mann–Whitney U-test or the Wilcoxon matched-pair signed rank test was used to compare the differences between the groups. For group-wise comparison, the patients were stratified based on smoking status, and further sub-divided based on treatment with TNFα-inhibiting antibodies. Patients were also stratified for aCCP and disease duration. The Spearman’s rank correlation test was used to calculate r- and p-values of correlating data. A p-value below 0.05 was considered statistically significant.GraphPad Prism v.7 for Mac OS X  to sPD-L1 levels were entered to the regression model that assessed their contribution to the model by the backward Wald method. Only complete cases were analyzed.The association between the dependent variable, sPD-L1, and the independent variables; smoking; IL-6, IL-1β, and ESR (systemic inflammation); DAS28 (disease activity) and disease duration, aCCP and RF (autoantibodies); MTX and TNF-α inhibiting therapeutic antibodies ; sex and age was tested using the Spearman’s correlation, to establish associations, and the Pearson’s correlation, to test linearity. Correlation plots were generated in R version 3.4.1 . Since sOur initial aim was to determine the relationship between the sPD-L1 production and inflammation in a cohort of 246 RA patients. Thus, an explorative analysis of associations between sPD-L1 and several inflammation and disease parameters was conducted. The results are presented in Figure r = 0.13, 95% confidence interval (CI) = −0.0023 to 0.25, and p = 0.048] and IL-1β , but RF was not correlated. The weekly dose of MTX displayed a negative correlation with the sPD-L1 levels , but there was no correlation with TNFi treatment. There was no association between log sPD-L1 and age or sex among RA patients and healthy controls.There was a significant correlation between the logarithm of sPD-L1 and serum levels of IL-6 , meaning that smoking was significantly associated with low sPD-L1 levels when potential confounders were accounted for. Other covariates that significantly contributed to the prediction of sPD-L1 were IL-1β  and aCCP .The independent association between smoking and sPD-L1 levels was analyzed by multiple logistic regression analysis, to control the confounders identified by univariate logistic regression analysis. The variables tested by multivariate analysis were smoking (p = 0.016). sPD-L1 levels remained high in patients with DD > 10 years .Further exploring the association between sPD-L1 and aCCP status, we subdivided the RA patients by their disease duration. The disease duration served as a rough approximation of the exposure time to aCCP. In early RA (DD < 3 years), aCCP-positive and aCCP-negative patients had similar levels of sPD-L1. In established RA (DD > 7 years), aCCP-positive patients had significantly higher levels of sPD-L1 . The production of sPD-L1 was negatively correlated with the aCCP concentration .To study the immediate effect of aCCP on the production of sPD-L1, we conducted an p = 0.018). However, this difference was reversed in patients with DD of 7–9 years. Here, TNFi-treated patients had higher sPD-L1 .We asked if RA-specific antibodies were unique in their ability to induce sPD-L1 in RA patients. To address this, we compared sPD-L1 levels in RA patients with different disease duration by stratifying patients on TNFi treatment status. The studied RA cohort contained 50 patients treated with TNFi. Among them, 38 patients were treated with infliximab, 5 patients with adalimumab, and 7 patients were treated with golimumab. Those 20 patients who were treated with etanercept, a Fc-fusion protein interacting with TNFα receptor, were excluded from the analysis. We observed that patients with DD < 3 years treated with TNFi had significantly lower sPD-L1 . In the 5 patients who left blood samples 2 weeks after the first infusion, 3 patients had elevated levels and 2 patients had suppressed levels of sPD-L1, compared to the pre-infusion sample . A reduction in sPD-L1 levels was measured in 6 of 7 patients after 2 infusions of infliximab at 6 weeks .We next analyzed sPD-L1 in smoking patients with and without treatment with TNFi. We saw that TNFi reversed the suppressive effect of smoking on sPD-L1 levels. Smokers treated with TNFi had sPD-L1 levels similar to those observed in non-smokers not treated with TNFi  FcγRIIIA, and of the inhibitory FcγRIIB, in human PBMC.p = 0.029).To investigate whether the expression of FcγRs was related to the release of sPD-L1, we compared their expression in IgG-stimulated PBMC cultures. We observed a higher expression of both FcγRIIIA and FcγRIIB mRNA in the cell cultures with higher sPD-L1  and FcγRIIB  compared to aCCP-negative healthy controls.Direct cell exposure to aCCP + IgG in culture had no significant effect on FcγR Figure B. Howevep = 0.00062).Finally, we wanted to learn if smoking interfered with the FcγRs expression. For this purpose we analyzed the expression of FcγRs in PBMC enriched with the antigen-presenting cells of 11 smokers and 16 non-smokers. Smokers had somewhat lower expression of FcγRIIIA mRNA, but did not differ from non-smokers in FcγRIIB expression. This resulted in a higher FcγRIIB/FcγRIIIA ratio, characteristic for the inhibitory receptor predominance, in smokers (Figure In the present study, we measured the soluble form of PD-L1 in serum of RA patients. We observed that sPD-L1 was connected to systemic inflammation, and its production was suppressed by MTX treatment. Several attempts were previously made to explain why PD-L1 fails to suppress inflammation in RA. It was suggested that the expression of PD-L1 in the RA synovia is sub-optimal to control joint inflammation . An alteThis study revealed an association between smoking and distinctly low levels of serum sPD-L1 in RA patients. We previously reported that the expression of its receptor, PD-1, was limited by smoking . TogetheTwo aspects need to be considered in the interpretation of low sPD-L1 levels in RA patients. On the one hand, we show that sPD-L1 levels may be low in absence of inflammation, or when inflammation is suppressed by immune modulating treatment. On the other hand, a well-defined function of PD-L1 is to limit the inflammatory response and protect against autoimmunity. Thus, low levels of sPD-L1 could indicate either suppressed inflammation, or insufficient control of the inflammation. To address this issue we conducted a multivariate analysis, which demonstrated that smoking was independently associated with low levels of sPD-L1, and not merely attributed to reduced systemic inflammation. Consequently, smoking is likely to prevent an increase of sPD-L1 levels when protective PD-L1 function is required. We speculate whether the suppression of this essential regulatory system increases the susceptibility to autoimmune reactions and RA. This suggestion is in agreement with the established association between RA and smoking .Anti-CCP antibodies seemingly had a stimulatory effect on sPD-L1 production. However, comparing patients with different disease duration, we observed that aCCP-positive patients in the early phase of the disease did not have higher sPD-L1 levels. We speculate whether the time of exposure to aCCP is of importance for their effect on sPD-L1. Naturally, disease duration is a crude estimation of aCCP exposure, based on the assumption that patients with longer disease duration have been positive for aCCP antibodies for a longer time. Earlier studies indicate that approximately 40% of the patients have aCCP before they develop RA, with a median of 5 years and maximum of 14 years before diagnosis . This imWe concluded that exposure to the IgG antibodies aCCP and TNFi generated similar effects with regards to sPD-L1. We, therefore, anticipated that the production of sPD-L1 was regulated by FcγRs. Indeed, a predominance of the stimulatory FcγRIIIA was found in PBMCs generating high sPD-L1 levels. PBMC subjected to short-term stimulation with aCCP in culture, and PBMC from patients chronically exposed to aCCP, showed opposing effects on their FcγRs transcription. We speculate that chronic exposure to aCCP influences the expression pattern of FcγRs, thereby explaining the differential effect of aCCP on the sPD-L1 production in these two situations. It was previously shown that aCCP levels in RA patients were associated with the protein expression of FcγRIII  and FcγRFinally, we demonstrated that smokers had a higher ratio of FcγRIIB/-IIIA. We propose that the predominance of the inhibitory FcγRIIB repressed sPD-L1 production, offering an explanation to how smoking and antibodies may interact to influence sPD-L1 levels. Possibly, the infusion of the TNFi antibodies provides enough stimulatory signals to override this misbalance in FcγR expression, and normalized sPD-L1 levels in smokers.To conclude, the present study shows that serum levels of sPD-L1 in RA are triggered by systemic inflammation. Smoking limits sPD-L1 response in RA smokers by changing the balance in the expression of stimulatory and inhibitory FcγRs. Activation of FcγR by aCCP antibodies and therapeutic TNFi antibodies has differential sPD-L1 production response in the early and late RA, which may mark a switch from acute to chronic inflammation.This study was carried out in accordance with the recommendations of the Regional Ethical Evaluation Board in Gothenburg, Sweden. The protocol was approved by the Regional Ethical Evaluation Board in Gothenburg. All subjects gave written informed consent in accordance with the Declaration of Helsinki.MB and CW contributed to conception and design of the study. MB, BL, AB, CM, and LE performed clinical evaluation of study participants and collected clinical material. SS recruited patients and organized the database. CW, ME, and RP conducted serological measurements and cell experiments. CW performed the statistical analysis. CW and MB wrote the first draft of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.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.


Amyotrophic lateral sclerosis (ALS) is characterized pathologically by the occurrence of phosphorylated TDP-43 (pTDP-43)-immunoreactive neuronal and glial inclusions in the central nervous system. Recent studies have shown that pTDP-43 aggregates also occur in the skeletal muscles in a certain proportion of ALS patients.The aim of this study was to clarify the distribution and incidence of pTDP-43 aggregates in the skeletal and cardiac muscles of patients with ALS, and also those of patients with neuromuscular diseases (NMDs) and non-NMDs.n = 30), NMDs (n = 13) and non-NMDs (n = 7).Five regions of muscle  were examined histologically and immunohistochemically in patients with ALS . The mean density of pTDP-43 aggregates in ALS was significantly higher than that in NMDs and non-NMDs. In contiguous sections stained with hematoxylin and eosin and anti-pTDP-43, muscle fibers with dense filamentous inclusions demonstrated single-fiber atrophy with vacuolar degeneration.The present findings indicate that pTDP-43 aggregates in skeletal and cardiac muscle are a myogenic pathological marker in multiple diseases including ALS. Amyotrophic lateral sclerosis (ALS) is characterized pathologically by loss of upper and lower motor neurons with consistent occurrence of phosphorylated TDP-43 (pTDP-43)-immunoreactive neuronal and glial inclusions in the central nervous system , 15. AltHowever, TDP-43 aggregates have been described in the skeletal muscles from patients with various diseases, including sporadic inclusion body myositis (IBM) , 26, ALSRecently, Cykowski et al. demonstrated pTDP-43 aggregates in the skeletal muscles of 19 among 57 patients (33.3%) with ALS, including sporadic and familial cases, being more common in the axial muscles (paraspinous muscles and diaphragm) than in the appendicular muscles . Among 2The growing list of reports on TDP-43 aggregates in the skeletal muscles from patients with ALS and various NMDs prompted us to investigate the significance of pTDP-43 aggregates as a pathological marker in muscle tissue. Using immunohistochemistry, we examined the skeletal and cardiac muscles of patients with sporadic ALS, and also patients with NMDs and non-NMDs. Here we report that pTDP-43 aggregates are present in both skeletal and cardiac muscles in various conditions, including ALS as well as NMDs and non-NMDs, and that these aggregates are associated with myogenic muscle degeneration.n = 16), NMDs , and non-NMDs   were examined to investigate whether pTDP-43 aggregates are present in cardiac muscle; these included cases of sporadic ALS , NMDs , and non-NMDs  ; these included cases of sporadic ALS , native TDP-43  and p62 . The sections were pretreated in an autoclave for 15 min in 10 mM citrate buffer (pH 6.0).To evaluate whether pTDP-43 aggregates are proteinase K (PK)-resistant, PK  in PK buffer  at 37 °C for 10 min was applied to selected sections.2 of section; “2”, detectable in 1–2 pTDP-43 aggregates per 1 cm2 of section; and “3”, detectable in > 2 pTDP-43 aggregates per 1 cm2 of section).We developed a semi-quantitative scale to score the density of pTDP-43 aggregates in skeletal and cardiac muscles. The total number of pTDP-43 aggregates was quantified in each section. The entire regions were surveyed at × 200 magnification using an eyepiece graticule and parallel sweeps of the microscope stage. We measured the whole area of each section using Image J software provided by the National Institutes of Health and calculated the density of pTDP-43 aggregates in each section  was investigated in each section.To determine whether pTDP-43 aggregates are more common to be found in ALS than in non-ALS groups (NMDs and non-NMDs), Kruskal-Wallis and Steel-Dwass tests were applied to differences in the density of pTDP-43 aggregates between the groups. To determine which region is more vulnerable to pTDP-43 pathology, Quade and Steel-Dwass tests were applied to differences in the density of pTDP-43 aggregates between the five muscle regions. Calculations were performed using Statcel software .As for ALS cases in Group B, the presence or absence of TARDBP and C9ORF72 gene mutations was analyzed in 29 cases for which frozen tissue samples were available (other than B-30) as described previously . ALS casImmunostaining with anti-pTDP-43 antibody revealed pTDP-43 aggregates in fibers of skeletal muscles  and cardiac muscle. Two types of pTDP-43 aggregates were distinguishable morphologically: dense filamentous Fig. a-d and spTDP-43 aggregates were found in at least one region of skeletal or cardiac muscle in 5 of 16 cases of ALS (31.3%), 3 of 5 cases of NMDs (60%), and 3 of 6 cases of non- NMDs (50%) in Group A  were examined Table . pTDP-43Since the incidence of pTDP-43 aggregates in ALS was higher than that in NMDs and non-NMDs, we statistically compared the density of pTDP-43 aggregates between these three groups. The mean density of pTDP-43 aggregates in ALS was significantly higher than that in NMDs and non-NMDs Fig. a. The meWe further compared the density of pTDP-43 aggregates in five types of muscle. The mean density of pTDP-43 aggregates in the diaphragm was significantly higher than that in the cervical muscle and myocardium in ALS  were evident in the skeletal muscles in all cases. No myogenic pattern was noted in skeletal muscles, and no obvious alterations were seen in the myocardium.In the patients with NMDs, myogenic pathology  was evident in the skeletal muscles. Moderate to marked fibrosis was seen in the myocardium in 3 cases  and mild fatty degeneration was evident in a case (case B-38).Among the patients with non-NMDs, mild neurogenic atrophy was seen in the skeletal muscles in a case (case B-44). No obvious changes were evident in the skeletal or cardiac muscles in positive cases .In contiguous sections of the skeletal muscles and myocardium stained with HE and anti-pTDP-43, muscle fibers showing single-fiber atrophy contained pTDP-43-positive short linear inclusions and/or dense filamentous inclusions Fig. a-d. DensTDP-43 (TARDBP) gene mutation or C9ORF72 repeat expansion was not observed in 29 cases of ALS in Group B.Recently, Cykowski et al. reported the presence of pTDP-43 aggregates in axial (paraspinous and diaphragm) muscles and appendicular (deltoid and quadriceps) muscles in 19 of 57 patients (33.3%) with sporadic and familial ALS, including C9ORF72 expansion-associated ALS . In the Cykowski et al. reported that similar aggregates were also present in patients with IBM, but not in non-ALS patients with neurogenic muscular atrophy . HoweverIn the present study, we have demonstrated that the mean density of pTDP-43 aggregates in ALS was significantly higher than that in NMDs and non-NMDs. Since TDP-43 is an RNA binding protein ubiquitously expressed in all tissues, it is possible that cytoplasmic TDP-43 aggregates may occur in the degenerating or regenerating muscle fibers. Vogler et al. suggested that increased assembly or decreased clearance of functionally normal myo-granules, one of stress granules in myofibers, could be the source of cytoplasmic TDP-43 aggregates in regenerating muscle fibers . HoweverTo our knowledge, this is the first time that pTDP-43 aggregates have been reported in the myocardium, being present in patients with ALS (12 of 30 cases), NMDs (4 of 13 cases) and non-NMDs (1 of 7 cases). Since atrophy of the myocardium is not a neurogenic process, the presence of pTDP-43 aggregates in cardiac muscle may imply the involvement of a myogenic mechanism. With regards to the abnormal protein deposition in cardiac tissues of neurodegenerative diseases, phosphorylated α-synuclein aggregates are observed in the cardiac sympathetic nerves, but not in the cardiac myofibers, in Lewy body diseases . In the The present study revealed two types of pTDP-43 aggregates (dense filamentous and short linear inclusions) in skeletal and cardiac muscles. Both types were also immunopositive for nTDP-43 and p62. These immunohistochemical features are similar to those of pTDP-43 aggregates (skein-like and round inclusions) found in the central nervous system of ALS patients , 13, 14.2 and the size of pTDP-43 aggregates in our samples was very small. Judging from the light microscopic findings reported by Cykowski et al. [The density of pTDP-43 aggregates in skeletal and cardiac muscles was very low in our samples, for example 1–2 aggregates per 1 cmi et al. , pTDP-43The present findings suggest that pTDP-43 aggregates in skeletal and cardiac muscle are a marker of myogenic degeneration in amyotrophic lateral sclerosis and various conditions associated or unassociated with NMDs.


Recent advances in graphene and other two-dimensional (2D) material synthesis and characterization have led to their use in emerging technologies, including flexible electronics. However, a major challenge is electrical contact stability, especially under mechanical straining or dynamic loading, which can be important for 2D material use in microelectromechanical systems. In this letter, we investigate the stability of dynamic electrical contacts at a graphene/metal interface using atomic force microscopy (AFM), under static conditions with variable normal loads and under sliding conditions with variable speeds. Our results demonstrate that contact resistance depends on the nature of the graphene support, specifically whether the graphene is free-standing or supported by a substrate, as well as on the contact load and sliding velocity. The results of the dynamic AFM experiments are corroborated by simulations, which show that the presence of a stiff substrate, increased load, and reduced sliding velocity lead to a more stable low-resistance contact. The exceptional electrical, thermal, chemical, mechanical, and optical properties of two-dimensional 2D) materials are continuing to find new applications in many different fields ,2,3,4. OD materiaMultiple efforts have been focused on understanding the effect of contact geometries and material properties on electrical contact at nano- and micro-scale junctions . Signifi2 surfaces, due to less interference of confined charges by the adjusted media [Conductive atomic force microscopy (AFM) is one of the traditional techniques for characterizing the electrical properties of surfaces . For 2D ed media .Here, using conductive AFM complemented by atomistic simulations, we explore how the electrical dynamic contact between graphene and a noble metal is affected by the contact load, substrate material and the sliding velocity. The major aspect distinguishing this work from previously reported graphene/metal contact studies ,34 is ou−1) and G peak (at ~1560 cm−1) confirm the single-layer nature of the film.To characterize changes in electrical contact, AFM measurements a,b were 3N4 sample using a conductive silver paint. This sample geometry was created to allow the electrical current to travel from the metal tip to the graphene, and then laterally across the graphene film to the contacting pads. The electrical resistance of the contact pads and silver paint used for grounding was confirmed to be less than 1 ohm, which is much less than the >500 ohms measured from the AFM experiments. Therefore, the contribution of resistance due to grounding could be eliminated from the measurements, and the effect of the dynamically actuated contact between the AFM tip and the graphene sample monitored directly.The samples were attached to the insulating quartz substrate using a ceramic paste, and the grounding connection was made on the edge of the graphene-on-SiAFM measurements were performed in ambient atmosphere conditions (relative humidity 30%) using a Bruker Multimode AFM in contact mode with a conductive platinum/iridium tip (spring constant of 0.1 N/m). Specifically, two types of measurements were performed. First, the evolution of the electrical contact between the Pt/Ir tip and free-standing and supported graphene was tested in static mode while increasing the applied load. The maximum load was limited to 13.5 nN, which remained well below critical loads required for free-standing CVD grown graphene rapture (order of 2000 nN) or for inelastic deformation under AFM nanoindentation ,37. The Although the supported and free-standing graphene exhibit some similar behaviors, there are differences between these two cases. Most significantly, a larger load is required to obtain substantial current on the free-standing graphene. For example, we observe significant current flow for the supported graphene at a load of 0 nN, while the free-standing graphene requires a load 8.1 nN to achieve this. We hypothesize that this difference is due to the stability of the contact, which is poorer for the free-standing graphene, due to the flexibility of the material.In order to further explore the stability of the metal/graphene contact, we performed dynamic electrical current mapping by scanning the conductive tip on the graphene surface, both on the supported and free-standing graphene areas. In the case of the free-standing graphene, the scanning area was selected to be close to the center of the membrane (100 nm × 100 nm). The load was fixed at 5.4 nN and the voltage bias at 0.5 V. The results demonstrate a similar trend of lower conductivity  on free-standing graphene than supported graphene compare a,c. AlsoTo investigate our hypothesis that the difference between the contact conductance for free-standing and supported graphene is due to the contact stability, we developed atomistic models of these two systems. The models consisted of a diamond tip  and a graphene layer that was either suspended by a gold substrate with 12 nm diameter hole (free-standing graphene membrane) or supported by a gold substrate without hole (supported graphene), shown in 0 = 0.00832 eV, r0 = 0.387035 nm and α = 1.25707. The Lennard–Jones potential was used to model interactions between C atoms in the tip and the graphene layer, where the parameters were taken from [The embedded-atom method (EAM) potential was applied to simulate the Au–Au interactions  and the ken from .The initial separation between the bottom of the tip apex and the fixed edge of the graphene layer in the z-direction was approximately 0.9 nm, a distance at which the interaction force between the tip and graphene layer was negligible. The model system was first equilibrated at 300 K for 20 ps. The temperature was controlled by applying Nosé–Hoover thermostat to all the non-constrained atoms. The tip was then moved at a speed of 10 m/s towards the graphene layer. At different vertical positions, the tip movement was stopped and the system was relaxed for 300 ps.The interaction force between the tip and graphene as a function of relaxation time for the tip at a representative vertical position is shown in In conclusion, the effect of the substrate on the electrical performance of graphene/metal contact at a range of applied contact loads was investigated. Our results demonstrate more stable contact when graphene is supported by the substrate. When the support for the graphene is removed, as in case of free-standing graphene, the contact loses stability and contact resistance increases. This was observed in both static and dynamic contact measurements. For static contacts, the contact resistance for free-standing graphene at low contact loads was on average 5 times larger than that of supported graphene. For dynamic contacts, the free-standing graphene contact resistance was up to 4 times larger at slow 50 nm/s scanning, and more than 10 times larger at faster 100 nm/s scanning, when compared to the substrate-supported graphene. To test the hypothesis that the stability of the contact could explain the difference between the conductivity on free-standing and supported graphene, we developed atomistic models of these two systems. The simulations results indicated that the same normal load could be achieved with constant contact for the supported graphene and intermittent contact with the free-standing graphene. This behavior could correspond to lower conductivity at the same load, as observed in the experiment. Overall, our study emphasizes the important role of the contact stability in determining graphene/metal electrical conductance, where the substrate can have a pronounced effect on stability and therefore conductance of such contacts. This study also highlights the effects of contact mechanical loading and sliding velocity on current transport at graphene/metal contacts, which are critical for applications with dynamic or periodically actuated electrical contacts.


L1) with a t-butyl pyrrole moiety as the recognition site was designed and synthesized. Photophysical studies show that L1 exhibits excellent sensitivity and selectivity towards Cu2+ to other metal ions in neutral acetonitrile aqueous media. Mechanism studies suggest that the recognition process may associate with a Cu2+ promoted hydrolysis reaction of L1. Furthermore, L1 has been successfully applied in fluorescence imaging of Cu2+ ion in living cells.The development of fluorescent methods for the detection of metal ions is of great importance due to their diverse environmental and biological roles. Herein, a rhodamine 6G-based off-on fluorescent probe ( For example, copper is integrated into various proteins and metalloenzymes that perform basic metabolic functions [The exploration of detection methods for environmentally and biologically important metal ions is of great interest to researchers currently , 2. Amonunctions . Copper unctions . Howeverunctions –8. Hence2+ was usually detected through fluorescence quenching of chemical sensors [Traditionally, inductively coupled plasma atomic emission spectrometry (ICP-AES), atomic absorption spectroscopy (AAS), and inductively coupled plasma mass spectrometry (ICP-MS) are used to analyze copper ions –11. Howe sensors , 23, whi sensors –29. In a sensors –40.t-butyl pyrrole moiety into the rhodamine fluorophore to form a turn-on fluorescent probe L1 for the detection of Cu2+. The molecular structure of L1 was verified by 1H NMR, 13C NMR, and MS spectra. With the addition of Cu2+ to L1 in a neutral CH3CN/H2O solution, metal-triggered ring-opening reaction of the spirolactam in L1 took place, resulting in sensitive colorimetric response and fluorescence emission. Mechanism studies suggest that the detection process may associate with a Cu2+ promoted hydrolysis reaction of L1. Furthermore, fluorescence microscopy experiments demonstrated that L1 could be used to image Cu2+ in living cells.Taking into account the criteria mentioned above, we herein incorporated a All chemicals are purchased commercially and used directly without further purification. Deionized water was used throughout, and the pH was adjusted using diluted sodium hydroxide solution or hydrochloric acid. The pH value was measured with a Rex pHS-3E pH meter. Silica gel (200–300 mesh) was used for column chromatography. NMR spectra were obtained with a 600 MHz Bruker spectrometer, and tetramethylsilane (TMS) was used as the internal standard. High-resolution mass spectra were measured on an Agilent LCMS 6500 spectrometer. Absorbance spectra were measured on a Shimadzu UV-3101PC spectrometer. Measurements of fluorescence spectra were performed on a Shimadzu RF-5301PC spectrometer. Both excitation and emission slit widths were set at 3 nm. All experiments were operated at about 298 K.L1 (2 × 10−4 M) was prepared in CH3CN. Stock solutions of metal ions (2 × 10−3 M) were prepared in deionized water from their chloride salts or nitrate salt (Ag+). Working solution of L1  was freshly prepared prior to spectroscopic experiments by diluting the high-concentration stock solution. In the sensing experiments, each time, a 3 mL working solution of L1  was put in a quartz optical cell of 1 cm optical path length, and appropriate amounts of stock solutions of metal ions were added by a pipette. Spectral data were collected 2 min after the addition of the ions.A stock solution of probe tert-butylpyrrole-2-carbaldehyde (3.3 mmol) were dissolved in 30 mL methanol. After addition of 0.1 mL acetic acid, the mixture was stirred and heated to reflux for 16 h. A pale yellow solid obtained was filtered off and washed using cold methanol. The solid was dried in vacuum and further purified by column chromatography . Yield: 88.6%. 1H NMR , δ (ppm): 10.93 , 8.16 , 7.84 , 7.50 , 6.94 , 6.30 , 6.18 , 6.05 , 5.77 , 5.04 , 3.12 , 1.85 , 1.20 . 13C NMR , δ (ppm): 163.12, 151.96, 150.64, 147.66, 146.61, 141.63, 133.12, 128.41, 128.31, 126.64, 126.50, 123.24, 122.60, 118.11, 112.32, 105.03, 104.64, 95.91, 65.00, 37.44, 31.38, 29.98, 16.96, 14.13. HRMS: m/z calculated for M+, C35H39N5O2, 561.3104. Found: 562.3192 (M + 1).To a 100 mL flask with three necks, rhodamine 6G hydrazine (2.8 mmol), which was synthesized according to reported methods , and 5-t2. Before the experiments, the MCF7 cells were washed with PBS and then incubated with 10 μM L1 at 37°C for 30 min. After removal of excess probe and washed with PBS, the MCF7 cells were incubated with 20 μM CuCl2 for another 30 min. The MCF7 cells were rinsed with PBS again and live-cell imaging was conducted using an EVOS FL Auto microscope.The human breast adenocarcinoma cells MCF7 were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C under an atmosphere containing 5% COL1 was easily synthesized from rhodamine 6G through a two-step reaction , L1 was obtained in an 88.6% yield. The molecular structure was verified by 1H NMR, 13C NMR, and MS spectra  in neutral CH3CN/H2O  solution is shown in L1 existed with the structure of spirolactam. Once the solution of Cu2+ was added, a new peak was detected at 525 nm. With the increase of Cu2+ concentration, the intensity of the peak was gradually enhanced, which could be interpreted as the transform from a spirolactam structure to a ring-opened form of L1. Correspondingly, the color of the solution changed from colorless to purple, which help to achieve the naked-eye recognition of Cu2+ ion. As shown in 2+, the absorption value of L1 (10 μM) was found to increase linearly in the range of 2 μM–14 μM at 525 nm. The selective sensory studies of L1 (10 μM) in neutral CH3CN/H2O solution were then extended to other metal ions . When 1 equiv metal ions were added into the relevant solution, only Cu2+ could induce a purple color and an obvious increase of absorbance at 525 nm  was also investigated. When no Cu2+ was added, the free L1 solution had little fluorescence at the excitation wavelength of 500 nm due to the spirocyclic form of its molecular structure. However, similar to the results of the absorption experiments, once the solution of Cu2+ was added, an obvious increase of the fluorescence intensity at 545 nm (L1 (10 μM) was linear with the concentration of Cu2+ in the range of 2 μM–26 μM, and the detection limit of Cu2+ was calculated as 0.38 μM (S/N = 3). The fluorescence quantum yield was calculated to be 0.19 in the presence of Cu2+ ion (26 μM) by using rhodamine 6G as the standard (Φf = 0.94 in ethanol) [L1 towards Cu2+ over other cations was satisfactory. On excitation at 500 nm, no obvious spectral change of L1  was observed with the addition of Cr3+, Al3+, Fe3+, Ca2+, Ba2+, Co2+, Fe2+, Hg2+, Mg2+, Mn2+, Ni2+, Pd2+, Zn2+, Ag+, Li+, K+, Na+, and Sn4+ (10 μM). In contrast, more than 500-fold fluorescence intensity increase was detected when the same amount of Cu2+ ion was added  . The selas added . In orde2+ of L1 was studied. As shown in L1 between pH 4.0 and 8.0, indicating that the spirolactam structure still dominated in this pH range. However, with the addition of Cu2+, fluorescence change of L1 was observed with different fluorescence enhancement efficiency under different pH values  Na2EDTA was added to the solution of L1 in neutral CH3CN/H2O solution  containing Cu2+ ion (10 μM). No obvious decrease in the fluorescent intensity or color change was observed after the addition, indicating that the detection of Cu2+ ion by L1 was an irreversible process , indicating rhodamine 6G as a final product (2+ into the solution of L1 in pure CH3CN resulted in no fluorescent emission or color change. Based on these experimental results, a mechanism involving Cu2+-promoted redox hydrolysis of L1 was proposed (2+ by rhodamine B hydrazide [In order to illustrate the interaction mechanism between Cu process . HRMS ex product . Moreoveproposed , which wydrazide .L1 in the detection of Cu2+, fluorescence imaging experiments were carried out in MCF7 cells. The MCF7 cells were incubated with L1 for 30 min and washed with PBS. As shown in 2 at the same conditions for another 30 min, strong fluorescence in the MCF7 cells was observed (L1 could pass through the cell membrane and be used for the detection of Cu2+ in living cells. The cytotoxicity tests were studied by MTT assay with the concentrations of L1 from 0 to 30 μM (L1 for 24 h at 37°C, indicating that L1 has low cytotoxicity to cells in this dosage range.For further studying the practical application of observed . These rto 30 μM . ExperimL1 for Cu2+ was developed through the combination of rhodamine 6G and a pyrrole moiety. As a turn-on fluorescent probe, L1 exhibited excellent sensitivity and selectivity for Cu2+ detection in acetonitrile aqueous media with a low detection limit. Moreover, fluorescence bioimaging experiments confirmed that L1 could be used to detect intracellular Cu2+ in living cells.In summary, a new colorimetric and fluorescent probe


In sharp contrast to monolayer graphene, bilayer graphene is expected to show radiation-induced magnetoresistance oscillations more similar to the results observed in the GaAs/AlGaAs 2D system. Such expectations for the radiation-induced magnetoresistance oscillations are presented here to guide future experimental studies in both of these modern atomic layer material systems.We examine the characteristics of the microwave/mm-wave/terahertz radiation-induced magnetoresistance oscillations in monolayer and bilayer graphene and report that the oscillation frequency of the radiation-induced magnetoresistance oscillations in the massless, linearly dispersed monolayer graphene system should depend strongly both on the Fermi energy, and the radiation frequency, unlike in the case of the massive, parabolic, GaAs/AlGaAs 2D electron system, where the radiation-induced magnetoresistance oscillation frequency depends mainly on the radiation frequency. This possible dependence of the magnetoresistance oscillation frequency on the Fermi level at a fixed radiation frequency also suggests a sensitivity to the gate voltage in gated graphene, which suggests an  Under microwave/mm-wave/terahertz photoexcitation, high mobility 2D electron systems confined, for example, within GaAs/AlGaAs heterostructures exhibit large amplitude “1/4 cycle shifted” magnetoresistance oscillations with resistance maxima in the vicinity of E = (j + 3/4)ħωc, resistance minima near E = (j + 1/4)ħωc, and nodes in the resistance oscillations in the vicinity of the cyclotron resonance, and integral and half-integral cyclotron resonance harmonics, i.e., E = jħωc and E = (j + 1/2)ħωc. Here, E = energy, ωc = eB/m*, e = electron charge, m* = electron effective mass, ħ = the reduced Planck constant, and B = the magnetic field. Most remarkably, at the lowest temperatures under modest photo-excitation, the deepest resistance minima saturate into zero-resistance states2, about B = 4/5Bf and B = 4/9Bf, of the characteristic field Bf = 2πfm*/e, where f is the electromagnetic-wave frequency83. That is, experiments suggest that the diagonal resistance can be switched off in the 2D electron system by photo-excitation in the presence of a small magnetic field. Such experimental reports have motivated the theoretical study of transport in photo-excited 2D semiconductor specimens83. Large amplitude magnetoresistance oscillations under photoexcitation also imply strong electrical sensitivity to electromagnetic waves, which could potentially be applied towards the realization of microwave/mm-wave/terahertz photodetectors87. Indeed, since the observations thus far have indicated Bf = 2πfm*/e, the implication is that radiation frequency f = eBf/2πm* can be determined from the characteristic field Bf of the magnetoresistance oscillations. The concurrent observed sensitivity to both the radiation-intensity and frequency has suggested the possibility of a tuned narrow band radiation sensor in the microwave/mm-wave/terahertz bands25.Carrier scattering gives rise to electrical resistance – a measure of frictional losses within semiconductor specimens55, strained Si/SiGe88, electrons on the surface of liquid Helium89, and the oxide MgZnO/ZnO 2DES system90 for scattering lifetimes, and effective masses. Thus, one expects that photo-excited magnetotransport studies of modern atomic layer 2D systems such as monolayer and bilayer graphene could potentially unveil new science and applications99. However, the expectations for device response of these materials is not known and such phenomena have not been observed thus far in the graphene system. As a consequence, a question of interest is: what will be the oscillatory resistance response of modern atomic layered 2D materials such as, for example, graphene under electromagnetic wave excitation in a magnetic field? Below, we examine the expected characteristics for both the monolayer and bilayer graphene systems. As mentioned above, our results suggest a strong sensitivity of the response in monolayer graphene to both the Fermi energy and the radiation frequency. However, bilayer graphene is expected to show a more GaAs/AlGaAs like behavior, characteristic of parabolically dispersed systems. The results are summarized below.Above mentioned radiation-induced magnetoresistance oscillations have already served to characterize material systems such as GaAs/AlGaAs heterostructuresEN = sgn(N)(2ħeBN)1/2vF98. Here, N is the Landau level index, and vF is the carrier velocity. Suppose that the Fermi level lies in the Nth Landau level, EN = EF, and the electromagnetic radiation induces transitions from N’th Landau level to the N + q’th Landau level61, such that EN + q = EF + hf, where h is Planck’s constant, and f is the radiation frequency. From studies of the GaAs/AlGaAs system2, it is known that nodes in the radiation-induced magnetoresistance oscillations appear when the radiation energy spans such an integral number of Landau levels, i.e., EN+q − EN = hf, where q denotes the order of the node. Consider It is well known that the Landau level dispersion for Dirac fermions in monolayer graphene is given by EN+q − EN = hf and EN+q + EN = 2EF + hf, Then, by substituting into Eq. Apply the identity mentclass2pt{minimq’th node of radiation-induced magnetoresistance oscillations in monolayer graphene and it suggests that such oscillations in monolayer graphene are also dependent upon value of the Fermi energy, EF, unlike in the GaAs/AlGaAs system41. In Fig. B, vs. the inverse of integers, 1/q, for several typical values of the Fermi energy in monolayer graphene. The figure shows a set of straight lines, which indicates that the expected magnetoresistance oscillations are periodic in B−1, just as in the GaAs/AlGaAs system. The characteristic frequency or field, Bf, of the radiation-induced magnetoresistance oscillations that appears in the empirical formula for the oscillatory magnetoresistance lineshape ΔR ≈ −exp(−λ/B) sin (2πBf/B)11, is found from the slopes in Fig. B vs. 1/q, which suggest increases of Bf with EF, at a fixed radiation frequency, f = 100 GHz. The inset of Fig. Bf of the radiation-induced magnetoresistance oscillation with EF at several radiation frequencies, f = 25, 50 100, 200 and 400 GHz. The inset conveys that Bf increases faster with EF at larger radiation frequencies f. Note that Bf increases linearly with f over this range of f since EF ≫ hf, see eqn. f = 100 GHz, hf = 4.125 × 10−4 eV is much smaller than a small practical value for EF such as EF = 5 × 10−3 eV.Thus, Eq. R ≈ −exp(−λ/B) sin (2πBf/B)11, in the monolayer graphene system are illustrated in Fig. R vs. B at EF = 0.5, 0.25, and 0.166 eV for photoexcitation at f = 100 GHz. Since the characteristic field Bf increases with EF, and the parameters B, f, e, and vF in Eq. EF.The expectations for the oscillatory resistance, ΔB, or, as is more typical for graphene, vs. the gate voltage VG. In monolayer graphene, the carrier density, nq, varies as the square of the Fermi energy, i.e., nq = (4π/h2)(EF/vF)2. Then,From the experimental perspective, measurements can be carried out as a function of the magnetic field, SiO2 on doped Si the relation between the gate voltage and the carrier density is nq = sgn(ΔVG)α|ΔVG| with α = 7.2 × 1010 cm2/V98. Upon inserting these relations, we obtain from eqn. Further, for graphene on top of 300 nm Bf of the magnetoresistance oscillations vs. the electron density, i.e., nq = n, and vs. the difference in the gate voltage with respect to the neutrality voltage, VN, i.e., ΔVG = VG − VN. The square root dependence observed in Eqs BF with respect to these parameters at a fixed radiation frequency, f, i.e., Bf ≈ n1/2 and f = 200 GHz. The Fig. B. Finally, the spacing between, say, successive oscillatory minima, increases with increasing ΔVG because the magnetoresistance oscillations are actually periodic in Bf of the magnetoresistance oscillations upon the Fermi energy when EF ≫ hf even at a fixed radiation frequency in monolayer graphene )1/2ω0B, where ω0 = eħ/m*, and m* = 0.037 m is the nominal effective mass of the carriers98. Here, the + and − describe the response of the electron and the hole systems, and N can take on positive and negative integers for electrons and holes, respectively. To determine the expected response for the radiation-induced magnetoresistance oscillations in bilayer graphene with electrons, we suppose that at a magnetic field, B, the Fermi level lies in the Nth Landau level, EN = EF, and the electromagnetic radiation induces transition from Landau level N to the N + q Landau level so that EN+q = EF + hf. Further, assume that nodes in the radiation-induced magnetoresistance oscillations will appear when the radiation energy spans an integral number of Landau levels, i.e., EN+q − Eq = hf, where q denotes the order of the node. To extract the characteristics for radiation-induced magnetoresistance oscillations in such a system, we examine:Unlike in monolayer graphene, charge carriers in bilayer graphene are massive fermions as a consequence of the parabolic band structure. Bilayer graphene is zerogap system in the absence of a transverse electric field and it develops a bandgap under a transverse electric fieldBy setting EF. Figure q vs. B for different values of the EF at f = 100 GHz for bilayer graphene. The remarkable feature in this figure is the relative insensitivity of the results to the value of the Fermi energy for bilayer graphene, unlike in the case of monolayer graphene (see Fig. Equation see Fig. . Thus, iB-positions, which are insensitive to EF in bilayer graphene, are examined for different f in Fig. q = 1, 2, 3…, shifts to higher B as f increases. Further, the frequency or characteristic field Bf of the radiation-induced magneto-resistance oscillations increases linearly with f in the limit where EF ≫ hf, as indicated in the inset of Fig. Bf-field should shift at the rate of 1.37 mT/GHz in bilayer graphene vis-à-vis the ≈2.35 mT/GHz shift observed in the GaAs/AlGaAs system. These calculations have assumed a fixed effective mass of m* = 0.037 m for bilayer graphene, while it is known that non-parabolicity could provide for variation in m* with EF98. Thus, studies of radiation induced magnetoresistance oscillations in bilayer graphene as a function of the carrier density could serve to characterize the non-parabolicity and determine the effective mass as a function of the energy.The expected nodal EF dependence of the radiation-induced oscillatory magnetoresistance characteristics. For monolayer graphene, the oscillations depend strongly on EF and therefore also the ΔVG. On the other hand, for bilayer graphene, there is a lack of sensitivity to EF and therefore also on ΔVG. This suggests that the characteristics of the observed magnetoresistance oscillations could also serve to differentiate between these two types of graphene.In comparing the expectations for monolayer and bilayer graphene, the striking feature is the great dissimilarity in the


However, the low carrier concentration and high resistance caused by vacancy defects, grain boundaries, and superposed folds in typical graphene films limit its application. In this study, we propose a method to increase both the conductivity and carrier concentration in single-layer graphene (SLG) by blending it with silver nanowires (AgNWs). AgNWs provide connections between grain boundaries of graphene to improve charge-carrier transport. The AgNWs in this study can reduce the resistance of SLG from 650 Ω/◻ to 27 Ω/◻ yet still maintain a transmittance of 86.7% (at 550 nm). Flexible organic light-emitting diode, with a maximum 15000 cd m Indium tin oxide (ITO) has been the most popular TCE for decades because of its excellent photoelectric properties. However, with the development of science and technology and increasing human needs, the trend for future electronic equipment is to became flexible, wearable and manufactured at low cost11. Because ITO is subject to potential shortages of indium and tends to be brittle i.e. non-flexible, there is a need for suitable alternative transparent electrode materials. In the past, the potential candidate materials such as metallic nanowires13, conductive polymers14, carbon nanotubes16 and graphene18 have been investigated. Among these, single-layer graphene (SLG), with its extremely high carrier mobility (>103 cm2·V−1·s−1) and transmittance (97%), represents perhaps the most promising alternative. However, large-area growth and transfer of graphene is difficult to accomplish efficiently. Many defects can occur, such as point defects, impurity defects on graphene sheets20 and grain boundaries21. Moreover, with massive boundaries, a single graphene is isolated into nanosheets. These defects and overlaps between graphene sheets can cause high resistance in SLG films. In order to improve its conductivity, several procedures, like stacking22, acidification23, chemical doping24 and physical mixing with other conductive materials25, were studied in the past. Han et al. successfully fabricated efficient white OLEDs using a four-layer stacked graphene anode, respectively, which was doped with AuCl3 and HNO36. Zhang et al. modified SLG with Al-TiO2 and used graphene/Al-TiO2 composite film as the cathode in organic solar cells26. Seo et al. used graphene-silver nanowire (AgNWs) hybrid structure as electrode in ultraviolet light emitting diodes27. Xu et al. used graphene–AgNWs hybrid films as electrodes for loudspeakers28. However, they paid more attention to the application of this structure, and did not deeply explore the interaction between the AgNWs and graphene and the principle of AgNWs to improve the conductivity of graphene. Dong et al. also studied the graphene-AgNWs structure and applied structure to OLED29. However, they also did not study the mechanism in depth, and their device performance had much room for improvement, the luminance was below 5000 cd m−2 at 13 V.Transparent conductive electrodes (TCEs) are important components in optoelectronic devices, such as touch-screen displays−2.In our work, we blend graphene with AgNWs to take advantage of beneficial composite structures of one-dimensional and two-dimensional materials to improve the electrical properties of graphene. AgNWs with very high length-diameter ratios were used to ensure a good combination of optical and electrical properties. The primary purpose of the nanowires is to reduce the effect of grain boundaries and overlap defects between graphene sheets by providing additional conductive pathways. Simultaneously, the carrier concentration of the SLG film can be increased. We also studied the interaction mechanism between graphene and AgNWs and considered the percolation theory of binary systems. To ensure suitability for optoelectronic devices, several flexible, green, organic light-emitting devices were fabricated with this novel transparent and flexible composite electrode. These devices reached a luminance of over 15000 cd m−1, 1592 cm−1 and 2684 cm−1. The G peak is characteristic main peak of graphene, which is caused by the in-plane vibration of carbon atoms (sp2). The 2D peak is the two-phonon Raman peak, which is used to characterize the stacking mode of carbon atoms in graphene samples. The D peak is commonly seen when defects are present in carbon materials. The normalized intensity ratio of the D and G peaks (ID/IG) can be used as an indicator of defect quantity. The normalized intensity-ratio of the 2D and G peaks (I2D/IG) can identify single-layer graphene (SLG). Figure D/IG, which means there are not many defects, and the I2D/IG ≈ 2 means the graphene we used was SLG30.Prior to fabricate the composite electrode, we first studied the graphene film via Raman spectroscopy. Figure Optical transmittance is a key property of transparent electrodes. Transmittance spectra of SLG and SLG/AgNWs composite films with different AgNWs concentrations are shown in Fig. s) of the sample films was tested with a four-point probe instrument. The Rs of SLG/AgNWs composite films with different concentrations of AgNWs  are shown in Fig. s of the as-prepared SLG can reach up to ~650 Ω/▯, as a result of a mass production size (21 cm × 29 cm). The high sheet resistance of SLG could be result from the two different types of defects: The first type of defects occurs on the surface of graphene sheets, such as dots and single vacancy defects. The electron-scattering centers form on the surface of graphene because of these defects. They cause electron scattering, affect the electron transfer, and, ultimately, decrease the conductivity of graphene31. The second type of defects occurs between graphene sheets. They are caused by wrinkles, grain boundary, overlap sections. Large-area graphene-growth in different regions does not guarantee a uniform extension of the crystal orientation. When the regional graphene grows to a certain size, cross-regions begin to appear, and different crystal orientations lead to the formation of graphene line defect, wrinkles, and overlapping stacks32. The above mentioned first defect needs mainly chemical modification. In this work, we used AgNWs to reduce Rs of SLG to create more accessible conductive pathway, which can solve the effects of second type.To investigate the conductive properties of the composite electrode, the sheet resistance  When the AgNWs do not form a two-dimensional film, the introduction of a very small number of AgNWs destroys the planar structure, which is equivalent to introducing impurity defects. (II) When the AgNWs have just formed the two-dimensional film, the density of AgNWs is not efficient to connect from each other, leading to little improvement compared with the contact resistance between SLG and AgNWs.Figure 34. Herein, AgNWs are simulated as thin rods with negligible diameter, and the rod length is in the range of 0.1~0.2 unit length, shown in Fig. Cp). We calculated that the percolation concentration is about 0.1 mg/ml, and the error is less than 5%. The simulated diagram of AgNWs at the percolation concentration used Monte Carlo method is shown in Fig. To investigate this critical factor, we conducted a simulation of the percolation using the Monte Carlo methods of graphene. Between overlapping sections of graphene, the carrier transport is blocked. Simulation results showed that AgNWs could effectively percolate on SLG in concentrations over 0.3 mg/ml, indicating that most graphene nanosheets were connected by AgNWs. AgNWs provide a better-accessible pathway for electron transfer than inter-layer hopping between graphene sheets - see Fig. At 0.3 mg/ml, the negative effects caused by the contact resistance and the positive “bridge effect” are just offset. The positive “bridge effect” is illustrated in Fig. s decreased to ~270 Ω/▯ for 0.5 mg/ml and reached ~27 Ω/▯ at concentration of 1.5 mg/ml. Even though the concentration of the AgNWs was over 1.5 mg/mL, the Rs improved only slightly, ~15 Ω/▯ at concentration of 2.0 mg/ml, this is because AgNWs have formed a dominant and independent conducting network.R37, which follows an inverse scaling function for electrical conductivity:Rs denotes the sheet resistance of the composite film. C denotes the concentration of the AgNWs. Cp denotes percolation concentration and k denotes the conductivity exponent, whose value ranges from 1~2 for two-dimensions. The formula is also applicable for composite electrode systems, and hence this study. This provided the theory supports to the ratio of length to diameter of the selected AgNWs (70 nm × 100 um).The experimental data are also in agreement with percolation theory for single material systems2·V−1·s−1, yet the carrier concentration was very low, about 1.5 × 1013cm−2. A low carrier concentration limits hole-injection, which causes unbalanced electron-hole recombination in the device. Figure 14cm−2 for an AgNWs concentration of 0.5 mg/ml. When the AgNWs concentration reached 2.0 mg/ml, the carrier concentration was as high as (5.0 ± 0.9) × 1017cm−2. This increasing trend, however, soon slowed down. The reason for this is that the AgNWs formed a separate conducting network whose carrier concentration approaches the upper limit. The 3D laser scanning microscope images in Fig. We also measured both carrier mobility and concentration of SLG/AgNWs composite films using a Hall-effect measurement system. The carrier mobility decreased sharply with increasing concentration, while the carrier concentration increased by an order of magnitude - see Fig. 0)/R0 was used to compare the flexibility data of these test samples. ITO showed a sharp increase in Rs when the curvature radius was less than 20 mm, see Fig. Figure After measuring and analyzing the electro-optical and mechanical properties of composite electrodes, it is necessary to analyze the surface roughness of composite electrodes. We used AFM to test the surface morphology of four different composite electrodes. Figure −2 at 6.0 V, and that of the ITO device reached the same intensity at about 4.0 V. This is because the lower current density than ITO reduced the carrier recombination at the same voltage. However, the current density of the composite electrode was considerably enhanced compared to graphene, the value increased from 55 mA cm−2 to 107 mA cm−2 at 9.5 V, which is consistent with the Hall-effect measurement that indicates that AgNWs successfully formed a conducting network to provide additional carriers for graphene. This consequently increased both current density and the luminance intensity. Figure Considering that a high concentration of AgNWs film might negatively affect both optical transparency and surface roughness of the electrode, we chose composite electrode with a 1.5 mg/ml concentrations of AgNWs to be used as the anode in organic light-emitting diodes. The J-V-L characteristics are shown in Fig. −2 luminance at 9 V driving voltage, were successfully fabricated with this composite electrode. In summary, we can confirm that combining AgNWs with SLG can not only effectively improve the conductivity of the graphene film but also meet the requirements to serve as effective transparent anode in flexible-OLED. These novel composite electrodes may open new possibilities for the next generation of efficient and flexible optoelectronic devices.In this work, we used AgNWs in conjunction with a graphene-based electrode to improve the common problem of poor connectivity and low carrier density of SLG. We studied the underlying mechanism of this improvement using percolation theory, and we determined the percolation concentration for this composite system. The SLG/AgNWs composite-electrode shows excellent photoelectric properties (Rs of 27 Ω/▯), high transmittance (86.7% at 550 nm), and it can bend over 1000 times with only small changes in conductivity. Flexible, organic light-emitting diodes, with 15000 cd mThe graphene in our work was prepared using a CVD method that contains 95% single-layer rate and its sheet resistance is about 650 Ω/▯. The preparation process of composite electrode is shown in Fig. 3 doped with 1% C545T/30 nm Alq3/1 nm Liq and 100 nm Al.To study its suitability as transparent electrode, we used this electrode to fabricate OLEDs. The structure of the OLEDs was the following: Anode (ITO or SLG/AgNWs composite electrode)/5 nm HAT-CN/40 nm TAPC/30 nm AlqRaman measurements were carried out using a Raman microscopy system equipped with a 532 nm wavelength laser. Optical transmittance was measured with a UV-Vis-IR spectrophotometer . Sheet resistance was measured using a four-point probe. The charge-carrier concentration was measured with a Hall Effect Measurement System (HL5500PC). A self-built bending test system was used to assess the bending properties of the composite electrode. The current-density–voltage–luminance (J–V–L) characteristics of the flexible-OLED were simultaneously measured using a source-meter (Agilent B2902A) and a Minolta luminance-meter (LS-110). The work function (WF) was measured with a Kelvin probe system. The surface roughness was measured with an atomic force microscope (Bruker Dimension Icon-PT).The simulation process is as follows. The AgNWs are considered as long line segments. At the beginning, place the AgNWs with length value in normal distribution and negligible thickness on the plane randomly. Each nanowire stick is given a random azimuthal orientation (θ), from 0 to π. Both of them are generated by random functions. There are three parameter matrices which mean x, y, θ and an array of the numeral orders, we can call them IDs. For each recursion, we will set the nanowires in contact with each other to the same ID. At this point, the entire plane is not conductive. Then the total number of AgNWs will be increased by recursion until critical density. The critical density is defined as that the same ID could be found on both boundaries of plane, left and right, bottom or top. It means that the system will become a conductor, and the resistance will drop rapidly after that. In one word, through this simulation we acquire percolation threshold of the AgNWs system. The sheet resistances of different density were measured by experiment, to acquire more data to determine the relationship between them.


Graphene shows great potential in biosensing owing to its extraordinary optical, electrical and physical properties. In particular, graphene possesses unique optical properties, such as broadband and tunable absorption, and strong polarization-dependent effects. This lays a foundation for building graphene-based optical sensors. This paper selectively reviews recent advances in graphene-based optical sensors and biosensors. Graphene-based optical biosensors can be used for single cell detection, cell line, and anticancer drug detection, protein and antigen–antibody detection. These new high-performance graphene-based optical sensors are able to detect surface structural changes and biomolecular interactions. In all these cases, the optical biosensors perform well with ultra-fast detection, high sensitivities, unmarked, and are able to respond in real time. The future of the field of graphene applications is also discussed. Foperties . Graphenhe world ,19,20. The world ,22,23.Due to the special electron band structure, graphene shows high carrier mobility and zero bandgap characteristics. Thus, the excellent optical properties of graphene, such as broadband absorption, saturation absorption, and fluorescence, gradually attract the attention of scientists ,26,27,28There are two main types of interactions between light and graphene from the energy band transition: Interband transitions and in-band transitions. In the far-infrared and THz spectral regions, the electronic response is mainly in-band transition (free carrier response), which can be well described by the Drude model. The graphene electron response in this band is similar to the free electron response in the metal, which can excite the surface. In the near-infrared and visible-light bands, the photoresponse is mainly interband transition. The absorption of light appears as a general absorption independent of wavelength, and the absorption coefficient is determined by the fine structure constant. In this case, due to the Pauli blocking principle, the light absorption of graphene can be regulated by adjusting the position of the Fermi surface. In the ultraviolet region, the interband transition is close to the saddle point, at which point the light absorption exceeds the universal absorption value and has an exciton effect. Graphene shows high optical absorption as a monoatomic layer material, but as a material, 2.3% optical absorption is still very low. This suggests that the interaction of light with graphene is still weak, and many of the optical properties of graphene are limited by this weaker optical absorption. Therefore, it is a prominent problem of graphene optics to find ways to enhance the interaction between light and graphene.Moreover, graphene also exhibits excellent biological ,37,38,393·H2O–NH4Cl (0.1 M) buffer solution (c) and the oxidation peak current (Ipa) of acetaminophen was studied by square wave voltammetry (SWV). The Ipa was linearly related to the paracetamol concentration over the range of 0.1–20 μM. The results showed that the graphene-modified glassy carbon electrode (GCE) could exhibit good reproducibility when detecting paracetamol. Cyclic voltammetry (CV) was used to compare the interference of dopamine acid (DA) and ascorbic acid (AA) on bare GCE and graphene-modified GCE, respectively. These results indicate that the developed sensor exhibits an excellent electrocatalytic activity towards the reducing and oxidation of paracetamol. Moreover, it has an excellent detection limit of paracetamol with a detection limit of 3.2 × 10−8 M, which has high sensitivity and selectivity for detecting paracetamol in medical applications and commercial fields. All in all, the electrode modified with graphene had good electrocatalytic activity to paracetamol.These excellent properties of graphene are used in many different types of sensing applications, including optical sensors, electric field sensors, electrochemical sensors, and gas sensors. First of all, the excellent electrical properties of graphene have aroused wide interest of scientists. Graphene has good conductivity, high current density of sustainability, high carrier mobility and relativistic carrier property . Thus, tIn short, in the field of electrochemistry, graphene has the opportunity to show its talents. The application of graphene on electrochemical sensors has the advantages of high sensitivity, fast response time, fast electron transfer, easy to fix proteins and maintain their activity. However, there are some shortcomings in graphene-based electrical sensors. Since transistor-based graphene sensors only measure current changes on the surface of graphene, electrical sensing is seriously restrained compared to the time and spatial resolution of optical sensing . In addiGraphene shows unique and ideal optical properties, such as broadband and tunable absorption ,69, and Graphene has excellent high-energy transfer efficiency, large surface area, and biocompatibility , and canspk) propagating at the metal-dielectric interface is described by the following equation [spω is the angular frequency of the surface plasmon, and mε and sε are the dielectric constants of the metal film and the dielectric medium, respectively.Surface plasmon is quantum of plasmonic oscillation generated by the interaction of freely vibrating electrons and photons on a metal surface, which was observed in Wood’s experiment in 1902 . SPR is equation ,(1)ksp=SPR biosensing was first applied in 1983, and since then, biospecific interactions have also begun to be detected . Since tRI0 = 52 and SRI1 = 53.2 for ΔP = 0.26, respectively. Besides, adding more graphene layers will widen the SPR curve and make it difficult to measure SPR. Furthermore, there is a linear relationship between the ΔSRIL/SRI0 and the number of graphene layers L, as shown in RIL/SRI0 will increase [RIL = SRIL − SRI0. Due to the optical properties of graphene, it has been found that coating more graphene layers can provide increased sensitivity [In 2010, Wu presented an SPR-based graphene biosensor by coating graphene over a gold film . Compareincrease , where tsitivity ,98. In sIn addition, optical focusing limits the sensitivity, resolution, dynamic range, and other functions of the spectral-based SPR instrument when measuring wavelength or angle ranges and produces significant defects in the real-time performance of the mentioned biosensor. There is an inverse relationship between the response speed of the SPR biosensor and its sensitivity. The intensity type SPR sensor performs a fast response speed but low sensitivity. While angle or phase type SPR sensors exhibit higher sensitivity at the expense of lower response speed . AlthougFiber optic sensor is an important sensor, and its basic working principle is similar to other sensors. The signal light emitted from the light source enters the sensing area through the optical fiber, where the parameters change, such as the concentration and refractive index of the objects pending measuring, and then modulates the optical properties of the signal light . The signal light enters the optical detector  through optical fiber and converts it into a readable signal to analyze the parameters of the objects pending measuring. In fiber optic sensors, light is used as a carrier for sensitive information, and optical fibers are used as a transmission medium for sensitive information . Fiber oGraphene is combined with fiber to obtain a graphene-based optical fiber sensor due to its excellent performance ,104,105,−5 RIU. The sensor can not only use the finite element method to obtain the maximum phase matching between modes, but also to detect the thickness of the biological layer.In 2013, Kim et al. firstly used chemical vapor deposition (CVD) to transfer graphene on the d-type region of plastic optical fiber, replacing the traditional metal film layer, to build a graphene fiber SPR sensor, and executed corresponding detection of double-stranded DNA and protein-streptavidin as target molecules. The graphene SPR sensor adsorbs the target molecule, and the resonance summit exhibits a characteristic traverse with the change of the target molecule concentration, which effectively proves that graphene can replace the metal film to realize the graphene fiber SPR sensor . Then, P1), graphene layer (RI = n2), and glucose solution (RI = n3). Among them, the graphene layer wraps the sensing area. In a fiber of uniform diameter, the evanescent field cannot interact with the surrounding environment of the fiber. However, for tapered fibers, the evanescent field can interact with the surrounding environment. In this experiment, the concentration of the glucose solution is increased alone, and the RI of the glucose solution is also increased. At the same time, this also leads to an increase in penetration depth and large consumption of energy, and the output power is lowered. In addition, it can be seen that the change in the concentration of the glucose solution affects the intensity of the output light. As the concentration of the glucose solution increases, the intensity of the output light gradually decreases. When the glucose solution concentration is increased from 1% to 40%, the output light intensity is also reduced from 29,500 to 27,500. It can also be seen that at each glucose concentration, the output light intensity exhibits a reasonable linear relationship with it. This work could provide a unique strategy for tapered fiber optic sensors with high reliability and repeatability. Then in 2015, Jiang et al. dipped graphene oxide into the u-shaped zone of quartz fiber and prepared u-shaped fiber sensor with evanescent wave absorption response for the detection of alcohol solution. The results show that the sensor with rotation-coated graphene oxide has good accuracy, repeatability, and sensitivity, and the response time and recovery time are within 5 s. The actual wine samples are tested, and the findings are consistent with the alcohol experimental data, laying a foundation for the practicability of the sensor [In 2014, Qiu and Jiang designed a novel graphene tapered fiber sensor for glucose detection by growing a single-layer graphene film on copper foil by CVD and then transferring the single-layer graphene film to the tapered region of the fiber. The tapered fiber b shows te sensor .In various photonic and optoelectronic devices, the interaction of graphene with light is also different, so the optical devices of graphene need to work under different structures. Among them, the sensor requires a strong interaction between graphene and light. Using a prismatic TIR structure, the interaction of graphene with light has the characteristic of polarization absorption. Under this structure, graphene shows the characteristics of polarization absorption and broadband absorption enhancement. The polarization absorption enhancement of graphene can be achieved by optimizing the refractive index around the graphene medium or by using a multilayer structure. Based on the polarization absorption effect of graphene under TIR, combined with microfluidic technology and biomedical knowledge, a series of sensitive new optical sensors, called polarization absorption enhanced biosensors, are designed and put into practical applications. The sensitive sensing of this sensor utilizes the polarization absorption effect of graphene. As the RI n of the upper medium of the graphene is changed, the optical power of the two polarization states changes. Therefore, by simultaneously reading the optical power of the two states of polarization in the reflected light, the RI n of the upper medium of the graphene can be sensed. These sensors have a similar structure to SPR sensors and provide a reliable RI sensing platform for complex fluid systems over a wider dynamic range. Sensitive sensing of the test sample can be achieved by measuring the change in RI produced by the flow of the sample through the microfluidic channel. It can be applied to the biological field to achieve sensitive sensing of a single cell, cell lines, anticancer drugs, specific proteins, and antigenic antibodies.Although graphene has an optical absorption of 2.3% as a two-dimensional material of a single atomic thickness, 97% light is wasted and not capable of interacting with graphene . This li1) with high RI and medium 2(n2) with low RI by a sandwich structure, and the RI of medium 1 and medium 2 is fixed, as shown in n is RI of graphene and k is extinction coefficient [d is relative to the number of graphene layers . This structure not only has strong optical absorption of TE waves under total internal reflection (TIR) but also increases the broadband absorption of graphene. The energy density distribution of TE and TM waves under the graphene sandwich structure is simulated. It can be seen that when the incident angle is greater than the critical angle, the simulation results show a large energy density. In addition, there is a significant energy density distribution in the graphene layer. The stronger the incident energy, the more the graphene layer absorbs, and the less the reflected energy. It can be said that the reflected energy is directly related to the degree of light absorption of the graphene layer, and higher precision is obtained by comparing reflection with transmission [Ye et al. designed a sandwiched graphene structure. Here, graphene is sandwiched at the interface of medium 1 of the capillary-based optofluidic ring resonator designed by Li and Fan [−9) of the label-free optical biosensor based on a free-space Young interferometer configuration is presented by Schmitt [1 and n2, respectively. As the incident angle θ1 approaches the critical angle θc, the resolution and sensitivity can be improved by taking advantage of graphene with optimal thickness. Xing et al. designed a graphene-based optical sensor by combining an optical sensor with a microfluidic channel to enable the detection of flow single cell . The gra and Fan , slightl Schmitt . In addiS is the sensitivity of RI sensing, L is the resolution of RI sensing, ΔR is the reflectance difference for TE and TM polarized light, P0 is the incident power of TE or TM wave, α is the response of the balance detector, and nV is the noise value. In the above formula, some parameters are known quantities. Among them, the power is fixed at 80 μW, the thickness of h-rGO is 8.1 nm, α is 0.00361 μW/mV, and nV is about 10 mV. The h-rGO-based RI sensor detects real-time voltage changes in water and different ultra-low concentrations of sodium chloride solution, as shown in Resolution and sensitivity are two parameters that are essential for this study and can be calculated by the following formulaThe graphene-based optical single-cell sensor (GSOCS) is shown to be extremely sensitive to single cells by detecting standard polystyrene (PS) microspheres. 7 mV/RIU and a resolution of 1.7 × 10−8. They used a GSOCS to detect lymphoma cells from normal lymphocytes and demonstrated that the sensor is highly sensitive. In addition, this high-performance ultra-high resolution and sensitivity sensor for RI measurements can be applied to other areas, such as biopharmaceuticals, medical monitoring, and environmental sensing [In short, they proposed a graphene-based RI sensor that measures the current change of a single cell on the graphene surface. The sensor is capable of a wide range of flow detection with a sensitivity of 4.3 × 10 sensing .Detection of unlabeled living cells has been challenging . The mosc) of 1.35 × 10−7 with an SNR of 5.3 in the experiment, and the corresponding sensitivity was increased to 1.2 × 108 mV/RIU. In addition to hypersensitivity, the sensor can also detect the ultrafast RI changes generated by the weak ultrasound within 260 ns. Due to the ultra-fast response characteristics of GOB, the transient response in living cells can also be accurately detected. Furthermore, the detection depth of the GOB is also very surprising, which is more than 2 μm from the graphene film, and this detecting depth can be used to characterize the height dimension of the entire cancer cell. Therefore, this sensor for living cell sensing has ultra-high sensitivity and high precision. The biosensor is capable of detecting whole-cell responses with the advantage that cell labeling is not needed.A graphene-based optical biosensor (GOB) consisting of polydimethylsiloxane (PDMS)/microfluidic channel/graphene glass/prism was designed , as showD and the sensitivity S of the GOB is as follows [dU is the change in voltage response and noiseN is the noise signal. The input of the pulse generator not only adjusts the intensity of the ultrasonic pressure but also verifies the detection limit D and the sensitivity S of the GOB. The ultrasonic generator produces 1 kPa of ultrasound. The relationship between the RI change of the water solution dn and the change in the ultrasonic wave pressure dP is dn = 1.35 × 10−10 dP. As shown in −7) produced by 1 kPa of ultrasound, with a corresponding voltage signal of 17 mV and an SNR of 5.3. The noise signal shown in the inset of −8 RIU. The duration of the entire signal is 2 μs. The illustration in It is extremely important to detect cancer cell responses using high-sensitivity RI sensors over a wide dynamic range. Furthermore, RI changes reflect the detection limits and sensitivity of GOB, which vary greatly within cancer cells. The limit of detection is usually estimated by measuring different sodium chloride concentrations. The ultrasound can accurately evaluate the detection limit, sensitivity, and response time of the GOB. The formula for calculating the detection limit  follows :(4)S=dU/−5 RIU. These results indicate that GOB can accurately measure the response of cancer cells to low concentrations of paclitaxel.The diameter of cancer cells is larger than normal cells . Two colk of the response is taken as a statistical parameter to facilitate calculation analysis. The right image of In order to further study the response of cancer cells to paclitaxel, the concentration of paclitaxel solution is used as a variable factor, and 80 nM and 6 μM paclitaxel solutions are used for experiments. The left image of In order to better analyze the changes in RI, three factors are summarized here. First, the polymerization and total mass changes of tubulin affected the changes in RI. Secondly, nuclear fragmentation and microtubule purpura affect the changes of RI in some cells of G2/M phase. Finally, after adding paclitaxel, interphase cells take a long time to become apoptotic or necrotic. This also affects the changes in RI. These three factors induce abnormal response curves.In summary, this biosensor enables ultrasensitive and real-time dynamic detection of the response of unlabeled cancer cells to paclitaxel, and new responses are observed during early drug delivery. Colorectal cancer cells show a stronger and faster response to low-concentration paclitaxel treatment compared to high-concentration paclitaxel treatment. This simple biosensor shows excellent performance for unlabeled living cells and is expected to be widely used as a sensor for cancer treatment, anticancer drug screening and other unlabeled living cell responses for cell-based research .Since graphene could be easily modified and functionalized for targeted fixation of biological receptor units ,127,128,2O as the base liquid, when a 0.011% NaCl solution is introduced, a potential difference between the two liquids is clearly observed. Therefore, the change in RI between H2O and 0.011% NaCl solution could be easily seen, indicating that this based RGO sensor is very sensitive to changes in RI. Biomolecular interactions  typically induce changes in RI. Since RGO is easy to modify and functionalize, graphene can distinguish between cancer cells and normal cells by detecting biomolecules in the case of TIR [The surface treatment of reduced graphene oxide (RGO) is more likely to enhance the surface function of the SPR sensor relative to the gold film . Furthere of TIR .Here, the optical sensor and RGO are combined and functionalized to develop an RGO-based optical biosensor that could be used for binding to specific proteins. The sensor is able to detect biomolecules. A balanced photodetector is capable of measuring changes in the polarization-dependent absorption corresponding to changes in RI induced by antigen–antibody interactions. The sensor responds well to minimal concentrations of immunoglobulin G (IgG) and has the advantages of real-time detection, no labeling, ease of integration, and low cost.The schematic of an RGO-based optical biosensor is shown in Sensitivity is an important parameter for biosensors. The sensitivity of the biosensor is investigated by injecting different concentrations of rabbit IgG solution into the microfluidic channel. The minimum concentration of 0.0625 μg/mL rabbit IgG solution can be distinguished with a voltage change of about 0.17 V, and the sensitivity of the sensor is well linear with the rabbit IgG concentration. Specificity is also an important parameter for biosensors. Bovine IgG specificity was also verified in the same conditions for detecting rabbit IgG. Goat anti-rabbit IgG was injected into the microfluidic channel and allowed to bind to the sensor surface, and then bovine IgG was injected until the detected signal was stable. Comparing the results of the test, it could be seen that the voltage change of bovine immunoglobulin M (IgM) is significantly smaller than the voltage change of rabbit IgG, thus indicating that rabbit IgG is not fully bound. Therefore, an RGO sensor modified with an antibody selectively detects an antigen. In other words, different antibodies bind to the biosensor and could match different antigens.In summary, due to the interaction of antigens with antibodies and the polarization absorption properties of graphene, they designed an RGO-based optical biosensor for detecting properties. Based on the experimental results, the sensor can measure the minimum reaction of the minimum concentration of 0.0625 μg/mL rabbit IgG, which is more sensitive than the previous SPR device. This sensor provides real-time, high sensitivity, and selectivity for the detection of specific proteins without labeling. In addition, considering the use of GO, the sensor can be produced at low cost and on a large scale. These factors should make this sensor a good potential candidate for biosensors.Graphene is a novel two-dimensional material, due to its unique structure and superior performance. Graphene-based derivatives are used in various fields, including biomedicine and sensing. Among them, the applications in biological imaging, biomolecular detection, cancer detection, drug treatment, etc. is particularly extensive. Although graphene-based electrical sensors are widely used, they can only measure current changes on the graphene surface and may damage living cells, which can affect the test results. Graphene-based optical sensors do not have the above problems, and the detection range is wide, which has potential application prospects in the sensing field. This paper focuses on the optical properties of graphene , the mechanism of light action and the method of enhancing interaction with light, and summarizes the research progress of graphene in the field of sensing, and the structure and characteristics of graphene used in these sensors. Moreover, the interaction between graphene and light under the prism TIR structure is proposed. Under this structure, the interaction between graphene and light is polarization-dependent, and under certain conditions can enhance the absorption of light by graphene. Additionally, under the condition of total internal reflection, the interaction of light with graphene is extremely sensitive to the refractive index of the medium, so the required sensitivity, selectivity, and reusability can be achieved according to graphene in biosensing applications. This paper reviews in detail the latest research on the application of graphene-based optical sensors in the biological field. Graphene-based optical biosensors not only detect whole-cell responses in the presence of unlabeled cells but also show high sensitivity and precision. These simple biosensors have the advantage of large depth detection, high sensitivity and accurate and rapid identification for unlabeled living cells and are expected to be widely used in cell-based research for anticancer drug screening, cancer therapy, and other unlabeled living cells response. These graphene-based optical biosensors have excellent sensitivity and selectivity for detecting single cells, cell lines, anticancer drugs, and specific proteins.At present, new biosensors and other applications using graphene as a sensing element have been developed, but there is still much work to be done. First, the shape, size, number of layers, electronic band gap structure, purity, and defects of graphene grown in the experiment are all uncertain. However, these properties can affect the conductivity of the sample, interaction with biomolecules, and fluorescence quenching, which, in turn, can affect the performance of graphene-based optical biosensors. Therefore, an efficient method of growing graphene should be explored to obtain high-quality graphene-based nanomaterials ,135. SecIn summary, because graphene materials are superior to traditional materials, detailed research on graphene-based materials will open a new direction for research in the field of biosensors. Graphene optical biosensors will also provide critical and breakthrough solutions for the world.