Symbiodinium communities in corals are inherited versus environmentally acquired is fundamental to understanding coral resilience and to predicting coral responses to stressors like warming oceans that disrupt this critical endosymbiosis. We examined the fidelity with which Symbiodinium communities in the brooding coral Seriatopora hystrix are vertically transmitted and the extent to which communities are genetically regulated, by genotyping the symbiont communities within 60 larvae and their parents  using high-throughput sequencing of the ITS2 locus. Unexpectedly, Symbiodinium communities associated with brooded larvae were distinct from those within parent colonies, including the presence of types not detected in adults. Bayesian heritability (h2) analysis revealed that 33% of variability in larval Symbiodinium communities was genetically controlled. Results highlight flexibility in the establishment of larval symbiont communities and demonstrate that symbiont transmission is not exclusively vertical in brooding corals. Instead, we show that Symbiodinium transmission in S. hystrix involves a mixed-mode strategy, similar to many terrestrial invertebrate symbioses. Also, variation in the abundances of common Symbiodinium types among adult corals suggests that microhabitat differences influence the structure of in hospite Symbiodinium communities. Partial genetic regulation coupled with flexibility in the environmentally acquired component of Symbiodinium communities implies that corals with vertical transmission, like S. hystrix, may be more resilient to environmental change than previously thought.Determining the extent to which  Symbiodinium has allowed corals to thrive in oligotrophic tropical seas through the utilization of symbiont photosynthates. Similar nutritional facilitation has been described for sap-sucking insects that rely on microbial partners to supplement their diets  or passed maternally into eggs or larvae , with the latter thought to be the most prevalent mode of transmission in brooding scleractinian corals  or associate with multiple partners, and superinfections of multiple Symbiodinium types and subtypes of varying abundances are common among species  is the parameter typically used to describe the degree to which variability in a trait is explained by genetic factors. Assuming that the Symbiodinium community associated with a coral can be represented as a complex trait, then an h2 value of 1 would imply that variability of the community is mostly due to host genetics. Conversely, an h2 value estimated at 0 would imply no genetic basis for variability in the community, thus the community would not be under selection and could not evolve  of this trait, we quantified the in hospite Symbiodinium communities of individual planula larvae and their parents across a spectrum of relatedness using high-throughput sequencing. Relatedness was based on a population genetic parentage analysis that assigned the likely paternal identity of each larva. In light of results on heritability and fidelity of symbiont transfer, we discuss the potential of larvae from brooding corals like S. hystrix to acclimate to novel environments.To examine Seriatopora hystrix broods sexually produced larvae following internal fertilization of eggs by sperm from surrounding colonies   were sequenced with the primers ITS2alg-F and ITS2alg-R  were analyzed using the USEARCH and UPARSE pipelines  and multiplicative (log2 fold) terms between or among treatments ) that incorporates OTU richness, evenness, and sequence similarity , adult colonies were divided into five size classes based on their mean diameter , 8 – <14 cm (n = 19), 14 – <20 cm (n = 13), 20 – <26 cm (n = 11), and 26–32 cm (n = 1). Differential abundance testing of Symbiodinium OTUs among size classes was performed as for larval communities.To determine if Symbiodinium OTUs associated with the 45 adult colonies of S. hystrix (ShA) that were genotyped across the 16 m × 40 m sampling area. Gradient-boosted models and linear models were run in the package “gbm”  . On average, adults contained 29.9 ± 0.6 (SE) OTUs and larvae had 22 ± 0.4 OTUs  Fig. . OverallTUs Fig. . Of the TUs Fig. . Unique ity Fig. . Of the ity Fig. . AlthougSymbiodinium, 8 of putative Symbiodinium taxonomy, Fig. Symbiodinium communities were characterized by up to 4–5.7 (2–2.5 log2 fold) times more D-types , and A-types (A1_OTU10 and A3_OTU8) compared to larvae  significantly higher abundances in adults  and the remaining C1 types had between 0.02–0.13 (−5.4 to −2.9 log2 fold)  although 95% probability regions overlapped  larvae was 0.43 ± 0.21 SD, with a posterior mode of 0.33  0.1–0.8; Fig. Leinster and Cobbold estimates of ies Fig. . The posp > 0.05; Fig. Symbiodinium C120/C120a were greatest in colonies that were closest to the lower left of the sampling area : p = 0.019), consistent with a gradual increase in distance down the reef slope, this pattern was not consistent along the reef slope (GBM: p = 0.00841). The abundance of D1 was significantly higher in the top-right and lower left side of the sampling area than in other aspects . D1a was least abundant in the top left and inner portion of the sampling area . Finally, although the variance normalized abundances of all three OTUs were significantly positively correlated overall , extremely low abundances of C120/C120a at x-coordinates >15 contrast markedly with high abundances of the two D-types in the same region   differ from those associated with their parents, providing experimental evidence that at least a portion of the Symbiodinium community is horizontally transmitted in a brooding coral. These results further demonstrate that coral-Symbiodinium symbioses align with well-characterized models of invertebrate symbioses. Results from previous studies suggesting exclusively vertical transmission in brooding corals may have been due to the lower detection capability of earlier methods and will no doubt change with the continued application of NGS technology to more brooding coral species. Overall, Symbiodinium communities were found to be moderately heritable, with only 33% of variability in larval symbiont communities under genetic regulation. Model selection also showed that, for larvae released from the same colony, sharing the same maternal environment did not significantly explain variability in Symbiodinium communities found among larvae from the same brood. This result, combined with the moderate heritability estimate, indicates that similarities in Symbiodinium communities among larvae of the same maternal brood were due to gene(s) inherited by these larvae.The availability of a full larval pedigree for Symbiodinium communities in S. hystrix (ShA). Much greater heritability of the Symbiodinium community was expected in this vertically transmitting coral, and in comparison with what is known of other important reproductive and fitness traits. For example, fertilization success, larval heat tolerance, protein content, settlement success, settlement substrate preferences, and juvenile growth and survivorship are all heritable traits  has some capacity to respond to changing environmental conditions. Thus, intervention efforts to facilitate such phenotypic change may be possible  could also be candidates for assisted Symbiodinium uptake.Heritability estimates reveal important information about the evolvability of a trait, such as the capacity of brooding corals to vary their symbiont communities in response to changing environmental conditions. If levels of heritability and genetic variance are low, then responses to natural or artificial selection (evolvability) would be limited  have a mixed-mode transmission strategy, in which dominant symbionts are transmitted vertically but additional background strains are acquired from environmental sources. Although adult diversity may have been under-sampled by only sequencing one branch of each parental colony, unique larval OTUs were not detected in any of the 45 adult colonies that were genotyped. Environmental uptake of novel Symbiodinium by larvae of this species is further supported by the appreciable amount of variation in the composition of larval Symbiodinium communities that was not under genetic control, according to our heritability model. These results validate the hypothesis of potential mixed-mode transmission initially raised by Byler et al.  contradicts previous assumptions that maternally transmitted symbiont communities are transferred to offspring with high fidelity in corals  larvae mirrors increased diversity of Symbiodinium communities detected in eggs of Montipora capitata and M. digitata compared to adults , vertical transmission of Symbiodinium that are locally adapted to the parental environment is likely to provide benefits for a species that is able to self-fertilize (Sherman S. hystrix (ShA), combined with a functional, established symbiosis upon release, may facilitate both local and long-distance dispersal . Theory suggests that competition among symbionts may preclude transmission of an exact replica of the parental symbiont community because conditions promoting growth for some symbionts may differ between life stages  larvae may provide benefits similar to those observed in insect symbioses, for example to provide larvae with the flexibility to host optimal symbiont types for the changing conditions through ontogeny  immune-related genes are being transmitted from parents to offspring and whether novel symbionts are a function of an under-developed immune response.Conversely, the greater variation and diversity found in larval compared to adult Symbiodinium communities in larvae versus adults found here further indicate that the re-shaping of the Symbiodinium community through ontogeny is an important developmental process in corals. Ontogenetic variability in microbial communities (both Symbiodinium and bacteria) is common in both vertically and horizontally transmitting cnidarian species   than in broadcast-spawning corals  are only partially genetically regulated by their host and that larvae retain the flexibility to associate with novel symbionts across generations. Our results reveal a mixed-mode transmission strategy for establishing Symbiodinium communities in larvae of a brooding coral, based on demonstrations that a subset of novel and unique Symbiodinium types are found in brooded larvae but not in adults . Importantly, this information aligns symbiosis transmission ecology in corals with well-known terrestrial invertebrate symbioses that typically exhibit mixed-mode transmission strategies. Advances in the understanding of heritable genetic mechanisms quantified here provide important insights into how Symbiodinium communities may be targeted for intervention strategies to increase reef resilience.On the basis of novel heritability and paternity analyses, we show that DNA sequences: All sequencing data will be made available through NCBI SRA SRP082130.Supporting Information


Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) is life-threatening. Several serum biomarkers, such as Krebs von den Lungen-6 (KL-6) and surfactant protein D (SP-D), are clinically used for evaluating AE-IPF, but these biomarkers are not adequate for establishing an early and accurate diagnosis of AE-IPF. Recently, the protective roles of the members of the peroxiredoxin (PRDX) family have been reported in IPF; however, the role of PRDX4 in AE-IPF is unclear.Serum levels of PRDX4 protein, KL-6, SP-D and lactate dehydrogenase (LDH) in 51 patients with stable IPF (S-IPF), 38 patients with AE-IPF and 15 healthy volunteers were retrospectively assessed using enzyme-linked immunosorbent assay. Moreover, as an animal model of pulmonary fibrosis, wild-type (WT) and PRDX4-transgenic (Tg) mice were intratracheally administered with bleomycin , and fibrotic and inflammatory changes in lungs were evaluated 3 weeks after the intratracheal administration.Serum levels of PRDX4 protein, KL-6, SP-D and LDH in patients with S-IPF and AE-IPF were significantly higher than those in healthy volunteers, and those in AE-IPF patients were the highest among the three groups. Using receiver operating characteristic curves, area under the curve values of serum PRDX4 protein, KL-6, SP-D, and LDH for detecting AE-IPF were 0.873, 0.698, 0.675, and 0.906, respectively. BLM-treated Tg mice demonstrated aggravated histopathological findings and poor prognosis compared with BLM-treated WT mice. Moreover, PRDX4 expression was observed in alveolar macrophages and lung epithelial cells of BLM-treated Tg mice.PRDX4 is associated with the aggravation of inflammatory changes and fibrosis in the pathogenesis of IPF, and serum PRDX4 may be useful in clinical practice of IPF patients. Idiopathic pulmonary fibrosis (IPF) is a fibrosing parenchymal lung disease that has chronic, progressive, and even fatal outcomes. The natural history of patients with IPF is extremely complex; moreover, the clinical course of such patients varies from relatively stable to sudden acute exacerbation, which often prove fatal. Recently, clinical trials have demonstrated that antifibrotic agents, such as pirfenidone and nintedanib, reduce the decline in forced vital capacity (FVC) and prolong progression-free survival in patients with IPF , 2; howeApproximately 5–10% of patients with IPF generally demonstrate acute exacerbation (AE) annually , 5, leadPeroxiredoxin (PRDX) is a recently identified antioxidant family that contains reactive cysteine in a conserved region near the N-terminus , 12. SixWe previously generated human PRDX4-transgenic mice (Tg mice) using C57BL/6 mice and reported that PRDX4 may have a protective role against the progression of atherosclerosis and nonalcoholic fatty liver disease via its antioxidant effect , 20.In the present study, we compared the serum PRDX4 protein level in patients with stable IPF (S-IPF), AE-IPF and healthy volunteers to evaluate the significance of PRDX4 in patients with IPF. In addition, we examined the pathogenetic roles of PRDX4 in pulmonary inflammation and fibrosis using Tg mice in a BLM-induced PF model.S-IPF and AE-IPF were diagnosed based on the criteria for IPF and AE-IPF , 21, 22,Serum and BALF PRDX4 protein levels in both humans and mice were assessed using enzyme-linked immunosorbent assay (ELISA)  according to the manufacturer’s protocol as previously described .The animal study was approved by the Ethics Committee of Animal Care and Experimentation, University of Occupational and Environmental Health, Japan  and performed in accordance with the National Institutes of Health guidelines. Male wild-type (WT) mice  and Tg mice  were selected and maintained on a regular diet . WT mice were obtained from Kyudo Co., Ltd. . PRDX4-Tg mice were generated and provided in our facility .2.0-mg/kg BLM  was dissolved in 40-μL sterile saline. This solution (BLM group) or 40-μL sterile saline alone  was intratracheally instilled in both WT and Tg mice after receiving intraperitoneal sodium pentobarbital. Body weights were recorded at 0, 3, 7, 14 and 21 days after the intratracheal instillation. On day 21, the mice were inhaled with 3% sevoflurane to initiate anesthesia and then deeply anesthetized with intraperitoneal injection of sodium pentobarbital (50 mg/kg). These mice were euthanized by cutting the inferior vena cava to induce exsanguination after collecting blood from the inferior vena cava. Separately, a group sacrificed on day 0 without any intratracheal instillation was provided in order to evaluate the human PRDX4 mRNA and protein levels at baseline (baseline group).n = 5), Tg mice at baseline (Tg-baseline) (n = 5), saline-treated WT mice  (n = 5), saline-treated Tg mice  (n = 5), BLM-treated WT mice (WT-BLM) (n = 14) and BLM-treated Tg mice (Tg-BLM) (n = 14). The numbers of mice that survived until day 21 among the saline- and BLM-treated mice were as follows: WT-saline (n = 5), Tg-saline (n = 5), WT-BLM (n = 12) and Tg-BLM (n = 7). Furthermore, to avoid the influence of BAL on other experimental results, the following numbers of mice were prepared for the BALF analysis: WT-baseline (n = 5), Tg-baseline (n = 5), WT-saline (n = 5), Tg-saline (n = 5), WT-BLM (n = 14) and Tg-BLM (n = 14). The number of mice that survived until day 21 among the saline- and BLM-treated mice for the BALF analysis were as follows: WT-saline (n = 5), Tg-saline (n = 5), WT-BLM (n = 12) and Tg-BLM (n = 6).The numbers of mice were as follows: WT mice at baseline (WT-baseline)  images of mouse lungs were evaluated on day 21 after instillation using a micro-CT system  using the following conditions: 90 kV, 88 μA; field of view, 36 mm; voxel size, 60 × 60 × 60 μm; and scan time, 4 min.BALF in mice was obtained by cannulating the trachea using a 20-gage catheter and by washing three times using 1-mL sterile saline. Cytospin was performed to evaluate the presence of BALF cells, and the obtained cell-free supernatants were stored at − 80 °C until PRDX4 protein assessment as previously described [2O, and were embedded in paraffin. Subsequently, 3-μm sections of embedded lung tissues were stained with hematoxylin and eosin (HE) and Masson’s trichrome. The Ashcroft score was assessed to evaluate PF as previously described [Left lungs of the mice were removed by incising at the anterior midline, were fixed with 15% formalin neutral buffer solution  at 25 cmHescribed . Each spThe mouse anti-human monoclonal fibronectin antibody , rabbit anti-human PRDX4 polyclonal antibody  , 23, 25,For immunofluorescence studies, the sections of right lung of Tg mice were embedded in the OCT compound , snap-frozen in liquid nitrogen, and stored at − 80 °C until use. To identify PRDX4-positive cells in lung tissues, 6-μm-thick cryosections were used for double immunofluorescence staining of polyclonal rabbit anti-human PRDX4 antibody  and were visualized using goat anti-rabbit IgG antibodies conjugated with Alexa Fluor 488®  combined with monoclonal rat anti-mouse Mac-2 , monoclonal mouse anti-rat thyroid transcription factor , and monoclonal mouse anti-human α-smooth muscle actin  antibodies visualized using goat anti-mouse IgG antibodies conjugated with Alexa Fluor 546® .The total RNA extracted from the homogenized right lung tissue using ISOGEN reagent  was reverse-transcribed. The expression of CC chemokine ligand 2 (CCL-2), collagen 1A1, connective tissue growth factor (CTGF), human PRDX4, interferon γ (IFN-γ), interleukin (IL)-1β, IL-4, IL-6, IL-13, IL-17A, tumor necrosis factor (TNF)-α, platelet-derived growth factor subunit B (PDGF-B), active tissue growth factor-β1 (TGF-β1), fibronectin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was quantified using real-time quantitative PCR with the ABI prism 7000 sequence detection system , as previously described , 26. TheFibronectin protein concentrations in mouse lung homogenates were measured using the ELISA kit  according to the manufacturer’s protocol.P <  0.05 were considered to be statistically significant. All calculations were performed using the StatFlex software version 6 .Data are presented as medians (interquartile range) or the number of subjects (%) in human data and as means (standard error of the mean) in murine data, unless otherwise specified. Continuous variables were compared using the Mann–Whitney U test or Kruskal-Wallis test, and categorical variables were compared using the chi-square test or Fisher’s exact test, as appropriate. Changes in human serum PRDX4 protein, KL-6, SP-D, and LDH levels were determined using the Wilcoxon signed rank test. The receiver operating characteristic (ROC) curves and Youden indices were used to determine the optimal cut-off of serum PRDX4, KL-6, SP-D, and LDH levels to distinguish AE-IPF from S-IPF. The survival probability of each group was estimated using the Kaplan–Meier method and compared using the global log-rank test. Values of 2/FiO2) ratio, significantly higher alveolar–arterial difference of oxygen (A-aDO2), significantly lower FVC and significantly lower diffusing capacity of the lungs for carbon monoxide (DLCO), and the overall survival of the AE-IPF group was significantly worse than that of the S-IPF group after the diagnosis.We analyzed the blood samples of 51 patients with S-IPF, 38 patients with AE-IPF and 15 healthy volunteers. Among them, BALF samples were also evaluated in 14 S-IPF and 10 AE-IPF patients. During the observation period, 9 of the 51 S-IPF patients developed AE, and the serum samples of these 9 patients were collected in both the stable state before AE developed (as S-IPF) and also at time of AE development (as AE-IPF). Table The serum levels of PRDX4 protein, KL-6, SP-D and LDH in patients with S-IPF were all significantly higher than those in healthy volunteers Fig. , and thop <  0.05)  days. For these patients, changes in serum PRDX4 protein, KL-6, SP-D, and LDH levels at S-IPF and AE-IPF were compared. Serum PRDX4 protein levels at AE-IPF were significantly higher than those at S-IPF (The area under the curves (AUCs) of serum PRDX4 protein, KL-6, SP-D, and LDH in AE-IPF were 0.873, 0.698, 0.675, and 0.906, respectively . In addition, the body weights of Tg-BLM significantly decreased compared with those of WT-BLM   and Tg-saline (0.2 ± 0.1) were almost normal; however, these scores in Tg-BLM were significantly higher than those in WT-BLM .


Altered expression of many genes and proteins is essential for cancer development and progression. Recently, the affected expression of metadherin (MTDH), also known as AEG-1 (Astrocyte Elevated Gene 1) and Lyric, has been implicated in various aspects of cancer progression and metastasis. Elevated expression of MTDH/AEG-1 has been reported in many cancers including breast, prostate, liver, and esophageal cancers, whereas its expression is low or absent in non-malignant tissues. These expression studies suggest that MTDH may represent a potential tumor associated antigen. MTDH also regulates multiple signaling pathways including PI3K/Akt, NF-κB, Wnt/β-catenin, and MAPK which cooperate to promote the tumorigenic and metastatic potential of transformed cells. Several microRNA have also been found to be associated with the increased MTDH expression in different cancers. Increased MTDH levels were linked to the tumor chemoresistance making it an attractive novel therapeutic target. In this review, we summarize data on MTDH function in various cancers. Chen etin situ (DCIS) of the breast  leads to increased MTDH and acts as a prognostic marker for breast cancer outcome  play important, if subtle, roles in the regulation of gene expression and translation in cellular differentiation and proliferation. Unsurprisingly, aberrant expression of oncogenic miRs (onco-miRs) and tumor suppressor miRs is implicated in cancer pathogenesis , 50. MTDmiR-21 . In contt cancer   tumor suppressor via NF-κB mediated pathways and contributes to the HER2-targeting therapy resistance . Thus, M+ mediated immune response inhibited cancer cell proliferation and lung metastases in a mouse breast cancer xenograft model (Immunotherapy targeting the tumor antigens has been an area of intense research. Expression of MTDH is increased in many different cancer types. Importantly, auto-antibodies against the MTDH protein have been detected in cancer patients confirming its immunogenicity and supports MDTH as a potential immunotherapy target . We receft model . Given tMTDH is pro-oncogenic factor playing multifaceted and diverse roles in cancer progression. Its association and central role in regulating signaling pathways such a MAPK, wnt/β-catenin, PI3K/AkT, NF-κβ pathways in various cancers shows that it plays a vital role in metastasis. MTDH contribution to chemo and radiotherapy resistance provides a new direction for the development of anticancer therapeutics. Multiple mechanisms converge to promote expression of MTDH in cancers. Further studies are therefore warranted to determine whether the elevated MTDH expression has prognostic value for development of the malignancy. Given its increased expression in many cancer types it would be important to determine whether MTDH represents a feasible target for cancer therapy, including immune-therapy. For these reasons there is now an urgent need to determine the clinical and therapeutic significance of increased MTDH expression in cancer.GD originated the idea for writing the review and wrote about MTDH association in different cancers. NS and MG wrote the following sections: micro RNAs regulation of MTDH expression and MTDH target for cancer therapy. ER, JL-R, and NM were involved in writing the MTDH regulation and molecular functions and preparing the original figures. RM and SK contributed to the clinical significance of MTDH expression in cancer. AR managed the different collaborations during the writing of the review and contributed to editing the manuscript. MB was overall responsible for coordinating and managing multisite collaboration, and writing the manuscript.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.


This AIE process was further configured into an immunoassay for analyzing drug residues in foodstuffs. In this approach, glucose oxidase (GOx) was used as an enzyme label for the immunoassay and triggered GOx/glucose-mediated H2O2 generation, which caused oxidation of TPE-HPro and a “turn-on” fluorescence response at 540 nm. To quantitatively analyze the drug residues in foodstuffs, we used amantadine (AMD) as an assay model. By combining the AIE-active “turn-on” fluorescent signal generation mechanism with conventional ELISAs, quantifying AMD concentrations in chicken muscle samples was realized with an IC50 (50% inhibitory concentration) value of 0.38 ng/mL in buffer and a limited detection of 0.06 μg/kg in chicken samples. Overall, the conceptual integration of AIE with ELISA represents a potent and sensitive strategy that broadens the applicability of the AIE-based fluorometric assays.A new fluorescent “turn-on” probe-based immunosensor for detecting drug residues in foodstuffs was established by combining the mechanism of aggregation-induced emission (AIE) and an indirect competitive enzyme-linked immunosorbent assay (ELISA). In this study, a luminogen, with negligible fluorescence emission (TPE-HPro), aggregated in the presence of H The enzyme-linked immunosorbent assay (ELISA) is an extensively used immunoassay to detect the concentration of protein biomarkers and small molecules for clinical diagnosis, environmental monitoring, and food analysis . AMD was purchased from Tokyo Chemical Industry Co., Ltd . Bovine serum albumin (BSA) was purchased from Amresco Inc. . Glucose oxidase-labeled goat anti-mouse IgG (gtAm-GOx) was purchased from Abcam , and horseradish peroxidase-labeled goat anti-mouse IgG (gtAm-HRP) was purchased from Jackson ImmunoResearch . Phosphate-buffered saline (PBS) and Tris-HCl were purchased from Solaribo . TMB substrate was purchased from Beyotime Biotechnology . TPE-HPro was produced in our laboratory as previously described  were mixed with 42 μL Tris-HCl buffer . Then the mixtures were added into the TPE-HPro probe solutions (400 μM in acetonitrile) at a 9:1 volume ratio. The solutions were kept to react at 37°C for 30 min. The fluorescence spectra of the solutions were recorded using a microplate reader.The cleavage of the phenyl boronic ester occurs only in high pH environment. In our previous work, we evaluated and optimized the pH effect on the AIE process  was mixed with the gtAm-GOx solutions at a 3:1 volume ratio and incubated at 37°C for 30 min. Subsequently, 120 μL of these solutions were mixed with 42 μL Tris-HCl buffer . Finally, 135 μL of the mixture was incubated with 15 μL TPE-HPro probe solutions (400 μM in acetonitrile). Fluorescence intensity was measured after incubation at 37°C for 30 min.For AIE-based gtAm-GOx sensing, gtAm-GOx was serially diluted with deionized water 0–14 μg/mL. Then, glucose . Samples were analyzed by the AIE-based and conventional methods after the following pretreatment: 1.00 g of the chicken muscle sample was exactly weighed and spiked with AMD at 0.5, 1.0, and 2.0 μg/kg. Then, 5 mL 1% acetic acid in acetonitrile was added and well homogenized for 2 min. Subsequently, the homogenate was centrifuged for 5 min at 4,000 g. Finally, 3 mL of the supernatant was evaporated under nitrogen to dryness at 40°C, and the residue was dissolved in PBS and analyzed using an indirect competitive ELISA  and incubated it for 30 min at 37°C. Finally, 42 μL Tris-HCl buffer  was added and the mixtures were added into the TPE-HPro probe solutions (400 μM in acetonitrile) at a 9:1 volume ratio. FL intensity was recorded by the plate reader after incubation at 37°C for 30 min. The calibration curve was analyzed by a four-parameter logistic equation using OriginPro 9.1 .For the AIE-based fluorometric immunoassays, 100 μL AMD-OVA conjugates were added to a 96-well plate and incubated at 4°C overnight. The plate was then blocked with 150 μL 1% BSA in PBS at 37°C for 1 h. The plates were washed three times with washing buffer (PBS containing 0.025% Tween-20) then 50 μL mAb 3F2 was added with 50 μL AMD of varying concentration or samples for competitive reactions. After incubation at 37°C for 30 min, the plate was washed four times and 100 μL gtAm-GOx in PBS (containing 1% BSA and 0.01% Tween-20) was added and incubated for 30 min at 37°C. The plate was washed three times with washing buffer and three times with deionized water. Then, we added 120 μL glucose  and a cis-trans isomerization process of C = N  can even be sensed with the fluorescence spectrophotometer. The sensitivity suggested that H2O2-triggered “turn-on” fluorescence could be further implemented to design a highly sensitive AIE-active “turn-on” sensor.As a proof of principle, we first demonstrated that Hntration , the calresponse  inset. M2O2-generating enzyme that can modulate the AIE process for fluorescent measurements. To achieve this goal, we employed commercially available gtAm-GOx to effectively generate H2O2, as in our previous studies  H2O2 was efficiently formed by the GOx-catalyzed oxidation reaction; (II) the AIEgen could be hydrolyzed by H2O2 even at 5 μM to produce strongly emissive AIE aggregates. Therefore, we expect to achieve highly sensitive detection by combining the AIE-active “turn-on” fluorescent sensor with conventional immunoassay technology.When developing an enzyme-mediated immunoassay, the main challenge is finding a H process . The FL 2O2 formation and subsequent of AIE induction and fluorescent “turn-on” signal generation. Compared with traditional ELISAs, the only difference was that glucose rather than TMB was introduced into the signal generation step. Therefore, the procedures of our newly established AIE-based fluorescence “turn-on” immunoassay were nearly the same, and thus were fully compatible and comparable with current ELISA platforms. Instead of the absorbance values of a traditional ELISA, the fluorescence “turn-on” from the AIE-based sensor further amplified the signal output and enhanced the detection sensitivity.We further employed this AIE-active “turn-on” fluorescent sensor for highly sensitive indirect competitive ELISAs . AMD-OVA2O2 was generated. Thus, no noticeable H2O2-induced “turn-on” fluorescence was obtained in the presence of high concentration of AMD. With decreasing AMD concentration of, fluorescence intensity gradually increased (blk)/(FL0-FLblk)%] was used to quantitatively evaluate AIE process level .To evaluate the sensitivity of our AIE-based indirect competitive ELISA, various concentrations of AMD were tested (0.06–13.5 ng/mL). Because of the competitive nature of this immunoassay platform, an increased AMD concentration led to intense competition, such that less primary and GOx-labeled secondary antibodies were bound on the ELISA plate; and subsequently less Hncreased . Relativ50 value of a conventional TMB/HRP-based ELISA was 0.93 ng/mL, while our newly established AIE-based fluorescence “turn-on” immunoassay provided a higher sensitivity; the IC50 value was 0.38 ng/mL. This suggests approximately 2.5-fold improvement in sensitivity. The limit of detection  was determined to be 0.06 ng/mL. This improved sensitivity relied on the following two principles: first, H2O2 was efficiently generated by the enzyme-catalyzed oxidation reaction; and second, the production of strong emissive AIE aggregates could be triggered by low concentrations of H2O2. These unique features make this “turn-on” fluorescent immunosensor an attractive immunoassay platform for highly sensitive detection.We next compared our newly designed AIE-based ELISA with a conventional ELISA . These r2O2 was generated by GOx, which decreased the AIE-based fluorescence “turn-on.” Because of the complexity of chicken muscle samples, a pretreatment step was required. The color intensity change obtained for negative and positive chicken samples could be visually distinguished using a hand-held UV lamp (Finally, having successfully shown that the assay could quantify AMD, the “turn-on” fluorescent immunosensor was further evaluated using real food samples. AMD-contaminated chicken samples were used as a model. Chicken muscle samples were spiked with AMD at concentrations of 0.5, 1.0, and 2.0 μg/kg. In the indirect competitive immunoassay, spiked AMD in chicken samples competed with coating antigen for binding of the primary antibody. Thus, as the level of competition increased, less H UV lamp . To quan UV lamp . Therefo2O2-stimulated TPE-HPro aggregation. This cascade reaction makes use of the catalytic ability of the enzyme and the AIE characteristics, which lead to the signal amplification of the immunoassay. The AIE-based fluorogenic ELISA has much higher sensitivity due to the efficient generation of H2O2, which leads to a high fluorescence emission at low concentrations of H2O2. Compared with conventional HRP/TMB-based ELISAs, this newly designed immunoassay offers a 2.5-fold enhancement in sensitivity. Furthermore, this novel sensing strategy can be directly adapted to the current indirect competitive conventional ELISA format using an H2O2-triggered AIE fluorescence “turn-on” as the signal generation mechanism. This represents an alternative to the existing commercial immunoassay. More importantly, this approach can be applied to quantifying AMD in real food samples. Therefore, the developed AIE-based fluorescence “turn-on” ELISA has excellent potential for use in the testing of other drug residues in food samples.In conclusion, we developed an AIE-based fluorescence “turn-on” immunoassay by combining a GOx-triggered AIE process with an indirect competitive ELISA for the highly sensitive detection of drug residues in foodstuffs. In this approach, the fluorescence “turn-on” is triggered by the GOx-catalyzed oxidation of glucose and HWY planned and supervised the experiments, processed the raw data, and wrote the manuscript. YL synthesized TPE-HPro. BX performed the experiments. MM, CC, CL, and XY discussed the results and processed the data. ZW, KW, BT, and JS revised and approved the manuscript. All authors reviewed the manuscript and approved it for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The paraffin-enabled transfer process could open realms for the development of high-performance ubiquitous electronics based on large-area two-dimensional materials.The performance and reliability of large-area graphene grown by chemical vapor deposition are often limited by the presence of wrinkles and the transfer-process-induced polymer residue. Here, we report a transfer approach using paraffin as a support layer, whose thermal properties, low chemical reactivity and non-covalent affinity to graphene enable transfer of wrinkle-reduced and clean large-area graphene. The paraffin-transferred graphene has smooth morphology and high electrical reliability with uniform sheet resistance with ~1% deviation over a centimeter-scale area. Electronic devices fabricated on such smooth graphene exhibit electrical performance approaching that of intrinsic graphene with small Dirac points and high carrier mobility (hole mobility = 14,215 cm The transfer process of as-grown graphene limits its electrical performance and reliability. Here, the authors develop a transfer approach using paraffin as a support layer and obtain wrinkle-reduced and clean large-area graphene retaining high mobility. Although mobility values as high as 200,000 cm2 V−1 s−1 have been reported for freely suspended graphene exfoliated from bulk crystal at 5 K2, the mobility values reported for large-area graphene is several orders of magnitude lower, regardless of the growth method or substrate employed7. This significant decrease in carrier mobility in large-area graphene can be attributed to four factors: (1) polycrystalline nature of graphene, (2) effect of surrounding medium, (3) contamination from transfer support layer, and (4) wrinkles present in graphene. Any of these factors can reduce the carrier mean free path in graphene, which could act as a source of extrinsic scattering, thus limiting the carrier mobility8. To address the first factor, there are considerable advances in the synthesis of wafer-scale single-crystal graphene domains10, with the latest achievement of 1.5-inch-large graphene monolayer synthesized on Cu–Ni alloys11. As for the second factor, the effects of the surrounding medium on graphene can be minimized by fully encapsulating the graphene layer within insulating substrates through van der Waals assembly approach12. For instance, a mobility value of 110,000 cm2 V−1 s−1 at 1.6 K was reported for an as-synthesized chemical vapor deposition (CVD) graphene flake sandwiched between hexagonal boron nitride (h-BN) flakes13. Furthermore, to fabricate graphene-based functional electronics, graphene must be transferred from the growth substrate to a destination substrate. Since graphene is only one atom thick, a support layer is required during the transfer process to prevent cracks from appearing and propagating in the graphene film. To date, after one decade of research efforts, the most widely used support layer for graphene transfer is still polymethylmethacrylate (PMMA). However, the PMMA-assisted transfer process engenders two major issues that severely degrade the carrier mobility in graphene: polymer contamination and graphene wrinkling (third and fourth factor mentioned above). To overcome these two issues, support layers made of other polymers and organic molecules have been attempted21, but none of these solutions fully solved both issues.Charge carriers in graphene exhibit ultrahigh mobility owing to their zero-mass nature in the linear E−k dispersion at low energy, where the conduction and valence bands meet at the Dirac points22. Here, we propose to use paraffin as a transfer support layer based on two rationales: (1) paraffin is an alkane with a simple unreactive chemical structure and (2) it has a high thermal expansion coefficient. Wrinkles form in graphene either natively during the growth process23  or during the graphene transfer process, where water (including vapor) can be trapped between the graphene and the destination substrate24. To address the graphene wrinkling issue, we scoop up the paraffin-supported graphene layer with a destination substrate from deionized water at a higher temperature of 40 °C, rather than the usual room temperature. At 40 °C, the paraffin support layer undergoes thermal expansion due to the small amount of heat from the water25. This expansion can stretch the graphene film underneath and thus effectively reduces wrinkles in graphene.We have shown earlier that paraffin can be used as a flexible substrate to transfer graphene onto itOur paraffin-enabled transfer technique can concurrently addresses both support layer contamination and wrinkling in graphene, enabling transfers of large-area graphene with homogeneous and enhanced electrical properties. Compared to PMMA, paraffin is found to leave much less residue on graphene, as it does not contain carbonyl (C=O) functional groups that react with electrophiles or nucleophiles. Moreover, paraffin radicals are not expected to form covalent bonds with graphene. Our density functional theory (DFT) calculations confirm that paraffin has lower adsorption energy with graphene compared to PMMA, which supports the effective curtailing of contamination observed in our paraffin-transferred graphene. Through atomic force microscopy (AFM) and Raman studies, we confirm that paraffin-assisted transfers substantially reduce contamination and wrinkling in graphene compared to PMMA, and moreover, the paraffin-transferred graphene has near-intrinsic levels of doping and strain. Consequently, field-effect transistors fabricated on paraffin-transferred graphene exhibit near-zero Dirac voltage and the electron mobility that is ~fourfold higher than that of our PMMA-transferred graphene. Our paraffin-enabled transfer technique presented in this work offers a solution for the development of high-performance large-area graphene-based electronics by minimizing the charge carrier scattering centers in graphene.26. As can be seen in Fig. f and Li denote the final and initial lengths of a material; α is the linear thermal expansion coefficient of a material 25; and Tf and Ti are the final and initial temperatures of a material, respectively. Thermal expansion of paraffin translated to tensile strain on the graphene film underneath, which stretched and diminished wrinkles in the graphene film . Subsequently, resistivity and Hall measurements were made on each paraffin-transferred graphene film in the presence of 2800 Gauss magnetic field to extract its sheet resistance, carrier concentration, and Hall mobility values. During the Hall measurements, the magnetic field was applied by attaching a piece of magnet underneath the sample with desired polarity. For comparison, PMMA-transferred graphene film on Si/SiO2 substrates were also fabricated with the same dimensions (see Methods for details) and measured. To avoid run-to-run variations, the graphene used in this study was synthesized on the same Cu foil in the same CVD growth cycle and transferred on SiO2/Si substrates that were diced from the same SiO2/Si wafer.We observed homogeneous and improved electrical properties in the polycrystalline CVD graphene prepared with our paraffin-enabled transfer technique. To determine the electrical properties, CVD graphene was cut into 15 × 15 mm2 V−1 s−1, which was 2.6 times higher than the PMMA-transferred graphene (1698 ± 377 cm2 V−1 s−1). Furthermore, the carrier concentration of paraffin-transferred graphene ranged from 0.9 to 2.6 × 1012 cm−2, which was lower than that of PMMA (7.2 to 12 × 1012 cm−2). The lower carrier concentration indicated that the paraffin did not dope graphene as heavily as the PMMA. We note that no annealing was performed on any transferred graphene in this study. For the same group of graphene samples, sheet resistance was also extracted using the van der Pauw method and plotted in Fig. 2, with 1 mm step size in both the x- and y-directions. The spatial sheet resistance maps of both PMMA- and paraffin-transferred graphene are plotted in Fig. 14.In Fig. 2 substrates and tested ; Lch and Wch the channel length and width; IDS the drain-to-source current; VDS the drain voltage; and VG the back-gate voltage. For the typical transistor fabricated on paraffin-transferred graphene in Fig. 2 V−1 s−1, while for the PMMA-transferred graphene, the extracted hole and electron mobilities were much lower, at 3,719 and 1,653 cm2 V−1 s−1, respectively. Transferring graphene with paraffin greatly enhanced the effective hole and electron mobilities by 3.8 and 4.5 times, respectively, due to reduced charge carrier scattering centers in the paraffin-transferred graphene. Figure 28.To examine the electrical performance of devices fabricated with paraffin-transferred graphene, more than 100 graphene back-gated field-effect transistors (FETs) were fabricated on Si/SiOted Fig. . SimilarVDirac) of the device fabricated on paraffin-transferred graphene was much smaller and closer to zero compared to that of PMMA. The VDirac of a graphene device is the specific back-gate voltage where the device operates with minimum conductivity, corresponding to the Dirac point or charge neutrality point where the graphene film’s conduction and valence bands meet, and intrinsic graphene exhibits minimum conductivity when gate voltage equals to zero. Hence, VDirac is a good indicator showing how intrinsic a graphene film is. Figure VDirac distribution of 100 graphene FETs fabricated with both PMMA- and paraffin-enabled transfer process. The VDirac of PMMA-transferred graphene had a spread from 24 to 56 V with a mean value of 38.7 V, while that of paraffin-transferred graphene had a much narrower distribution between 4 and 12 V with a mean value of 7.4 V, which is five times smaller than the PMMA-transferred graphene. The lower mean value for VDirac signified that the paraffin-transferred graphene exhibited electrical properties that were closer to intrinsic graphene compared to that of PMMA, although the paraffin-transferred graphene was also contaminated by some PMMA residues during the device fabrication process. The improved process of transferring graphene might play a vital role here in determining the electrical performance of large-area graphene devices, even though the device fabrication process was not optimized to prevent contamination by PMMA residues.Remarkably, the back-gate measurements also indicated that paraffin did not induce significant doping effects on graphene, consistent with the Hall measurements in Fig. 2 substrates. As can be seen, our paraffin-transferred graphene sample has no C=O and O=C–OH peaks, and the C–OH peak intensity is relatively weak, indicating lower levels of polymer contamination. Figure 2 substrate using PMMA and paraffin support layers, respectively. We note that all graphene samples were not annealed after the transfer process. Compared to PMMA-transferred graphene, paraffin-transferred graphene had a much cleaner surface  are not observable on the graphene surface. For the removal of graphene wrinkles, we inferred that the paraffin support layer could effectively smoothen these wrinkles through thermal expansion and releasing the compressive strain generated in graphene during the cooling step in the CVD growth process. To ascertain this inference, we collected 3600 Raman spectra from each type of the transferred graphene and analyzed the strain-doping relationship. Supplementary Fig. 2 substrate. The characteristic D band peak, typical in the presence of defects, was absent in paraffin-transferred graphene, indicating the transferred graphene was of high quality. In Fig. 2D) for the PMMA-transferred graphene. The black circle represents the G and 2D peak positions of an intrinsic graphene, where graphene has neither doping nor strain , we envision the integration of paraffin support layer in industrial scale production of wrinkle-free, pristine two-dimensional materials in near future.2 substrate) was then used to scoop the paraffin-supported graphene sample from the 40 °C deionized water from only one end. After that, the sample was baked at 40 °C in an oven for more than 24 h to minimize the residual water. In the event of incomplete removal of residual water, minor cracks randomly generated in the graphene  was contacted with the PMMA-supported graphene film and the sample was initially dried with nitrogen gun blowing, followed by an oven baking at 80 °C for at least 8 h. Subsequently, the sample was soaked in acetone at room temperature for 6 h to remove the PMMA support layer. The sample was then rinsed with IPA followed by nitrogen blow dry. We note that all process parameters chosen for the PMMA-supported graphene transfer in this work were adopted from earlier reports26. A separate experiment was conducted to further verify the advantageous of paraffin support layers over PMMA  950 A5 (Microchem Inc.) was spun at 2500 rpm for 1 min on the graphene synthesized above Cu foil. The PMMA-coated graphene was then baked in an oven at 80 °C for 1 h. Next, the sample was floated on top of Cu etchant  for 30 min to remove the growth substrate. The PMMA-supported graphene film was then rinsed with deionized water multiple times. Subsequently, the destination substrate (i.e. Si/SiO2 using either paraffin or PMMA support layers. The sample was then spin-coated with a 300 nm thick layer of polymethylmethacrylate (PMMA) 950 A5 (Microchem Inc.) and baked at 180 °C on a hotplate for 180 s. The source/drain contacts on graphene were delineated using electron beam lithography (EBL) and metallized with 20/30 nm of Ni/Au, followed by a 12-hour lift-off process in acetone at room temperature. Subsequently, the graphene film was delineated into a 5 µm wide ribbon using EBL followed by oxygen plasma etching. Finally, the sample was soaked in acetone for more than 12 h at room temperature to remove the PMMA electron beam resist. The dimensions of all graphene devices were kept constant in this work. The graphene channel and contact widths are 5 µm, while the channel length ranges from 1 to 11 µm. Ni was chosen as electrode as it is one of the metals that has been shown to provide reliable metal contacts to graphene31. No annealing is performed on any graphene devices prior to electrical measurements.Graphene synthesized on Cu foil was first transferred on an oxidized degenerately p-doped silicon substrate with 285 nm thick SiO−1, respectively. The laser beam size was ~500 nm and the laser power on the sample was adjusted to around 0.75 mW to avoid laser-induced heating. Atomic force microscopy (AFM) topography image was obtained using Veeco Digital Instrument Nanoscope III in tapping mode.All Raman spectra and maps were taken using a WITec Alpha300R system. The laser excitation wavelength and grating density were 532 nm laser line and 600 mm−1 from 0 to 100 °C in a nitrogen gas atmosphere.The measurement was run at a constant heating rate of 10 °C min32. The B3LYP functional was employed together with the 6-31 G* basis set to compute the optimized geometries, energies, and orbitals. All calculations were performed in implicit solvent using the conductor-like polarizable continuum (CPCM) model and the basis set superposition error (BSSE) was corrected with the geometrical counterpoise (gCP) method.DFT calculations were performed using the ORCA computational packageThe dual descriptor of the Fukui function, a local conceptual DFT reactivity descriptor, was determined to spatially locate where eventual chemical reactions would take place. Originally introduced by Parr and Yang, the Fukui function can be interpreted in two ways: the sensitivity of the chemical potential of a system to an external perturbation at a particular point, or the change of the electron density at each point around the molecule when the total number of electrons is altered. For our purposes, the dual descriptor of the Fukui function detailed the different locations where each molecule would undergo nucleophilic or electrophilic attacks upon chemical reaction. The Fukui function was determined from electron density differences between the neutral and the positively- and negatively-charged PMMA or paraffin molecule, according to the following expression:The chemical hardness, a global conceptual DFT descriptor, characterizes the polarizability of molecules as a measure of the resistance of a chemical species to changes in its electronic configuration. It can be interpreted as the global tendency of a molecule to give or accept electrons. Molecules with low polarizability are considered to be “hard” while molecules with high polarizability are conversely “soft”. The chemical hardness is formally calculated from the ionization energy and the electron affinity. Based on Koopman’s theorem, the first ionization energy can be approximated to the energy of the HOMO, while the electron affinity can be approximated to the energy of the LUMO. Thus, the chemical hardness was approximated from the energy “gap” between the HOMO and LUMO of the molecule,Supplementary InformationPeer Review File


We use large area chemical vapor deposited (CVD) graphene and MoS2, enabling large scale realization of these devices. These devices can emulate the most vital properties of a neuron, including the all or nothing spiking, the threshold driven spiking of the action potential, the post-firing refractory period of a neuron and strength modulated frequency response. These results show that the developed artificial neuron can play a crucial role in neuromorphic computing.With the ever-increasing demand for low power electronics, neuromorphic computing has garnered huge interest in recent times. Implementing neuromorphic computing in hardware will be a severe boost for applications involving complex processes such as image processing and pattern recognition. Artificial neurons form a critical part in neuromorphic circuits, and have been realized with complex complementary metal–oxide–semiconductor (CMOS) circuitry in the past. Recently, metal-insulator-transition materials have been used to realize artificial neurons. Although memristors have been implemented to realize synaptic behavior, not much work has been reported regarding the neuronal response achieved with these devices. In this work, we use the volatile threshold switching behavior of a vertical-MoS SNNs, the third generation of neural network models, have been found to be more hardware friendly and energy efficient; thus making them more biologically realistic compared to other neural networks2. The key components of an SNN are artificial neurons and synapses where the synapses connect the pre-neurons and the post-neurons. SNN uses discrete rather than continuous spikes along with the incorporation of the concept of time. It involves efficient transfer of information based on precise timing of a sequence of spikes. Important synaptic behaviors such as synaptic plasticity, long term potentiation and depression (LTP and LTD), and Spike Time dependent Plasticity (STDP) have been studied using emerging devices such as memristors for information processing, pattern recognition and learning methods of SNN5. But, neuronal response has mostly been realized using complex CMOS circuitry, which requires several transistors to mimic a single neuron. These circuits involve a large number of active components, increasing the power dissipation and making high density integration difficult7. To overcome these issues, various emerging devices have been used to emulate a biological neuron behavior. Insulator to metal transition (IMT) in materials such as vanadium dioxide (VO2)9 and niobium dioxide (NbO2)10, chalcogenide-based phase change materials11 and magnetoelectric switching of ferromagnets such as bismuth ferrite (BiFeO3)1, have been considered to develop various types of artificial neurons. VO2 has a very low critical temperature of ~340 K above which its switching behavior disappears, severely limiting the operational range of devices and making them unsuitable for on-chip integration12. In case of phase change materials-based neurons, the degree to which the neuronal characteristics can be tuned with nominal circuitry is limited by the fact that switching properties arise from the physics of crystallization11. For the same reason, the switching speed in phase change neuron is slow. Threshold switching in memristors can be a natural choice for realizing artificial neurons. However, the exploitation of threshold switching in memristors to realize artificial neurons is relatively unexplored. Recently, a Ag/SiO2/Au threshold switching memristor (TSM) was used to demonstrate an integrate-and-fire neuron13. The observation of volatile threshold switching in MoS2 stimulates the possibility of employing this phenomenon for the realization of artificial neurons14. However, the switching in a lateral MoS2 film mediated by the grain boundaries occludes scaling of lateral device dimensions. Hence, realizing such characteristics using a vertical structure can play a critical role in improving scaling opportunities.Understanding the complex and overwhelming functioning of the human brain has always fascinated scientists and researchers for biological fields and beyond. The way the human brain can process huge amounts of data and handle pattern recognition with relative ease is immensely compelling and its complexity is continuously evolving. The building blocks in the brain consist of interconnected neurons and synapses, which cooperate efficiently to process incoming signals and decide on outgoing actions. Inspired by the operation of the human brain, various neuromorphic devices, circuits, and systems have been developed that can work analogous to the brain. Owing to the low power dissipation and characteristics that are similar to biological neurons, spiking neural networks (SNN) have garnered huge interests for mimicking and closely resembling the neuro-biological system2 (v-MoS2) layers. A novel device structure was fabricated by growing v-MoS2 on a CVD-graphene monolayer as the bottom electrode and with Ni as the top contact to v-MoS2. The key characteristics of a biological neuron including an all or nothing spiking, a threshold driven spiking, a post firing refractory period and an input strength modulated frequency response were observed. The developed v-MoS2/graphene TSM artificial neuron can operate over a wide range of temperature and can potentially be scaled down to nanometer scale cross bar structures. This platform provides an attractive prospect for neuromorphic circuits, which can be seamlessly integrated with existing fabrication technologies enabling large scale realization of such devices.In this paper, we have constructed an integrate-and-fire artificial neuron by exploiting the threshold switching behavior in CVD-grown vertical MoS+, K+, Cl− and Ca2+ in and out of the neuron cell. The potential difference between the interior and the exterior of the neuron is referred to as the membrane potential1. As the membrane potential (VM) builds up, the neuron produces an output spike (Iout) when the potential reaches a particular threshold voltage (Vth)16. This phenomenon of neuron spiking is shown in Fig. 17. After the refractory period, the neuron starts integrating the input again and prepares for the following spike.The schematic of a biological neuron connected to multiple synapses is shown in Fig. 18. An IF artificial neuron fires as a function of the electrical potential developed across it, thus mimicking the crucial behavior of a biological neuron. Here, this behavior has been realized with a v-MoS2/graphene TSM as shown in the conceptual scheme in Fig. 2/graphene neuron integrates the input signals received with the help of a capacitor. The capacitor integrates the charge and as soon as the voltage across the capacitor increases beyond the threshold value of the TSM, the neuron fires and an output spike is produced.One of the most commonly used neuron models in computational neuroscience is the integrate-and-fire (IF) model2/graphene device is shown in Fig. 2 substrate19, followed by patterned growth of v-MoS2 on graphene. Nickel contacts were deposited on the graphene and on the v-MoS2. The optical image of the device is shown in Fig. 2/graphene Threshold Switching Memristors (TSMs) is shown in Fig. 2 film over graphene in the device. Two bands corresponding to the in-plane E12g mode and the out-of-plane A1g mode were observed at 383 cm−1 and 411 cm−1 respectively. The presence of these two distinct high intensity peaks and the difference of about 28 cm−1 between their two positions signifies the presence of good quality multilayer MoS220. On pristine graphene, as shown in the inset of Fig. viz. the G band at 1590 cm−1, and the 2D band at 2690 cm−1. The peak intensity ratio I2D/IG is about ~2, indicating that the monolayer graphene used for our devices is of good quality21. After sulfurization, the spectrum, presented in Fig. −1, the G band at 1590 cm−1 and the 2D band at 2690 cm−1. A significant decrease in intensity of the 2D band with respect to the G band is revealed. This reduced value of the peak intensity ratio of the G and 2D peak and the large D band indicate the introduction of defects in the graphene layer during sulfurization22. The detailed analysis of the v-MoS2/graphene 2D-2D van der Waal’s heterojunction was carried out by High Resolution Transmission Electron Microscopy (HRTEM). Figure 2 on graphene/Si/SiO2. It can be clearly observed that MoS2 grows vertically on the graphene surface with high density of exposed edge planes. The vertical orientation of 2D MoS2 atomic layers was obtained by the sulfurization of thick Mo films, which essentially minimizes the strain energy arising as a result of volume expansion during Mo to MoS2 conversion23. This eventually leads to the formation of polycrystalline MoS2 structure with vertically orientated grains perpendicular to the substrate. The thickness of the CVD grown v-MoS2, as determined by using Atomic force microscopy (AFM)  until a voltage higher than the threshold voltage is applied. At the onset of the threshold voltage (V1), the device undergoes an abrupt transition from the HRS to a low resistance state (LRS). During the reverse sweep, the device reverts to HRS as the voltage reduces below a particular value (V2). It is interesting to note that the device retains the volatile threshold switching behavior for all the applied current compliances.To assess the switching characteristics with respect to the current compliance, the v-MoS2/graphene device, we now concentrate on the demonstration of an artificial neuron exploiting the volatile switching. The schematic of the circuit used for realizing the artificial neuron with the v-MoS2/graphene TSM is shown in Fig. p = 1 MΩ and a capacitor Co = 100 nF, which are connected in series to a resistor Ro = 10 kΩ. A load resistor RL = 1 MΩ is connected in series with the TSM to measure the output voltage. The resistors and the capacitor are connected to the device externally through a breadboard. The parallel resistor ‘Rp’ is connected to drain excessive current away from the device, thereby protecting the device. Its value is chosen to be 1 MΩ which is less than the RLRS = ~3 MΩ and greater than Ro such that Co does not discharge through Rp. The input source is a train of constant amplitude voltage pulses applied to the left node of the circuit with frequency 5 kHz and TON = 100 µs. The output voltage is measured across the RL. In Fig. ON = 100 µs are applied to the circuit, the capacitor starts charging and the voltage starts increasing at node A (black line) till the capacitor gets completely charged. Initially, the TSM is in its HRS and the device resistance ‘RHRS’ is high (~10 MΩ) compared to Ro. The charging time constant (RoC) is ~100 times lower than that for the discharging time constant [(RP||RHRS|| RL) Co], hence the leakage through the device is almost negligible. Therefore, on application of the input pulses to the circuit, the capacitor (Co) gets charged. As the charge across the capacitor increases and the voltage at node A reaches the threshold voltage of the TSM, the device switches from the HRS to the LRS. Since the TSM is now in the LRS state, the capacitor starts discharging through the device and an abrupt increase in the current appears at the rightmost node (output). This corresponds to the increase in voltage at t ≈ 300 ms, observed in Fig. After confirming the volatile threshold switching behavior of v-MoS13. Once the refractory period is over, the device reverts from the LRS state to its HRS and the capacitor ‘Co’ starts integrating again. The refractory period from the v-MoS2/graphene TSM is shown in Fig. During the timeframe when the neuron is firing, the net charge integration across the capacitor is essentially zero. This is because any input voltage pulse applied is being directly drained by the TSM device (it being turned on). This period emulates the post firing refractory period of a biological neuron13. To emulate this behavior using the developed artificial neurons, a train of pulses with the same frequency 5 kHz (Ton = 100 µs) but two different amplitudes are applied to the v-MoS2/graphene neurons. On application of input pulses of 7.5 V to the circuit  and from the LRS to the HRS (V2) are plotted over the 40 cycles shown in Fig. 1 and V2 is maintained over the cycling period. Also, it is observed that V1 and V2 are probabilistic in nature and follow a stochastic distribution. Figure 2/graphene TSM, which, in turn, produces the neuron spiking probability. Additionally, the stochastic switching behavior of the v-MoS2/graphene TSM originating from device level stochasticity makes these probabilistic spiking neurons viable for applications in the field of hardware security, such as Random Number Generators (RNGs)25.We studied the DC characteristics of the TSM for over 40 cycles to shed light on the nature of the switching voltages. It is observed that the device retained the abrupt transition from the HRS to the LRS over this period as shown in Fig. 2 can be attributed to the grain boundaries (GBs)14. The switching behavior of our v-MoS2/graphene devices can be attributed to the multiple grain boundaries in the polycrystalline films of v-MoS2. To further comprehend the switching mechanism in the v-MoS2/graphene devices, the DC measurements were carried out in vacuum. It is interesting to note that in the absence of oxygen, the fabricated devices do not show the volatile switching behavior  demanding high input voltages for the circuit operation, the v-MoS2 stack can be engineered in the future by modifying its thickness and growth conditions to allow larger currents to flow through the circuit. This will consequently reduce the voltage requirements of the circuit as well.In summary, we have demonstrated a novel 2D material-based TSM comprising v-MoS2 (300 nm SiO2) substrate. The graphene was then patterned by photolithography and etched by oxygen plasma. The sample was again patterned and 10 nm thick Mo films were deposited on the etched graphene using an electron beam (e-beam) evaporation system . The Mo films were subsequently sulfurized to MoS2 in a low-pressure CVD furnace. 50 nm Ni contacts on both graphene and MoS2 were patterned, e-beam evaporated and lifted off.First, commercially purchased CVD-grown monolayer graphene was wet transferred over a Si/SiO2 substrate were loaded in the center of a quartz tube CVD furnace with a ceramic boat having sulfur powder at the upstream of Mo sample. The furnace was pumped down to a base pressure of ≤1 mTorr and then purged with argon (Ar) gas. The furnace was heated up to a temperature of ∼650−700 °C with a constant flow of 100 SCCM (standard cubic centimeters per minute) Ar gas at a pressure of ∼100 mTorr. After a 30-min reaction time, the furnace was naturally cooled down and the substrate was taken out of the furnace. The change of color of the substrate from blue to dark green, indicates the sulfurization of Mo on the graphene.The patterned Mo film on graphene/Si/SiOThe Raman spectra were obtained using a confocal Raman system (WITEC Alpha 300RA) in ambient conditions with a 532 nm laser source for excitation. The laser was focused with a Zeiss 50× objective lens  with power ~5.7 mW and integration time 5 s. Each raman spectra were taken over 10 scans. The AFM topography was acquired on an Anasys NanoIR2 system in tapping mode using Anasys tapping mode AFM probes (Model No. PR-EX-T125-10). The TEM imaging was carried out using JEOL ARM-200F equipped with an aberration corrector which provides spatial resolution down to ~1 Å at an operating voltage of 200 kV.The electrical measurements were carried at room temperature in Micromanipulator 6200 probe station using a Keysight B1500A semiconductor device analyzer along with WGFMUs for pulse I-V measurements. The vacuum measurements were carried out in a Janis cryo-probe station. A Tektronix DPO 2024B oscilloscope was used to measure the node voltage.Supplementary Information


Sarcoidosis, an inflammatory multi-organ disease with a wide variety of clinical manifestations, affecting people of working age. Patients suffer from a broad spectrum of physical symptoms of varying severity that impact function including cognitive impairment and disabling fatigue. The Dutch Sarcoidosis Society identified a knowledge gap in various facets related to work ability. The aim of this study was to assess sarcoidosis patients’ perceived problems related to work performance, employer, and disability evaluations.A cross-sectional web-based anonymous survey was conducted among Dutch sarcoidosis patients recruited through sarcoidosis patient societies and outpatient sarcoidosis clinics. This investigation queried work performance, employer support, and disability evaluations.n = 328) had undergone disability evaluation and were significantly more likely to experience extrapulmonary symptoms, severe fatigue, reduced exercise capacity along with memory problems and concentration problems with higher mean FAS and SFNSL-scores. Of these 328, 37% (n = 121) perceived they had not been listened to or taken seriously at assessments, and 38% (n = 124) disagreed with the outcome of disability assessments by benefits authorities; 75% (n = 93) appealed or requested re-assessment.The study sample included 755 patients of whom 43% (A better understanding of sarcoidosis-related impact on work ability and quantification of disease burden is needed. Education for medical examiners and employers on sarcoidosis may improve quality of assessments and work accommodations. Development of guidelines for benefit authorities, which consider the broad impact of sarcoidosis beyond that of reduced pulmonary function, including extra-pulmonary assessment like fatigue, cognitive difficulties, as well as other organ involvement are needed. Sarcoidosis generally occurs among the relatively young, working population. Sarcoidosis is a heterogeneous multi-organ system disease whereby the immune system launches a response to an unknown antigen resulting in granulomatous lesions occurring most anywhere in the body interfering with resident organ function. The lungs, eyes, and skin are the most commonly recognised; but cardiac, brain, spinal cord, and hepatic involvement are not uncommon .Depending on granuloma burden, location, and sensitivity of the organ involved ; symptoms related to sarcoidosis vary in type, severity, and extent of disability . Apart fThese impairments can be organ-related, such as dyspnoea or exercise intolerance related to pulmonary or cardiac involvement; but might also be non-organ related, non-specific. Apart from major organ involvement, sarcoidosis can involve reduced muscle strength, loss of physical condition, pain, extreme fatigue, and small fiber neuropathy (SFN)-associated symptoms. Psychological factors are pervasive including anxiety, concentration and memory difficulties, and depressive symptoms; and may not correspond with inflammatory disease activity nor respond to sarcoidosis treatment , 6. Theswww.sarcoidose.nl) [Sarcoidosis is associated with a high number of illness and health visit related sick-days and associated with large yearly income loss –11 that dose.nl)  reportedThe aim of this study was to assess difficulties sarcoidosis patients may have experienced regarding their work including performance, absenteeism, environment, employer policies, and disability evaluations. These experiences were examined in relation to disease burden.In cooperation with the Dutch Sarcoidosis Society, Sarcoidose.nl  and the Patients were recruited through membership of the Dutch Sarcoidosis Society, Sarcoidose.nl  via the Surveymonkey (www.surveymonkey.com) [Patients were provided the specific link to the survey through the online questionnaire tool key.com)  which qukey.com)  and the key.com) .The FAS is a ten-item self-report fatigue questionnaire rated on a five-point scale  providing a total score ranging between 10 and 50, and mental and physical sub-scores. A score > 21 indicates fatigue with > 34 indicating extreme fatigue. The FAS demonstrated good reliability and validity in sarcoidosis .The SFNSL is a 21-item self-administered screening questionnaire for SFN-related symptoms on a five-point scale . Scores range from 0 to 84, with scores between 11 and 48 indicating probable to highly probable SFN and > 48 indicating SFN .t tests, depending on type of variables (continuous or dichotomous). A probability (p) value of < 0.05 was considered to be statistically significant.All statistical analyses were performed using SPSS version 20 for Windows. Standard descriptive statistics were computed. After subdivision into assessed- and non-assessed for disability benefits, these two sarcoidosis patient samples were compared using Chi-square test and independent n = 328) reported having undergone disability assessments by the benefits authorities; 427 patients reported no assessment , and also had significantly more extra-pulmonary symptoms, extreme fatigue, reduced exercise capacity, and memory and concentration problems. Moreover, mean FAS and SFNSL-scores were significantly higher in the assessed group.Of those assessed, 75% were on treatment compared to almost two-thirds of the non-assessed sample (n = 267) at the time of the survey, 69% reported frequently finding work too strenuous, and reported sarcoidosis-related absence for shorter or longer periods. Among those assessed (n = 328), 37% perceived not having been listened to or taken seriously during assessment, while 10% had mixed experiences. Thirty-eight percent (n = 124) of those assessed disagreed with the assessment outcome; of which 75% (n = 93) appealed or requested re-assessment. Table At the time of the survey 17% of the total study sample were on sick leave, 28% were declared fully unfit for work, and 13% declared partially unfit for work. Among those employed  used only medical examiner perceptions to generate a global rating of workability [However, only few of these standardised methods are available and being used to measure impairments in claim assessments. The aforementioned systemic review demonstrated that only six of thirteen insurance-based studies administered one or more specific work-ability instruments; with no instrument reported as validated. Most studies , or other muscle and exercise testing in assessment guidelines may increase the accuracy of these assessment outcomes. A recent patient survey reinforced the need for clinical indicators to be used in tandem with patient-centred healthcare, including attention to work ability and other supportive measures .Furthermore, this discordance underscores the limited knowledge that clinicians generally possess regarding sarcoidosis as a disease of extra-pulmonary involvement. Fatigue is a central concern in sarcoidosis , 15, ourSarcoidosis patient organisations are an immense and rich resource to provide integral guidance on and development of effective methods of measuring and incorporating QoL and functionality outcomes with a view to improve sarcoidosis outcomes and management strategies . In lineThe development of a disease specific core set for sarcoidosis under the framework of “the International Classification of Functioning, Disability and Health (ICF)”  with inpA major limitation of the study might be selection bias with symptomatic patients being more likely to be members of a patients’ society or referred to a sarcoidosis clinic, and thus possibly influencing the rate of symptomatic respondents. To date, we recently published a study evaluating self-reported symptoms in three European Countries: Denmark, Germany, and the Netherlands whereby the German and Dutch cohorts were gathered through patient associations, the Danish cohort were patients from a sarcoidosis clinic. The German and Dutch cohorts had similar patient-reported severity as the Danish cohort, that had detailed objective severity data. This finding provides some reassurance regarding the Netherlands self-reported patient association data in this study .Another limitation is that data is self-reported and without verification of diagnosis nor correlation to other objective functional measures. Having hospital established severity data would have enhanced the design—but this is a preliminary study that founds the need for more involved studies. However, symptom burden and perceptions of disability evaluations processes are both intrinsically patient experiences and never before quantified.Although this study was based on self-reported symptoms and data on functional status was not collected, it is clear patients are plagued by multiple symptoms, most prominently fatigue. Earlier studies identified discordance between fatigue and lung function testing or 6MWD.The present study only scratches the surface of many important aspects of work ability, setting the stage for future studies delving into work accommodations, clinical operations and disability assessments . Future Sarcoidosis patients may be more severely disabled than current disability claims assessment protocols for sarcoidosis are equipped to measure; a sufficient extent of objective parameters appear to be lacking. This study showed that a high proportion of the sarcoidosis patients who had undergone a disability evaluation felt their concerns dismissed with many disagreeing with the assessment outcome. Medical examiners’ lack of education regarding scope of sarcoidosis disease burden beyond being a pulmonary disease could explain these perceptions. There is an urgent need for sarcoidosis-related education enhancing work-related medical examinations supported by guidelines that account for extent of sarcoidosis impact on functionality, and therefore work capacity.


Graphene and graphene oxide (GO) structures and their reduced forms, e.g., GO paper and partially or fully reduced three-dimensional (3D) aerogels, are at the forefront of materials design for extensive biomedical applications that allow for the proliferation and differentiation/maturation of cells, drug delivery, and anticancer therapies. Various viability tests that have been conducted in vitro on human cells and in vivo on mice reveal very promising results, which make graphene-based materials suitable for real-life applications. In this review, we will give an overview of the latest studies that utilize graphene-based structures and their composites in biological applications and show how the biomimetic behavior of these materials can be a step forward in bridging the gap between nature and synthetically designed graphene-based nanomaterials. Carbon materials form one of the most abundant groups of materials, and are used in applications that range from electronics to biotechnology ,2. Not o3 bonds return back to a sp2 configuration [Graphene oxide (GO) is a chemically modified graphene that is typically prepared by oxidation and exfoliation of graphite-bearing oxygen functional groups, such as carboxyl (-OOH), hydroxyl (-OH), or epoxy (-O), on their basal planes and edges  with theguration . By chooguration . Robust 2·g−1) [2·v−1·s−1) [−1·K−1) [Graphene (G) exhibits unique thermal, electrical, and mechanical properties arising from its strictly two-dimensional 2D) structure, and offers immense potential for technical applications. Graphene has a large theoretical specific surface area 2630 m2·g−1) , high inv−1·s−1) , a high v−1·s−1) , high th[−1·K−1) , high op[−1·K−1) , and goo[−1·K−1) . The var[−1·K−1) , tissue [−1·K−1) ,20,21, dD structu[−1·K−1) ,23, bios[−1·K−1) ,25, gene30 m2·g−1[−1·K−1) ,28,29,30[−1·K−1) ,32,33.3 [3 are 7.5 × lighter than air [Aerogels are porous solids systems (solid foams) with a predominance of open pores in which the dispersed phase is a gas . Aerogel3 , which a3 . Graphenthan air , possessthan air , a lowerthan air  or nuclethan air . The porthan air . Typicalthan air  to tissuthan air .With regard to the extent of the large possibilities of graphene-based structures, we endeavor to give a brief overview of their use in drug delivery for anticancer therapies, in tissue engineering, and as imaging agents. We start by briefly exploring the toxicity of these materials.The growing biomedical field of applications of graphene-based materials raises questions about their short- and long-term (cyto)toxicity ,45. It i4 cells) did not weaken the cell activity significantly, as shown by an MTT assay [3 plasma [It is worth mentioning that cytotoxicity was observed to be cell-dependent, e.g., a loss in viability was observed for the human lung adenocarcinoma cell line A549) and an increase in viability for the human colon cancer (Caco2) and monkey kidney (Vero) cell lines ,50. The 9 and an TT assay ,56. InstTT assay . Moreove3 plasma . The gro3 plasma . Interes3 plasma .The cytotoxicity aspects of graphene-based materials should not be considered alone. Another important aspect to consider is the degradability of the formed structures, which will also intrinsically depend on the type and size of the used raw material. This issue is still being actively pursued in the field, for example by Mukherjee et al. , who shoTo ultimately reach final applications, graphene-based materials need to be properly assessed in vivo. One of the most basic in vivo studies shows that GO administered at a dose of 0.25 mg via tail-vein injection does not affect the lifespan of mice, while a dose of 0.4 mg is too much and four out of nine mice died within 7 days post-injection. The autopsy showed GO granuloma localized in the lung, liver, spleen, and kidney, but no GO was detected in the brain . FurtherThe biocompatibility of pure-graphene-based structures can be further increased by G/GO surface functionalization or coatings. One of the most frequently used polymers is poly(ethylene glycol) (PEG), which has been shown to decrease cytotoxicity both in vitro  and in iThe aforementioned results emphasize the important role of the surface physicochemical characteristics of graphene and graphene-based materials in their interactions with biomolecules and cells . In partGraphene (G) is highly hydrophobic, whereas GO is decorated by oxygen-containing hydrophilic groups . This unAn ageing population, smoking, stress, and a lack of physical activity are major contributors to the rapid increase in the cancer mortality rates of many types of tumors . It remaN-isopropylacrylamide) (PNIPAM) was used as a polymer matrix for GO nanosheets, which resulted in a hybrid, self-healable, supramolecular hydrogel [+CD133+ cells, which are characterized by high tumorigenicity [Bxl-2 [Another strategy that is utilized in cancer treatment is the formation of injectable hydrogels with GO-based nanofillers that can bind and subsequently release typical hydrophobic anticancer drugs . One exahydrogel . Subsequhydrogel  preparedgenicity . These cy [Bxl-2 .2) functionalized GO nanosheets on a patterned gold surface. Interestingly, the authors observed that the same chip without the GO did not yield the same positive results; however, no discussion about the importance of GO was given. Nevertheless, such studies demonstrate a significant accomplishment towards the development of graphene-based diagnostic chips that can be used as sensors for targeting biomolecules from cancer patients’ samples, eventually leading to cheap cancer sensors.Functionalized GO chips for the highly sensitive capture of circulating tumor cells (CTCs) in the blood of cancer patients were also presented in another study . CTCs arGraphene quantum dots (GQDs) are another form of G used as carriers for cancer drug delivery applications. In one of the studies, GQDs were functionalized by a commonly used arginylglycylaspartic acid (RGD) peptide sequence, which binds to the “αvβ3” integrin site in a cancer cell. When Dox drugs were loaded onto the GQDs-RGD, their enhanced uptake by the PC-3 and DU-145 human prostate cell lines was observed in comparison to pure Dox . Ko et aOverall, many multimodal approaches that combine G/GO structures have been proposed for anticancer therapies. However, we still lack the full in vivo characteristics of the proposed G/GO nanomaterials, and it remains a challenge to assess their biodistribution, biodegradability, and the targeted effects in many types of cancers. Future work in the field will need to focus not only on multimodal approaches but mainly on the applications and feasible sites. In particular, all proposed structures will need to explicitly prove their safety in extensive trials prior to their proper use. Taking into consideration the fact that these materials could one day save someone’s life, their purity, firstly, will need to be of the highest level. To do so, the characterization techniques will need to be properly standardized and regulations will need to ensure that there are proper definitions for the materials and their properties.Engler et al. revolutionized the tissue engineering field in 2006 by showing explicitly the effect of substrate stiffness on the differentiation of mesenchymal stem cells (MSCs) into different cell lineages . They acOne of the interesting approaches for forming composite GO structures is their covalent crosslinking with biopolymers. One such approach was the formation of GO–chitosan hydrogel scaffolds that were prepared by covalent linkage of chitosan amino groups with carboxylate groups of GO . These G2 substrates [Currently, tissue damage remains one of the most crucial aspects that contributes to human death. In this regard, a number of studies have been performed that explore the use of graphene for stem cell engineering and musculoskeletal tissue engineering ,99,105. bstrates . One of bstrates .Human-adipose-derived stem cells (hASCs) have also been extensively studied in tissue engineering due to their being easy to obtain from patients. Chung et al. have investigated the effects of GO-coated substrates on the fate of hASCs . As othe2), poly-m-aminobenzene sulfonic acid- (–NH2/–SO3H), and methoxyl- (–OCH3)) on neurite outgrowth and branching [2 groups) was found to be more beneficial for neurite outgrowth and branching.Graphene-based materials were also explored in musculoskeletal tissue engineering using mouse myoblast C2C12 cell lines . The oxyranching . SurprisOverall, the abovementioned studies highlight the importance of understanding the physical interactions in biomaterials and substrates for biological purposes and emphasize the possibilities of modulating biological responses for tissue engineering by both chemical and physical approaches. Even though there exist a large number of studies on the effect of graphene-based materials, the exact elucidation mechanisms, due to the many combinations of protein–G/GO interactions or the endless chemical functionalization of G/GO, are yet to be revealed. Perhaps the growing power of artificial intelligence and multi-algorithm-based approaches will soon be able to screen the whole library of cell-influencing factors simultaneously to generate ideal tissue engineering platforms for regenerative medicine.Two-dimensional (2D) graphene-based material coatings on substrates and layered materials are insufficient for tissue engineering, and these need to be further developed in order to mimic a proper, functional 3D extracellular matrix (ECM) environment. Indeed, recent advances in this field include the exploitation of graphene foams (GFs) as three-dimensional (3D) scaffolds for a neural stem cell (NSC) culture  and humaAnother approach in tissue engineering is to use pure 3D G foams that are produced via metal-free methods . Pure G Another approach is to use hybrid 3D GFs with additional coatings. One such case was reported in which a commercially available hydrophobic pristine 3D GF was coated by a well-established biocompatible collagen that was cross-linked with genipin to form a hybrid 3D GF porous, hydrophilic, and conductive scaffold for the culture and differentiation of mouse mesenchymal stem cells . The obtObviously, 3D structures that mimic the tissue’s extracellular matrix are highly desirable in the tissue engineering field. Due to their intrinsic structural nature (very high aspect ratios), G and GO have so far mainly been used as coatings and substrates of flat surfaces and materials. However, there is a large trend to scale the results to the 3D world, either by simply incorporating the 2D flakes in a 3D biopolymer matrix or by forming hybrid foam-like structures that can better mimic the desired properties and characteristics of the chosen tissue. We envisage the use of such materials in real-world applications and wait for the future outlook, which will most likely consist of changes to the mass production of large quantities due to the increasing use of additive manufacturing techniques. In particular, we see a large rise in the 3D printing of graphene-based inks that, in the near future, may replace the complex 3D structure of tissues with enhanced properties ,116,117.Bio-imaging, which focuses on imaging selected biological entities, remains a crucial aspect of regenerative medicine . ImagingThe most often used imaging technique involves fluorescence. This has become the gold standard technique that is used in biology and regenerative medicine. In particular, Forster Resonance Energy Transfer (FRET) imaging  is most 2 [Some techniques, including computer tomography (CT), PET and MRI, and ultrasound imaging are already well-established and can be used in human studies. The EPRI technique is a relatively new technique under strong development. By using this method, one is able to detect and quantify multiple biological parameters in a tumor microenvironment in vivo in small animal models, including: pO2 ,135,136,2 ,137, and2 ,138. Usu2  or other2  are used2 . Raman s2 , ex vivo2 .Structures such as G, GO, and the related G-based composites that are used in bio-imaging are usually treated as platforms for the modification of existing contrast agents ,145. HowThe next and simultaneously higher level of bio-imaging development is the fabrication of multimodal materials (nanoparticles) that can simultaneously be used in more than one way, e.g., in photothermal and photodynamic therapy and tracking (imaging) ,156,157.Graphene-based materials in the form of flakes, layers, foams, nanofillers, nanodots, and other structured materials have had a profound impact on the regenerative medicine and biomedical fields. The cytotoxicity of these materials is strongly dependent on the intrinsic flake sizes that are used in the final material, the structure and shape of the material, its surface chemistry, and the type of cultured cells. The fundamental interactions between the surface and surface edges of materials with the biomolecules, drugs, and cells are driving the biological pathways, in many cases in still-unknown ways. The toxicity aspects of the structure of graphene/graphene oxide are to this day debatable and remain of high interest to the scientific community. Likewise, the structure of graphene/graphene oxide-based materials influences their possible application fields, which were briefly reviewed here and include anticancer drug delivery therapies, tissue engineering, and multimodal bio-imaging.Almost all of the drug delivery anticancer approaches rely on the successful adsorption of the drug on the surface of the graphene/graphene oxide structure. Many efforts have been focused on the use of a well-established anticancer drug, such as doxorubicin (Dox), and the use of versatile graphene-based and graphene-based hybrid vehicles for its delivery or co-delivery and imaging. However, to this day, the in vivo work has shown virtually no improvement in using such vehicles in comparison with pure drugs, or there have been no attempts to perform in vivo evaluations. This shows a challenging problem with anticancer therapeutics where the drug/material’s biodistribution, biodegradability, and the targeted effects are still unknown. Moreover, little has been done to derive new drugs using graphene-based materials as platforms. We see particular promise in the graphene quantum dots that have been used in multimodal therapies, which may, simultaneously, have selective sensitivity, be effective contrast agents for fluorescence imaging with a high quantum yield, and be used for photothermal and photodynamic therapy. Indeed, small doses of materials with proper characteristics were shown to be acceptable for cell culture growth and bio-imaging in terms of the cytotoxic effects.Interestingly, graphene-based materials remain highly successful in the tissue engineering field for the regeneration of a variety of tissues. However, most of the successful work has been performed on 2D substrate systems, which could prove that G/GO-based flake coatings are useful for various biomedical devices. On the other hand, G/GO have been used as nanofillers in a variety of hydrogels or soft-matter materials that add a desired functionality to the native materials, such as the possibility of adsorption for certain proteins or an electrically active percolation network and/or surface that triggers cell proliferation, adhesion, or differentiation. There is a lot of work that focuses these days on using G/GO in 3D systems, either as a pure network or as a hybrid with other materials, which holds great promise for the use of these materials in real-world biomedical applications. Based on the literature survey presented in this review, we believe that there remain many crucial aspects to consider before this field can progress further, namely: nanomaterial purity and characterization and proper definitions to ensure data reproducibility and appropriate data correlation.